In addition, participants accomplished a heart beat perception task and two selective attention tasks. Results revealed no differences between meditators and controls with respect to performance in duration reproduction or attentional capacities. Additionally no group difference in heart beat perception scores was found. Across all subjects, correlational analyses revealed several associations between performance in the duration reproduction tasks and psychophysiological changes, the latter being also related to heart beat perception scores. Furthermore, former findings of linearly increasing cardiac periods and decreasing skin conductance levels during the auditory duration estimation task (Meissner and Wittmann, Recent research suggests that bodily signals and interoception are strongly related to our sense of time. Mindfulness meditators train to be aware of their body states and therefore could be more accurate at interval timing. In this study,  The diversity of psychological and neurophysiological models of time perception is indicative of the fact that the neural substrates and the processes accounting for the experience of time are still unknown  during a duration reproduction task, with intervals of 8, 14, and 20 s, showed not only a linear increase of cardiac periods during the encoding of temporal intervals, but also revealed a positive relationship between the slope of this increase in cardiac periods and duration reproduction accuracy  and education (± 1 level of 5). Participants were recruited by advertisements in meditation centers, on online platforms of the universities, and by word of mouth. Meditators were included when reporting to regularly practice a form of meditation, which had a dominant orientation toward awareness of the present moment. Therefore, we included individuals if they participated in forms of mindfulness meditation, Vipassana meditation, or Soto Zen. Control subjects were required to have no experience with any form of meditation including Yoga or Tai-Chi. Age was restricted to the range between 21 and 50 years in order to constrain age-related effects in the psychophysical tasks and psychophysiological measurements. Participants had to be fluent in German and were additionally required to report good health and no known medical or psychological problems as assessed with a detailed screening questionnaire. All participants signed an informed consent and received a moderate financial compensation (€20) for participation.Twenty-two experienced mindfulness meditation practitioners with at least three years of continuous practice and at least 2 h of practice a week over the last 8 weeks were recruited, as well as The heart beat perception task consists of four heart beat counting intervals , during which participants are asked to attend to their own heart beats and count them silently  a visual and (2) an auditory stimulus with intervals of 8, 14, and 20 s duration. In each trial, a green square (resp. a sinus tone of 440 Hz) was first presented for one of the three durations. After the pause of either 4.5, 5, or 5.5 s duration a second yellow square (resp. a sinus tone of 500 Hz) appeared. It had to be stopped by pressing the space bar when participants felt that this second stimulus had reached the duration of the first. The reproduction task contained six presentations of each of the three durations summing up to 18 trials. At the beginning and the end of each stimulus, an onset resp. offset marker signal was sent from the Matlab program to the physiological acquisition software. Participants were requested not to use mental strategies such as inner counting but to rely on their subjective feeling of elapsed time. To further prevent mental counting, participants were additionally given a secondary working-memory task. Before each trial four numbers were presented. At the end of each trial one number appeared and the subjects responded whether the number was one of the four previously presented by pressing the left or right arrow button, respectively for “yes” and “no.” The accuracy of duration reproduction can be calculated from the temporal reproduction performance. In the following, the mean of the six reproduced time intervals of each of the three durations is referred to as “reproduced duration.” A duration reproduction score (referred to as “duration reproduction accuracy”) was calculated for each participant and each of the three durations from six trials according to the following equation:Similar to the heart beat perception score high scores (maximum 1) indicate accurate duration reproduction.Heart rate and electrodermal activity were continuously recorded using a BIOPAC MP 150 device  with AcqKnowledge 4.1 software for data acquisition. The sampling rate was 500 Hz for the ECG signal and 15.625 Hz for the electrodermal signal.Heart rate was measured using three disposable Ag/AgCl electrodes which were positioned in an Einthoven Lead I configuration and connected to the BIOPAC amplifier module ECG100C. Skin conductance was measured using two disposable Ag/AgCl electrodes which were attached to the thenar and hypothenar portions of the left hand and connected to the BIOPAC amplifier module GSR100C.Intervals between successive R peaks (cardiac periods) were extracted from the electrocardiogram signal using the peak-detection function implemented in AcqKnowledge 4.2. Cardiac periods were examined and screened for artifacts based on the procedure developed by Porges and Byrne . Cardiact-tests for normally distributed data, Mann–Whitney U-tests for non-parametric data, and Chi-Quadrat tests for categorial data. Group differences for performance in the auditory and visual duration reproduction tasks were explored by mixed-design ANOVAs, with the between-subjects factor “group”  and the within-subjects factor “interval” . To evaluate changes over time in cardiac periods and skin conductance levels separate mixed-design ANOVAs were performed for the encoding and reproduction phase of each duration interval. Due to individual differences in the length of the individually reproduced intervals, the analyses for physiological changes during the reproduction intervals for auditory and visual reproduction tasks were restricted to seconds that were available for all subjects. In the auditory reproduction task we analyzed the first 6, 8, and 12 s of the 8, 14, and 20 s intervals in the reproduction phase of the task, respectively. In the visual reproduction tasks the analyses were restricted to the first 6, 8, and 15 s for cardiac periods and 6, 8, 12 s for skin conductance levels of the 8, 14, and 20 s intervals in the reproduction phase of the task, respectively. Greenhouse-Geisser corrections for repeated measures ANOVAs were applied where appropriate.Differences between study groups where analyzed by Student-Individual linear slopes of cardiac periods and skin conductance levels for each of the 18 encoding intervals were determined using linear regression. In order to normalize the data, individual time series were converted into z-scores before performing the linear regression analyses. Individual slopes were then averaged for each participant across the six presentations for each of the three durations. Group differences between slopes were explored by mixed-design ANOVAs with “interval” as the within-subjects factor and “group” as between-subjects factor.Spearman-Rho's correlations were used to explore relationships between behavioral results in the duration reproduction tasks, associated psychophysiological changes, heart beat perception scores, and results in the attention tests.p ≤ 0.05. Whenever multiple tests were conducted, the significance level was Bonferroni-corrected.For statistical tests the significance level was set to On average, the mindfulness meditators had a meditation experience of 10.4 years (± 7.5 SD) and had practiced 7 h per week (± 5 SD). Meditation and control groups did not differ with respect to age, education, body mass index, physical activity, or current stress levels. As expected, meditators showed significantly higher values of self-reported mindfulness in the “Freiburg Mindfulness Inventory” (FMI) in both subscales of “presence” and “acceptance,” as well as in the sum score  in meditators and 0.78 (± 0.15 SD) in controls, with no significant difference in-between ; p = 0.0163], in that meditators showed a sharper decline of skin conductance levels than controls .Since no differences between groups were revealed the correlational analyses were performed for the whole study sample , while the three components of the ANT alerting, orienting, and executive function and ANT reaction times showed no significant association (results not shown). Correct answers in the TAP subtest for divided attention also correlated positively with reproduced duration during the 8 s-encoding interval of the auditory reproduction task , and with reproduced duration and reproduction accuracy during the 20 s encoding interval of the visual reproduction task . Alerting, orienting and executive function in the ANT as well as reaction times did not show such correlations (results not shown).Correct answers in the TAP subtest for divided attention correlated positively with heart beat perception scores across all subjects  and with the slope of skin conductance levels during the 8 s-encoding interval of the visual task  , did not differ between the two groups studied. Although mindfulness meditators learn to focus on their bodily sensations, they did not differ from carefully matched controls with regard to interoceptive awareness in the heart beat perception task. Two recent studies have also failed to show that experienced meditators are more accurate in the heart beat perception task  the found number of correlations cannot be judged as high. That is, one cannot conclude that the specific psychophysiological measures employed  are sufficient indicators of subjective time. Rather these physiological measures may contribute to the feeling of passing time in a similar way as they are selective indicators of the present condition of the body Craig, . SubjectThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To identify the main severe potential drug-drug interactions in older adults with dementia and to examine the factors associated with these interactions.This was a cross-sectional study. The enrolled patients were selected from six geriatrics clinics of tertiary care hospitals across Mexico City. The patients had received a clinical diagnosis of dementia based on the current standards and were further divided into the following two groups: those with severe drug-drug interactions (contraindicated/severe) (n=64) and those with non-severe drug-drug interactions (moderate/minor/absent) (n=117). Additional socio-demographic, clinical and caregiver data were included.® database.Potential drug-drug interactions were identified using Micromedex Drug Reax 2.0 A total of 181 patients were enrolled, including 57 men (31.5%) and 124 women (68.5%) with a mean age of 80.11±8.28 years. One hundred and seven (59.1%) patients in our population had potential drug-drug interactions, of which 64 (59.81%) were severe/contraindicated. The main severe potential drug-drug interactions were caused by the combinations citalopram/anti-platelet (11.6%), clopidogrel/omeprazole (6.1%), and clopidogrel/aspirin (5.5%). Depression, the use of a higher number of medications, dementia severity and caregiver burden were the most significant factors associated with severe potential drug-drug interactions.Older people with dementia experience many severe potential drug-drug interactions. Anti-depressants, antiplatelets, anti-psychotics and omeprazole were the drugs most commonly involved in these interactions. Despite their frequent use, anti-dementia drugs were not involved in severe potential drug-drug interactions. The number and type of medications taken, dementia severity and depression in patients in addition to caregiver burden should be considered to avoid possible drug interactions in this population. Anatomical and physiological changes and impairments in multiple organs and systems associated with aging can affect the pharmacokinetics and pharmacodynamics of drugs A high associated comorbidity has been previously reported for these patients, of whom 61% have three or more diseases. Of them, musculoskeletal, cardiovascular, metabolic/endocrine and gastrointestinal disorders are the most common Over time, new drugs (psychotropic and anti-dementia drugs) will emerge for the treatment of dementia patients, giving rise to new interactions and challenges for clinicians.The factors associated with potential DDIs in elderly patients without dementia have been previously reported and include age, female gender, polypharmacy and high comorbidity To our knowledge, there are limited data available on the factors contributing to severe potential DDIs caused by drugs prescribed to dementia patients. Thus, it is important to identify the main severe potential DDIs and the factors associated with these interactions in older adults with dementia.We used the database from the “Validation of the Quality of Life in Alzheimer's Disease (QOL-AD) scale in Mexican patients with dementia” We included data from 181 outpatients who were 60 years of age or older and had a clinical diagnosis of dementia. Dementia  had been previously diagnosed by a group of geriatricians dedicated to the study of dementia according to international criteria; these criteria have been described in a previous publication versus “non-severe”.Potential DDIs were identified using Micromedex Drug Reax 2.0® database. We focused our study on the worst potential DDIs because they tend to have a greater clinical impact on patients' well-being. For this analysis, potential DDIs were classified into the following 2 general categories: contraindicated/severe potential DDIs were combined in a “severe interactions” category to represent the worst possible outcomes of a given drug combination and moderate/minor/absent potential DDIs were combined to form a “non-severe interaction” group. The groups were labeled and compared as “severe” A list of medications taken by the elderly subjects was generated based on a review of the clinical records and it was confirmed by the physicians. The quantitative variable in this study was the total number of medications taken.We used a Spanish version of the Charlson index. This scale includes a list of 19 diseases and their complications. Comorbidities were determined by caregiver interviews and a review of the clinic's records and the total score was used for this variable Caregiver burden was determined by the Screen for Caregiver Burden . It has been adapted and validated for use in the Mexican population. We analyzed the total score achieved on this test (ranging from 0 to 100) Another measure employed was a Spanish version of the Dysexecutive Questionnaire (DEX), a 20-item questionnaire that uses a Likert scale ranging from 0 to 4 points. For this measure, we assessed the total score (ranging from 0 to 80), with the primary caregiver as the interviewed party (proxy) The Mini-Mental State Examination (MMSE) was used as an indirect indicator of dementia severity. We used a version that has been previously validated for use in the Mexican population The Barthel index was applied to assess the Spanish version of the Basic Activities of Daily Living (ADL) scale. We analyzed the total score (ranging from 0 to 100) The Lawton Scale was used to evaluate instrumental activities of daily living (IADL). This scale contains eight items, and we analyzed the cumulative score (ranging from 0 to 8) To evaluate the presence of depression, we used the depression subscale of the Neuropsychiatric Inventory (NPI-D). The frequency score was classified as presence=1 or absence=0 for a 12-item inventory validated for use in the Mexican population that, was completed by the caregivers The original project was approved by the National Scientific Research Committee of IMSS . All patients and caregivers provided written informed consent.2 test  for qualitative variables. The mean ± SD and Student's t-test  were used to analyze quantitative variables. The variables associated with potential DDIs (p<0.05) were included in a multivariate model using multiple logistic regression. We used STATA® version 11 software  to perform analysis.Descriptive analyses were performed using frequencies, percentages and Pearson's ChiWe evaluated 181 outpatients from the original study, including 57 males (31.5%) and 124 females (68.5%) with a mean age of 80.11±8.28 years. The patient characteristics are displayed in The top ten potential DDIs are included in The independent variables were compared according to the severity of the potential DDIs (severe or non-severe). Comorbidity, depression, polypharmacy, dementia severity and caregiver burden were the most significant factors and were thus used in comparative analysis .The significant variables were included in the final regression model. This model revealed that the variables associated with severe potential DDIs were depression, the number of drugs taken, dementia severity and caregiver burden. Depression was by far the most significant, followed by the number of drugs taken. In addition, caregiver burden and dementia severity exhibited positive but diminished associations with the severe potential DDIs .This study provides insights into the frequency of severe potential DDIs in older people with dementia (35.3%). The frequency observed in this study is higher than that of a previous report 26.6%) 6.6% 2,15One of the main objectives of this study was to identify the main severe DDIs because they are the most likely to have poor outcomes in the clinical setting; however, the results of studies based on Micromedex reports should be considered with caution. For example, the main potential DDI  determinConflicting results have been reported regarding the increased risk of the second most frequent potential DDI observed in this study (clopidogrel/omeprazole). Studies based on Micromedex reports have indicated that their concurrent use may result in an increased risk of bleeding. However, a recent clinical study found that in healthy volunteers omeprazole, esomeprazole and lansoprazole caused a relative decrease in exposure to the active metabolite of clopidogrel by of up to 50%, leading to modest decreases in its antiplatelet effect Regarding the factors associated with severe potential DDIs, the final regression model and comparative analysis revealed that the number of medications taken is a risk factor for the presence of severe potential DDIs, in agreement with other studies analyzing these interactions in older adults with and without dementia A secondary objective of this study was to identify different factors associated with the presence of potential DDIs; in this regard, depression was the main neuropsychiatric symptom associated with severe potential DDIs. One of the most widely used treatments for depression is the selective serotonin reuptake inhibitors (SSRIs). SSRIs are currently recommended as the first-line treatment for depression in older adults according to several clinical guidelines A modest association was found between caregiver burden and severe potential DDIs  and 4. PA greater number of diseases affected the patients with a lower MMSE score than those with a higher MMSE score; therefore, the increased numbers of prescription drugs taken by the patients in this study may be explained by the successive selection of patients attending memory clinics and may not be directly related to their cognitive statuses. Therefore, longitudinal studies should be conducted to confirm this relationship.There were several limitations of this study. It is important to note that the hospitals at which the outpatients were recruited are specialized institutions, and this study did not include institutionalized or community-dwelling older adults. Thus, our results can only be applied to institutions with similar characteristics. In addition, this was a cross-sectional study, and further research examining the longitudinal association between caregiver burden and other factors and severe potential DDIs is necessary.Another limitation was the lack of data on the outcomes of the potential DDIs identified, which is attributed to the nature of the study. The scores of the MMSE, which was used as a measure of the severity of dementia, should be considered with caution due to the limitations associated with its use in older adults with a low education level.Despite these limitations, we believe that this study contributes to current knowledge of the frequency of potential DDIs and the relationships between severe potential DDIs and the contrubuting factors in dementia patients.Bogetti-Salazar M assisted with the data collection, protocol development and manuscript preparation. González-González C, Juárez-Cedillo T and Sánchez-García S contributed to the protocol development and manuscript preparation. Rosas-Carrasco O contributed to the data collection, protocol development, statistical analysis and manuscript preparation.


Mitochondrial dysfunction is a hallmark of aging, and underlies the development of many diseases. Cells maintain mitochondrial homeostasis through a number of pathways that remodel the mitochondrial proteome or alter mitochondrial content during times of stress or metabolic adaptation. Here, using yeast as a model system, we identify a new mitochondrial degradation system that remodels the mitochondrial proteome of aged cells. Unlike many common mitochondrial degradation pathways, this system selectively removes a subset of membrane proteins from the mitochondrial inner and outer membranes, while leaving the remainder of the organelle intact. Selective removal of preexisting proteins is achieved by sorting into a mitochondrial-derived compartment, or MDC, followed by release through mitochondrial fission and elimination by autophagy. Formation of MDCs requires the import receptors Tom70/71, and failure to form these structures exacerbates preexisting mitochondrial dysfunction, suggesting that the MDC pathway provides protection to mitochondria in times of stress.DOI:http://dx.doi.org/10.7554/eLife.13943.001 Our cells contain compartments called mitochondria, which provide energy and serve as a home for many metabolic pathways that are critical for life. Changes in mitochondrial activity can contribute to aging and to the development of several age-associated diseases. However, our cells contain systems that detect changes in the performance of mitochondria and can act to re-establish a healthy state. These systems respond to stress or changes in metabolism by degrading particular proteins in the mitochondria. This enables damaged or unwanted proteins to be replaced with new proteins, but it is not clear how these mitochondrial defense systems work to keep mitochondria healthy as an organism ages.Budding yeast is a good model in which to study the aging process because the performance of this yeast’s mitochondria change in a characteristic way as the yeast ages. Previous studies have shown that these changes are caused by alterations in another cell structure called the lysosome, which can store nutrients and is where many proteins are degraded.Here, Hughes et al. used yeast cells to investigate how mitochondrial defense systems operate during aging. The experiments reveal an entirely new mitochondrial protein degradation system that helps to keep the mitochondria healthy as the yeast cells age. This process is rapidly triggered by changes in lysosome activity and results in certain proteins in each mitochondrion being sorted into a small compartment made from a part of the mitochondrion. This compartment is released from the mitochondrion and then travels to the lysosome where the proteins are destroyed. Inhibiting the formation of these compartments results in mitochondria being more sensitive to cellular stress.The next steps following on from this work are to find out exactly what role this mitochondrial defense pathway plays in cells and why it targets only a small set of all the proteins found in mitochondria.DOI:http://dx.doi.org/10.7554/eLife.13943.002 Mitochondria play a central role in cellular metabolism. Metabolic pathways that occur within mitochondria include the TCA cycle, oxidative phosphorylation, amino acid metabolism, and biosynthesis of lipids, heme, and iron-sulfur clusters . Because+-ATPase proteases within all mitochondrial compartments that promote the degradation of unassembled protein complex subunits, oxidatively damaged proteins, and tail-anchored membrane proteins mistargeted to the mitochondrial outer membrane , which deliver oxidized mitochondrial proteins to the lysosome for degradation . PINK1/PA key area in which many of these pathways function to promote mitochondrial homeostasis is during the aging process. There has long been a strong association between cellular aging and mitochondrial dysfunction . HoweverS. cerevisiae, defined by the number of times an individual yeast cell produces a daughter cell, functions as a model system for understanding fundamental aspects of cellular aging, including age-dependent mitochondrial dysfunction . However, vacuolar proteases rapidly degrade proteins taken up by autophagy and thus frequently prevent protein detection in the vacuole. Therefore, we re-assessed the presence of vacuolar Tom70-GFP in aged cells lacking  vacuole . In youn vacuole . However vacuole . Tom70-G4△ cells .10.7554/ATG5, a gene essential for all forms of autophagy   , and the-ATPase) . Loss of-ATPase) . To test-ATPase) , recapit-ATPase)  (Hughes -ATPase) . Consist-ATPase)  using thPase)  phenylhydrazone (FCCP). Although treatment of cells with these inhibitors caused mitochondrial depolarization equal to or greater than conc A , we did In addition to mitochondrial depolarization, loss of vacuolar acidity also causes oxidative stress in cells through an undefined mechanism . TherefoDuring the analysis of mitochondrial protein degradation in young cells, we noticed a distinct structure containing Tom70-GFP that formed prior to the appearance of Tom70-GFP foci within the vacuole , white *dnm1∆ cells formed MDCs normally at two hours after conc A addition, at later timepoints, MDCs grew very large in these cells and were not released from mitochondria to the vacuole  formed normal MDCs after two hours of conc A treatment  system . As illutom70△ and tom70△ tom71△ cells . Loss of TOM70 alone severely blocked MDC formation, and the loss of both TOM70 and TOM71 was completely inhibitory . This strain was created by transformation and insertion of NotI-digested plasmid pAG306-GPD-eGFP-ATG8 chr I, which is described below.All yeast strains are derivatives of ae S288c  and are ae S288c . Oligos ae S288c . pBS34 wae S288c . A colleae S288c  to the yae S288c  using a ae S288c . StrainsGFP-ATG8 from the constitutive GPD promoter. pAG306-GPD-eGFP-ATG8 chr I was constructed in two steps. First, we created pAG306-GPD-eGFP-ccdB chr I, a plasmid for high expression of N-terminal GFP fusion constructs from the GPD promoter that can be integrated into chromosome I (199456–199457) after NotI digestion. We generated pAG306-GPD-eGFP-ccdB chr I by ligation of a SmaI-digested fusion PCR product that contained two ~500 base pair regions of chromosome I flanking a NotI site into AatII-digested pAG306-GPD-eGFP-ccdB (Addgene plasmid 14308) (ATG8 into pAG306-GPD-eGFP-ccdB chr I from donor Gateway plasmid pDONR201-ATG8 (HIP ID ScCD00011665) using LR clonase according to manufacturer’s instructions  (pAG306-GPD-eGFP-ATG8 chr I is a plasmid that can be integrated into an empty region of yeast chromosome I after digestion with the restriction enzyme NotI. The plasmid expresses d 14308) . We geneham, MA) .6 cells/ml before the start of all aging and MDC assays. This period of overnight log-phase growth was carried out to ensure vacuolar and mitochondrial uniformity across the cell population. Cells were cultured in YEPD  for all experiments. Yeast Complete (YC) medium used during construction of the Tom70-mCherry GFP strain collection was previously described . Cells were harvested by centrifugation, and imaged live in all experiments. The number of cells with MDCs or Tom70-GFP in vacuole-localized autophagosomes was quantified in each experiment at the appropriate timepoint. For screening of the Tom70-mCherry/GFP collection to identify MDC substrates, all strains were grown in batches of 20 following the same procedure used for all other MDC assays.7 cells from a 15 hr YEPD log-phase culture twice in phosphate buffered saline, pH 7.4 (PBS) and resuspended in PBS with 3 mg/ml Sulfa-NHS-LC-Biotin (Thermo Fisher Scientific) at a final concentration of 2.5 x 107 cells/ml. Cells were incubated for 30 min at room temperature (RT), followed by two washes in PBS and one in YEPD. Biotinylated cells were resuspended in 10 ml of YEPD at 2.5 x 106 cells/ml and recovered with shaking for 2 hr at 30°C. These cells were used to seed cultures at a density of 2 x 104 biotinylated cells/ml in YEPD for aging experiments. To initiate the MEP aging program, β-estradiol (1 μM) was added to cultures and cells were grown at 30° for an appropriate time to obtain cells of a desired age . Cell densities never exceeded 4 x 106 cells/ml. 1 x 108 total cells were harvested for purification and microscopy analysis at each timepoint.For aging experiments, we used the Mother Enrichment Program (MEP)  coupled 8 cells/ml. Cells were then incubated for 30 min at RT with 25 μl of streptavidin-coated magnetic beads . Cells were then washed twice in PBS, resuspended in 8 ml of PBS, and loaded onto a LS MACS column (Miltenyi Biotec) that had been equilibrated with 5 ml of PBS. Cells on the column were washed twice with 8 ml of PBS. Columns were then removed from the magnetic field and aged cells were eluted by gravity flow with 8 ml of PBS. Cells were centrifuged to concentrate them for microscopy analysis.For purification after aging, cells were washed twice with PBS, and then resuspended in 500 μl of PBS at a density of 2 x 106) (Thermo Fisher Scientific) staining was carried out as previously described  (Thermo Fisher Scientific) staining was carried out exactly as DiOC6 staining, except that cells were incubated with 50 nM TMRM.3,3’-dihexyloxacarbocyanine iodide  staining of bud scars. For this analysis, 5 μg/ml calcofluor was included in the first post-staining wash step prior to imaging. For each experiment, cells were grouped into 3 categories based on age range: Young (0–4 budscars); middle-aged (7–12); and old (>17).2 CCD camera  and quantified and processed using Metamorph version 7.1.1.0 imaging software. For DiOC6 experiments, cells were scored as reduced if they exhibited at least a 2-fold decrease in mean fluorescence intensity. Microscopy analysis in For fluorescence microscopy analysis in all figures except those noted below, cells were visualized under 60X oil magnification using a Nikon Eclipse E800 with the appropriate filter set. Images were acquired with a CoolSNAP HQ2 CCD camera (Photometrics). Images were deconvolved with SoftWoRx 6.5.2 image analysis software  and images of the vacuole and mitochondria were IsoSurface rendered in Imaris 8.2.0 software .For 6 or TMRM staining, cells were analyzed on a BD LSRFortessa X-20 equipped with the appropriate filter sets. At least 10,000 events were analyzed for each sample. Statistical analysis for After DiOC7 log-phase cells were resuspended in 100 μl H2O. An equal volume of 0.2 M NaOH was added to the cell suspension, and cells were incubated 5 min at room temperature. Samples were then centrifuged at 20,000 x g for 10 min at 4°C. Pellets were resuspended in SDS-lysis buffer  containing protease inhibitors  for western blot analysis. Immunoblotting was carried out exactly as previously described  and four reviewers, one of whom, Andrew Dillin, is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "Selective sorting and destruction of mitochondrial membrane proteins in aged yeast" for consideration by The following individuals involved in review of your submission have agreed to reveal their identity: Suzanne Pfeffer, Thomas Nyström, and Liza Pon (peer reviewers).The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.As you will find, all three reviewers and the Reviewing editor are enthusiastic about the body of work presented. The authors put forth very compelling and interesting data to suggest that mitochondria in yeast might possess a quality control system to selectively remove outer membrane proteins in a vesicle like structure that is ultimately destined for the lysosome. The idea of a mitochondrial derived compartment that plays a role in mitochondrial integrity is exciting and provides an explanation for selective degradation of some mitochondrial clients.While there are differences among some of the reviewers’ comments, a shared theme of all includes better characterization of the MDC. This is particularly relevant since this is the first description of this compartment and the similarities/differences of mitochondrial derived vesicles (MDVs) needs to be better flushed out. Along these lines, the origination of the MDC with the autophagy machinery needs a deeper look and each reviewer has suggested a few experiments. Finally, the analysis of which mitochondrial perturbations can generate the MDC needs a little more analysis and suggestions to include mitochondrial toxins is a good place to start. Overall, the reviews are very favorable and please contact the Reviewing editor if you need additional input.Reviewer #1:In the manuscript submitted by Hughes et al. entitled "Selective sorting and destruction of mitochondrial membrane proteins in aged yeast", the authors put forth very compelling and interesting data to suggest that mitochondria in yeast might possess a quality control system to selectively remove outer membrane proteins in a vesicle like structure that is ultimately destined for the lysosome.The data to support such events include:1) Tom70-GFP is found inside the vacuole  of old cells and conditions where the vacuole acidity is altered (conc A treated cells).atg5 dependent.2) The trafficking to the vacuole appears partially dependent upon the autophagy pathway since it is atg32 mutant cells.3) Trafficking to the vacuole is independent of mitophagy. Experiment shows Tom70-GFP in vacuole in 4) The origin of the vesicle is suggested to require the fission machinery since Dnm1 mutations block Tom70-GFP accumulation in vacuole.5) During aging, the vacuole becomes fragments and the appearance of small vesicles containing Tom70-GFP is found.6) Tom70-GFP vesicles appear shortly after depolarization of mitochondria before entry to vacuole.7) Cargo is selective to outer membrane proteins that require Tom70/71. Tom20 not required.8) Trafficking to the vacuole seems to include both newly synthesized and older proteins, suggesting it is not a back-up of non-translocated proteins.In summary, the paper is very fascinating and suggests a new avenue for mitochondrial quality control that could be distinct from mitophagy and mitochondrial derived vesicles (MDVs) found in metazoan cells.To compare and contrast MDVs from MDC:Cargo for MDVs include oxidized proteins, lipids, outer membrane proteins and the target is lysosomes and peroxisomes. MDVs require PINK1/Parkin, DRP-1 independent and autophagy independent. The stress that causes MDVs to form include antimycin and other harsh mitochondrial toxins.The cargo of MDCs contains outer membrane proteins and import carriers that are targeted to the lysosome. Dnm1 dependent and autophagy independent. Yeast do not have a PINK1/Parkin pathway, so it is unknown if they could be involved if the MDC is conserved in metazoa. The aging process and de-acidification of the lysosome allows observation of MDCs.The major questions to address form this work:1) While the RITE experiment is very clever and suggests that newly made and older proteins can be found in MDCs , how sure are the authors that fused proteins of interest are not sitting in the cytoplasm due to the inability to be translocated to the mitochondria? Because all perturbations tested resulted in membrane depolarization and the Tom70/71 complex is essential for this process, which requires membrane polarization for import, could this be a clearance mechanism for non-transported proteins? For example, could a cytoplasmic localized Tom70-GFP be found in the lysosome using the conditions described here?2) Do other mitochondrial stresses also induce MDC? Oxidative stress or starvation?ATG32 experiments are very interesting. However, my understanding of the literature is that mitophagy mediated by ATG32 only occurs during starvation. It is not clear if the negative results showing that ATG32 is not required were done under vegetative or starved conditions where ATG32 is required.3) The 4) Because there is close correlation of "fizzed" mitochondria with appearance of the MDC, does blocking fusion give the same result?pep4 deletions strain. However, without having a similar tool to see the trapped cargo  it might be premature to rule out that the MDCs do not travel to other compartments or might even fuse with the plasma membrane and allow deposition of cargo to the outside of the cell.5) The authors correctly claim that the MDC travels to the vacuole because they can see the cargo accumulate there in a 6) The idea of the MDC is very compelling, but EM data to describe its structure would be more informative (i.e. does it have a membrane?).Reviewer #2:eLife will very much appreciate.This is a very well written and very interesting paper that describes a new pathway for the turnover of mitochondrial nutrient transporters that is distinct from classic mitophagy and may resemble the formation of mitochondrial derived vesicles seen in mammalian cells by McBride and coworkers – however this pathway requires mitochondrial fission. The authors have identified a select group of Tom70/Tom71 import substrates that are targeted to the vacuole upon loss of vacuole acidification or loss of mitochondrial membrane potential. Using a clever tagging approach they show that already synthesized and apparently translocated proteins participate in this process, implying that they may be organized by Tom70/71 after synthesis. The data are of high quality and the findings of broad interest that readers of Comments.1) The authors refer to this pathway as a quality control pathway that degrades mitochondrial components from aged/damaged mitochondria, but since it also is induced upon loss of vacuole acidification, couldn't it just as well be described as a pathway to regulate mitochondrial function when nutrients are less available (and metabolic need is less) that is distinct from the Atg32 pathway? Please discuss and consider modifying text/Abstract/impact statement2) Please comment on whether the levels of Tom70/71 and/or their substrates change upon vacuole neutralization or mitochondrial membrane depolarization.Overall, well done and so nicely presented such that non-experts can appreciate it.Reviewer #3:ATG5-dependent but ATG32-independent degradation of MDCs by the vacuole. The authors also provide evidence that MDCs are distinct from MDV.The authors identified a mechanism for mitochondrial outer membrane proteins and mitochondrial carrier proteins from the inner membrane are selectively removed from mitochondria in old cells and in all cells in response to loss of vacuolar acidity. Protein removal occurs by DNM1- and Tom70/71-dependent formation of a mitochondria derived compartment (MDC) and The work is novel and of clear interest to the field. Major concerns regarding this work are outlined below.PEP4-dependent degradation of Tom70-GFP to free GFP in old compared to middle aged cells?1) The resolution of fragmented vacuoles and Tom70-GFP in DNM1 is required for MDC formation. If the authors want to make the claim that MDCs are released from mitochondria by Dnm1-dependent mitochondrial fission, they need to provide evidence for a direct role of Dnm1 in MDC formation. For example, does Dnm1 localize to MDCs as they bud from mitochondria?2) The authors find that ATG5 is required for degradation of MDCs by the vacuole, and propose that MDC degradation occurs by macroautophagy. If they want to make the claim that MDCs are enveloped in autophagosomes prior to degradation in the vacuole, they need to test whether autophagosome marker proteins are associated with MDCs and whether deletion of DNM1 affects this localization.3) The authors also find that the 4) The finding that select proteins are excluded from MDCs in conc A-treated cells is interesting and important.A) Since mitochondria are generally more fragmented in old yeast compared to young yeast, it is not clear that the punctate Tom70-GFP-containing structures observed in old yeast are MDCs. Is Tim50 excluded from Tom70-GFP-containing structures in old yeast?B) To confirm that there is degradation of a select set of proteins in MDCs, it would be interesting to see if Tim50 is not degraded by autophagy in conc A-treated cells.tom70/71∆ cells are more sensitive to conc A because they have defects in protein import or defects in MDC formation. The text should be revised accordingly.5) It is not clear whether 6) Although loss of vacuolar acidification does result in a decline in mitochondria membrane potential, it is not clear that the proteins that are removed in MDCs are dysfunctional. In fact, the authors speculate that MDC-dependent remodeling of mitochondria alters mitochondrial function in metabolite transport. Therefore, it is possible that the MDC pathway is not a quality control pathway, but a pathway for control of mitochondrial function. The manuscript would benefit from studies to distinguish between these two possibilities. For example, is loss of mitochondrial membrane potential or function sufficient to trigger MDC formation? Either outcome would be of interest to the field.Reviewer #4:pep4 mutant, that the mitochondrial protein Tom70 is degraded in old yeast cells by the vacuole in an Atg5-dependent manner indicating a role for autophagy in the delivery of Tom70 the vacuole. In addition, the delivery of Tom70 was abrogated in dnm1 mutant cells suggestion that fission from mitochondria is required for proper rerouting of Tom70 to degradation. It is further shown that loss of vacuolar acidity, which the authors have shown previously occurs early during mother cell aging and causes mitochondrial dysfunction, triggers the destruction of Tom70 via the route discovered. Prior to delivery to the vacuole, Tom70 congregated into large foci, named mitochondria-derived compartment (MDC) and this assembly of Tom70 was independent on both Dnm1 and Atg5.In this paper, the authors report on the discovery of a new mitochondrial quality control system operating in yeast. They first show, taking advantage of Most interestingly, MDC was then demonstrated to be specific for mitochondrial proteins lacking the cleavable presequence and the authors show that preexisting mitochondrial proteins  are targeted to MDC in a Tom70-dependent manner.This is a very interesting and well-written paper, which reports on a new concept of mitochondrial quality control in yeast. As the authors themselves point out, there are several issues that remain to be elucidated, for example the mechanisms behind MDC formation and its specificity, how MDCs are mechanistically transferred to the vacuole, and the physiological significance of MDC in yeast aging/rejuvenation. Nevertheless, the paper entails a very important discovery and the experiments have taken this discovery to a level that warrants publication. I have a few comments and questions that the authors might want to consider for further clarifying some points of the paper:Fis1 cannot compensate for the lack of Dnm1 as the dnm1 deletion completely blocked Tom70 delivery to the vacuole. Nevertheless, it would be interesting to see if a fis1 deletion have some kind of defect also as Fis1 is required for proper localization of Dnm1. Also, as dnm1 deletion mutants display a defect in endomembrane trafficking one wonders if such effects could be part of the problem in delivering MDCs to the vacuole? Cannot come up with a simple genetic way/experiment to separate mitochondrial fission defects form endomembrane trafficking defects but the authors could perhaps comment on this.1) Concerning the requirement for fission. It is clear from the data that 2) In the experiments using conc-A to trigger loss of vacuolar acidity, the delivery of Tom70 to the vacuole is interesting for several reasons and one wonders if this could be discussed a bit further. First, loss of vacuolar acidity is effectively blocking endomembrane trafficking to the vacuole. Does this mean that the Atg5-dependet route is "immune" to such pH effects in the vacuole or does it indicate that MDCs are transferred from the mitochondria to the vacuole by a more direct route: mitochondrial-vacuolar contacts?The other interesting possibility (as the phenomenon is seen in old cells and during conc-A treatment) is that a loss of vacuolar acidity is, in fact, required for triggering the MDCs formation and delivery. Could it be tested perhaps if drugs affecting mitochondrial function but leaving the pH of vacuole intact can trigger MDC formation and delivery? In other words, approach the generality of the system discovered upon mitochondrial dysfunction.dnm1 strain – perhaps a better version could be added? Also, as the experiment uses Vph1 as a reporter, does not this indicate that the vesicle-like structures are derived from the vacuole (vacuolar fragmentation?) rather than formed by fission from mitochondria? At present, it is a bit unclear what these structures are, how they were formed, and how they relate to the MDC route discovered.3) It is stated that the formation of small vesicle-like structures (Tom70 associated) in the cytoplasm of old cells require mitochondrial fission (Dnm1) indicating that these membrane structures are derived from mitochondria. It is very hard to see these structures in the picture of the atg5 and dnm1, but not atg32, mutants display a similar loss of potential upon conc-A treatment?4) The authors suggest that MDC formation helps protect mitochondria from vacuole-induced stress as a greater loss in membrane potential is seen in tom70/71 mutants subjected to conc A treatment. Is it possible that this loss of potential in tom70/71 mutants could be related to other shortcomings than the formation of MDC? Also, did dnm1 and fis1 deletion mutants display a somewhat longer replicative lifespan, is it possible that a reduction in mitochondrial fragmentation (positive effect) in these mutants offsets the negative effect of an abrogated MDC response? Is the lifespan on tom70, atg5, atg32 mutants known?5) Since aging is the entry port in this paper, I wonder if the authors could discuss briefly the possible significance of MDC in yeast longevity? As  Reviewer #1:1) While the RITE experiment is very clever and suggests that newly made and older proteins can be found in MDCs , how sure are the authors that fused proteins of interest are not sitting in the cytoplasm due to the inability to be translocated to the mitochondria? Because all perturbations tested resulted in membrane depolarization and the Tom70/71 complex is essential for this process, which requires membrane polarization for import, could this be a clearance mechanism for non-transported proteins? For example, could a cytoplasmic localized Tom70-GFP be found in the lysosome using the conditions described here?We appreciate the reviewer’s concerns that the MDC may be a collection of newly synthesized, unimported proteins, and we specifically discuss this possibility in the paper. However, several pieces of data in the manuscript strongly support the conclusion that the MDC degrades preexisting mitochondrial proteins. The first is the inclusion of cycloheximide in combination with the RITE tag experiment in 2) Do other mitochondrial stresses also induce MDC? Oxidative stress or starvation?In response to the reviewers’ suggestion, we tested whether mitochondrial depolarizing agents FCCP and Antimycin A could induce MDC formation. We also tested hydrogen peroxide and amino acid starvation as well. All four treatments failed to induced MDC formation, suggesting the MDC is triggered by an unidentified signal originating from loss of vacuole acidity. The results from the FCCP, Antimycin A, and hydrogen peroxide experiments showing their impacts on mitochondrial membrane potential and structure but failure to trigger MDCs have been included in the manuscript as new 3) The ATG32 experiments are very interesting. However, my understanding of the literature is that mitophagy mediated by ATG32 only occurs during starvation. It is not clear if the negative results showing that ATG32 is not required were done under vegetative or starved conditions where ATG32 is required.ATG32 in the process were conducted in old cells or cells treated with concanamycin A to induce MDC formation, which are not conditions in which ATG32 has been previously implicated or tested. Our results show that ATG32 is not required for MDC formation, and the point of including this mutant is to show that the MDC pathway is distinct from the previously characterized Atg32-dependent mitochondrial autophagy pathway.The experiments addressing the role of 4) Because there is close correlation of "fizzed" mitochondria with appearance of the MDC, does blocking fusion give the same result?FZO1 does not cause MDC formation. However, the interpretation of this experiment is not straightforward, because loss of FZO1 causes yeast cells to lose mitochondrial DNA. Loss of mitochondrial DNA has previously been shown to interfere with vacuole-mitochondrial signaling (PMID: 23502676). Thus, although our results were negative, they may be complicated by the lack of mitochondrial DNA. Because of this, we did not include this data in the revised manuscript.In response to the reviewer’s suggestion, we performed experiments testing whether MDCs are constitutively formed in mitochondrial fusion mutants. We found that deletion of the fusion protein 5) The authors correctly claim that the MDC travels to the vacuole because they can see the cargo accumulate there in a pep4 deletions strain. However, without having a similar tool to see the trapped cargo  it might be premature to rule out that the MDCs do not travel to other compartments or might even fuse with the plasma membrane and allow deposition of cargo to the outside of the cell.We agree with the reviewer that there may be other fates of MDCs beyond the vacuole that we cannot visualize at this time. If other fates exist, they may be revealed in longer term studies. The text of the manuscript has been modified to reflect this uncertainty.6) The idea of the MDC is very compelling, but EM data to describe its structure would be more informative (i.e. does it have a membrane?).We agree with the reviewer that EM data describing the MDC will be very informative. However, these studies are non-trivial to perform and while ongoing, have yet to be achieved. We believe that the present study already provides a great deal of support for an unexpected discovery as presented.Reviewer #2:1) The authors refer to this pathway as a quality control pathway that degrades mitochondrial components from aged/damaged mitochondria, but since it also is induced upon loss of vacuole acidification, couldn't it just as well be described as a pathway to regulate mitochondrial function when nutrients are less available (and metabolic need is less) that is distinct from the Atg32 pathway? Please discuss and consider modifying text/Abstract/impact statement.We agree with the reviewer that the MDC pathway may not necessarily be a quality control pathway, but rather, a pathway involved in metabolic homeostasis. With the current set of data, we cannot exclude either possibility. As suggested, we have gone through the manuscript and modified our reference to the MDC pathway as a quality control pathway, and instead discuss it as a protein degradation pathway that responds to changes in the pH of the vacuole and modifies the mitochondrial proteome for reasons that remain to be determined.2) Please comment on whether the levels of Tom70/71 and/or their substrates change upon vacuole neutralization or mitochondrial membrane depolarization.We would expect that the levels of these proteins would change under conditions of MDC activation because they are targeted for degradation and removed from mitochondria. However, as can be seen in the western blot in Reviewer #3:1) The resolution of fragmented vacuoles and Tom70-GFP in atg5△, and dnm1△ cells taken with a DeltaVision microscope showing mitochondria (Tom70-GFP) and vacuole (Vph1-mCherry). As can be seen in the atg5△ cells  The authors find that DNM1 is required for MDC formation. If the authors want to make the claim that MDCs are released from mitochondria by Dnm1-dependent mitochondrial fission, they need to provide evidence for a direct role of Dnm1 in MDC formation. For example, does Dnm1 localize to MDCs as they bud from mitochondria?At the reviewer’s suggestion, we tested whether Dnm1-GFP colocalizes with Tom70-mCherry labeled MDCs, and found that Dnm1-GFP foci localize at or around MDCs in 88% of cells. This new data is included as 3) The authors also find that the ATG5 is required for degradation of MDCs by the vacuole, and propose that MDC degradation occurs by macroautophagy. If they want to make the claim that MDCs are enveloped in autophagosomes prior to degradation in the vacuole, they need to test whether autophagosome marker proteins are associated with MDCs and whether deletion of DNM1 affects this localization.In response to the reviewer’s suggestion, we tested whether Tom70-mCherry-labeled MDCs colocalize with the classic autophagy marker GFP-Atg8. GFP-Atg8 localizes to the pre-autophagosomal structure (PAS) in yeast, which is near the vacuole surface and marks sites of autophagosome formation. We found that the majority (68%) of MDCs localize at or near the PAS. Those that did not were away from the vacuole surface. These results suggest that MDCs form at the mitochondria away from vacuoles, and then are recruited to the PAS to be engulfed by autophagy. This data has been included in the revised manuscript as 4) The finding that select proteins are excluded from MDCs in Conc A-treated cells is interesting and important.A) Since mitochondria are generally more fragmented in old yeast compared to young yeast, it is not clear that the punctate Tom70-GFP-containing structures observed in old yeast are MDCs. Is Tim50 excluded from Tom70-GFP-containing structures in old yeast?We agree with the reviewer’s comment that MDC substrate selectivity is important, and indeed in our previous version of the manuscript, we do in fact show that Tim50-GFP is excluded from the MDC (marked with Tom70-mCherry) in aged cells (B) To confirm that there is degradation of a select set of proteins in MDCs, it would be interesting to see if Tim50 is not degraded by autophagy in Conc A-treated cells.We agree with the reviewer’s suggestion, and have now included western blot data showing that unlike Tom70, Tim50 is not processed by Pep4 in conc A-treated cells. This new data is included as 5) It is not clear whether tom70/71∆ cells are more sensitive to Conc A because they have defects in protein import or defects in MDC formation. The text should be revised accordingly.As the reviewer suggested, we added text to this section of the manuscript suggesting that the enhanced sensitivity to conc A in these mutants may be unrelated to their function in the MDC pathway.6) Although loss of vacuolar acidification does result in a decline in mitochondria membrane potential, it is not clear that the proteins that are removed in MDCs are dysfunctional. In fact, the authors speculate that MDC-dependent remodeling of mitochondria alters mitochondrial function in metabolite transport. Therefore, it is possible that the MDC pathway is not a quality control pathway, but a pathway for control of mitochondrial function. The manuscript would benefit from studies to distinguish between these two possibilities. For example, is loss of mitochondrial membrane potential or function sufficient to trigger MDC formation? Either outcome would be of interest to the field.Reviewers 1 and 2 raised the same important points, and as suggested, we addressed these issues both experimentally and by changing the text throughout the manuscript. Please see our detailed response to these suggestions in Reviewer 1’s section, question 2, and reviewer 2’s section, question 1. Briefly, we added a new Reviewer #4:1) Concerning the requirement for fission. It is clear from the data that Fis1 cannot compensate for the lack of Dnm1 as the dnm1 deletion completely blocked Tom70 delivery to the vacuole. Nevertheless, it would be interesting to see if a fis1 deletion have some kind of defect also as Fis1 is required for proper localization of Dnm1. Also, as dnm1 deletion mutants display a defect in endomembrane trafficking one wonders if such effects could be part of the problem in delivering MDCs to the vacuole? Cannot come up with a simple genetic way/experiment to separate mitochondrial fission defects form endomembrane trafficking defects but the authors could perhaps comment on this.FIS1, and found that loss of FIS1 blocks MDC release from mitochondria and subsequent delivery to the vacuole, exactly as previously observed in cells lacking DNM1. This new data has been added to the paper as part of FIS1 is also required (which phenocopies loss of DNM1 and is not known to play a role in other endomembrane trafficking reactions), and that Dnm1-GFP foci localize around the MDC, strengthens the argument that fission is required for release of MDCs from the mitochondria. Thus, we chose not to include an extra statement regarding a potential separate role for Dnm1 in this process.At the reviewer’s suggestion, we examined the MDC pathway in strains lacking 2) In the experiments using conc-A to trigger loss of vacuolar acidity, the delivery of Tom70 to the vacuole is interesting for several reasons and one wonders if this could be discussed a bit further. First, loss of vacuolar acidity is effectively blocking endomembrane trafficking to the vacuole. Does this mean that the Atg5-dependet route is "immune" to such pH effects in the vacuole or does it indicate that MDCs are transferred from the mitochondria to the vacuole by a more direct route: mitochondrial-vacuolar contacts?VAM3 prevents the appearance of Tom70 within the vacuole of conc A treated cells. Vam3 is part of the vacuolar homotypic fusion machinery, and is required for fusion of autophagosomes with vacuoles. This data has been added to We share the reviewer’s view that the role of vacuolar acidity in this process is very interesting, and that the mechanism of exactly how the MDC gets transferred from the mitochondria to the vacuole isn’t completely clear . Our current data suggests that the MDC or pieces of it gets released from the mitochondria by fission, and then degraded by autophagy in autophagosomes. This suggests that MDCs follow a traditional autophagy degradation route to the vacuole through autophagosomes. In support of this, we added data to the paper showing that loss of The other interesting possibility (as the phenomenon is seen in old cells and during conc-A treatment) is that a loss of vacuolar acidity is, in fact, required for triggering the MDCs formation and delivery.PEP4, does not causes accumulation of Tom70-GFP in the vacuole of young cells, nor does it cause formation of the MDC. Instead, the addition of conc A to these cells triggers both the formation of the MDC and accumulation of Tom70-GFP in the vacuole. Thus, this pathway is not constitutively on in young cells, and must be induced by a signal originating from loss of vacuolar acidity. We have added a sentence further clarifying this point in the revised manuscript.Yes, our current data supports the idea that loss of vacuolar acidity actually triggers formation of the MDC. This is most clearly illustrated in Could it be tested perhaps if drugs affecting mitochondrial function but leaving the pH of vacuole intact can trigger MDC formation and delivery? In other words, approach the generality of the system discovered upon mitochondrial dysfunction.This point has been addressed in our response to all three other reviewers above. Again, we now include data showing that loss of mitochondrial membrane potential is not sufficient to activate this pathway in new 3) It is stated that the formation of small vesicle-like structures (Tom70 associated) in the cytoplasm of old cells require mitochondrial fission (Dnm1) indicating that these membrane structures are derived from mitochondria. It is very hard to see these structures in the picture of the dnm1 strain – perhaps a better version could be added? Also, as the experiment uses Vph1 as a reporter, does not this indicate that the vesicle-like structures are derived from the vacuole (vacuolar fragmentation?) rather than formed by fission from mitochondria? At present, it is a bit unclear what these structures are, how they were formed, and how they relate to the MDC route discovered.atg5△, and dnm1△ cells taken with a DeltaVision microscope showing mitochondria (Tom70-GFP) and vacuole (Vph1-mCherry). As can be seen in the We agree with the reviewer that these structures are hard to see in the originally submitted manuscript. As addressed in response to reviewer 3’s comments above, we have now included 4) The authors suggest that MDC formation helps protect mitochondria from vacuole-induced stress as a greater loss in membrane potential is seen in tom70/71 mutants subjected to conc-A treatment. Is it possible that this loss of potential in tom70/71 mutants could be related to other shortcomings than the formation of MDC?Yes, we agree with the reviewer that the loss of membrane potential in the tom70/71 mutants could be unrelated to their role in the MDC pathway. We have now included a sentence in the manuscript addressing this alternative possibility.Also, did atg5 and dnm1, but not atg32, mutants display a similar loss of potential upon conc-A treatment?At the reviewer’s suggestion, we tested the membrane potential in these three mutant strains in untreated and conc A treated cells. We conducted this experiment by staining these cells with the membrane potential dye DioC6, and analyzing them by flow cytometry exactly as carried out in the paper. However, we found that all three of these mutants had membrane potentials lower than the WT strain even in untreated cells, which made the concA changes uninterpretable. Therefore, we did not include this data in the paper and instead added the qualifying statement mentioned in the point above.5) Since aging is the entry port in this paper, I wonder if the authors could discuss briefly the possible significance of MDC in yeast longevity? As dnm1 and fis1 deletion mutants display a somewhat longer replicative lifespan, is it possible that a reduction in mitochondrial fragmentation (positive effect) in these mutants offsets the negative effect of an abrogated MDC response? Is the lifespan on tom70, atg5, atg32 mutants known?dnm1△ mutants have an increased lifespan, we raised the possibility that the Dnm1-dependent mitochondrial fragments that form in old cells could negatively impact lifespan. The lifespan of tom70△ and atg32△ mutants has been shown to be longer than WT in dietary restriction conditions, but not necessarily normal growth conditions . Lifespan data on atg5△ has not been published. However, two other strains lacking genes required for general autophagy, atg1△ and atg8△, have been shown to have an extended lifespan . Based on all of this data, it is very hard to say whether the MDC pathway has any influence on lifespan. It may be the case the formation of the MDC is protective, and it doesn’t necessarily need to be degraded for protection.Following the reviewer’s suggestion, we have added a brief discussion of the lifespan point regarding the fission mutants and MDC in the discussion of the paper. Because


A fusion between the EML4 (echinoderm microtubule-associated protein-like) and ALK (anaplastic lymphoma kinase) genes was identified in non-small cell lung cancer (NSCLC) in 2007 and there has been rapid progress in applying this knowledge to the benefit of patients. However, we have a poor understanding of EML4 and ALK biology and there are many challenges to devising the optimal strategy for treating EML4-ALK NSCLC patients. In this review, we describe the biology of EML4 and ALK, explain the main features of EML4-ALK fusion proteins and outline the therapies that target EML4-ALK. In particular, we highlight the recent advances in our understanding of the structures of EML proteins, describe the molecular mechanisms of resistance to ALK inhibitors and assess current thinking about combinations of ALK drugs with inhibitors that target other kinases or Hsp90. Lung cancer accounts for around 13 % of all cancers diagnosed worldwide, and accounts for around 1.3 million deaths per year . The proMost NSCLC patients are treated with cytotoxic chemotherapy using a DNA-binding drug such as carboplatin/cisplatin in combination with a topoisomerase type II inhibitor such as etoposide or an anti-mitotic such as vinorelbine. Standard treatment also includes immunotherapies that target angiogenesis. Genetic analysis of lung cancers has revealed their underlying complexity, with many different driver mutations that are mutually exclusive in most cases. Molecular subtyping is now routine in NSCLC and patients with an EGFR mutation are treated with an appropriate tyrosine kinase inhibitor (TKI) such as erlotinib in first line therapy. Another reasonably common mutation identified in two landmark studies in 2007 was the first somatic oncogenic gene translocation in lung cancer, involving fusion of two genes, EML4 (echinoderm microtubule associated protein-like 4) and ALK (anaplastic lymphoma kinase) , 7. The Sea urchin EMAP (echinoderm microtubule-associated protein) was the first member of the EML family of proteins to be identified . SubsequRecent structural studies have established the molecular architecture of this family of proteins Fig. b. CrystaEML proteins interact with soluble tubulin and microtubules, and a functional association with the mitotic spindle has been shown for several family members , 20–23. ALK is a member of the insulin receptor kinase superfamily, a large grouping which includes other tyrosine kinases of relevance to cancer such as MET and RON –28. ALK D. melanogaster and C. elegans have been identified and the contribution of ALK to the development of specific tissues has been elucidated   ; however to 1 μM . This wa to 1 μM .ALK activation is driven by the TD of EML4, which drives self-association of the kinase domains. The TAPE domain is not critical for ALK activation because deletion within the TD and basic regions (aa31-140) of EML4-ALK v1 reduced kinase activity and transforming activity, but deletions within the TAPE domain had little or no effect . Indeed,The intracellular localisation of the most common EML4-ALK variant proteins has been studied in model cell lines and in cancer cells derived from patients. Using recombinant proteins expressed in NIH3T3 cells, EML4-ALK v1 and v2 were localised to the cytoplasm, whereas v3 localised to the nucleus, using an ALK antibody to label the fusion proteins . YFP-fusNSCLC patients harbouring EML4-ALK or EGFR mutations are younger, have more advanced disease and are more likely to be non-smokers or light smokers than the average . FurtherThe first research papers on EML4-ALK used non-specific inhibitors such as WHI-P154  or TAE68Many of the available ATP competitive ALK inhibitors, including the clinically approved drugs and those under investigation, are based on three chemical scaffolds Fig. a–c—aminoEML4-ALK patients who initially respond to TKIs such as crizotinib inevitably relapse as the tumour acquires resistance to the drug. Many different resistance mechanisms have been identified, the most common of which is alterations in the EML4-ALK gene, which occurs in approximately a third of patients. These alterations are primarily EML4-ALK gene amplification, mutations within the TK domain of ALK Table , or someDrug-resistant mutations in the ALK TK domain cluster around the ATP binding site and enhance ATP binding, reduce crizotinib binding or both Fig.  80]. Th. Th80]. The macrocylic inhibitor PF-06463922 is more potent against WT ALK and ALK mutants than crizotinib and retains substantial activity against ALK mutants that are resistant to crizotinib and second-generation inhibitors . AlthougThe development of ATP-competitive inhibitors has improved outcomes for patients and there is scope for further improvements using the newest ALK inhibitors. It is, however, inevitable that drug resistance will emerge for these compounds and alternative strategies are under investigation that could be applied as second-line to or in combination with ALK inhibitors.Molecular chaperones are a structurally diverse group of proteins that function in protein folding and quality control , 92. HspThe first evidence that Hsp90 inhibitors might be useful in EML4-ALK patients came from a phase II study that assessed the efficacy of IPI-504 after EGFR TKI therapy in patients with advanced NSCLC . AlthougNot all EML4-ALK fusion variants are equally sensitive to Hsp90 inhibition as v1 and v2 but not v3a were found to be sensitive to 17-DMAG . FurtherBecause they act through a different mechanism, Hsp90 inhibitors also induce the degradation of ALK fusion proteins that are resistant to ALK inhibitors. For example, ganetespib is equally potent against 15 different drug-resistant mutants of NPM-ALK fusions as wild-type NPM-ALK . FurtherCytotoxic chemotherapy treatment is usually delivered as a combination of drugs, which maximize cancer cell death through additive effects or synergism between mechanisms of action. By this rationale, it should be possible to find combinations of ALK inhibitors with other therapeutics that improve patient outcomes. There are many ways to identify combinations of drugs, starting from a rational basis or through screening approaches.Synergy between ALK and Hsp90 inhibitors has been shown in Ba/F3 cells, but was dependent on the EML4-ALK fusion variant with v1 showing the least effect . The comInsulin-like growth factor 1 receptor (IGF-1R) was identified as a potential target for combinatorial drug treatment of EML4-ALK NSCLC, based on the exceptional response of a patient to IGF-1R mAb plus erlotinib and an in-depth study of EML4-ALK cell lines . The patSeveral signalling pathways are activated in EML4-ALK cells, and dual targeting of ALK with these other pathways may be beneficial, particularly in pathways that are not entirely blocked by ALK inhibition. For example, treatment of H2228 cells with TAE684 does not markedly affect phosphorylation of ERK at concentrations at which ALK phosphorylation is inhibited , 61. IndA recent paper provides evidence that upfront dual inhibition of ALK and MEK may be advantageous, at least in some EML4-ALK patients . Cell grThe combination of ALK and MEK inhibitors was also effective in a ceritinib-resistant cell line derived from a patient and, in this case, the effectiveness of the combination may have been due to the presence of an activating mutation in MAP2K1 . HoweverThe identification of the EML4-ALK fusion in a subset of NSCLC patients, and the response of these patients to crizotinib is a clear example of the value of molecular genetics aligned with targeted approaches to cancer drug discovery. The ability of researchers to develop second-generation ALK inhibitors that have substantially improved pharmacological properties is a testament to the power of medicinal chemistry, backed by rational drug design approaches. However, as exciting as these advances are, we are still a long way from being able to offer patients a programme of treatments that cures their disease, or at least keeps it at bay for a decade or more. Therefore, the key challenge ahead is to identify rational combinations of drugs based on molecular and genetic analysis of an individuals tumour that prolong the duration of response to treatment, and to identify appropriate strategies to overcome resistance. Here we have described the current state of the art, from which improved programmes of treatment will develop. We believe that the answers to the following questions will be critical if we are to achieve dramatically improved patient outcomes:What are the missing connections in the signalling pathways that connect EML4-ALK to cancer proliferation and survival, and how can we exploit these in therapy? Over the next few years, we will discover much more about this intriguing oncoprotein and how the combination of different portions of EML4 and ALK affect its behaviour. In particular, we will learn much about the signalling pathways and mechanisms of resistance from clinical studies on second-generation ALK inhibitors alone and in combination with other therapeutics.What is the best strategy for ALK inhibitors in the clinic? Second-generation ALK inhibitors show benefit in patients who have relapsed on crizotinib, and have been approved by the FDA for treatment of these patients. We do not yet know which of them are the preferred options in crizotinib-resistant patients and whether, in time, they ought to replace crizotinib as the first-line therapy.Do we need to develop further ALK inhibitors, and what properties should they have? Unfortunately, drug resistance to advanced ALK inihibitors is inevitable. Because these inhibitors are more potent against ALK, and retain effective potency against key ALK mutants, we would expect a higher proportion of mutations that activate bypass pathways versus further mutations in ALK or ALK overexpression. In this context, the off-target effects of crizotonib, such as inhibition of MET, AXL and RON, may be beneficial in preventing the activation of bypass resistance pathways [pathways . In consHow are bypass pathways selected for during ALK inhibitor treatment, can we predict which path of drug resistance a tumour is likely to exploit, and how do we target these pathways on an individual patient basis? Initial studies suggested a number of bypass pathways, and we have to build a clear picture of the overall signalling network. Addressing this, for example through next generation sequencing, will require significant effort over the next few years as clinical samples of patients treated with second-generation ALK inhibitors become available.Which therapeutics should be used in addition to ALK inhibitors and how? There are many suggestions for therapeutics that could be used following ALK inhibitory therapy, or perhaps in combination. These include Hsp90 inhibitors and inhibitors of other RTKs. It is also possible that cancer immunotherapies will form part of the treatment programme [rogramme .How is the EML4 part of the fusion significant? In our view, the presence of a misfolded, partial TAPE domain in most EML4-ALK variants is a defining feature of these oncoproteins. This feature underlies exquisite sensitivity to Hsp90 inhibitors and the exposure of the HELP motif promotes RAS signalling. While EML4-ALK v3 localises to microtubules, the presence of a partial TAPE domain in other variants prevents microtubule association and may confer localisation to discrete cytoplasmic structures. This may contribute to oncogenic signalling by promoting co-localisation with other signalling molecules. Further work is required to elaborate the contribution of the EML4 portion of the fusion protein and exploit this in the clinic.Will the treatment of EML4-ALK patients be stratified by variant type? EML4-ALK variants are inhibited differently by ALK and Hsp90 inhibitors in vitro, but it remains to be seen whether these differences will be observed in the clinic. However, given the gross differences in the molecular properties of some variants, such as v3 or v5 compared with the others, it seems likely that some differences will be observed in patient response and drug resistance mechanisms. Most patients harbour one of the three most common variants and, provided that the variant is determined during clinical trials, it will be possible to identify any differences of clinical significance. However, it will be difficult to obtain sufficient data for the rare variants and we believe that it is crucial to develop models that provide a rational basis for planning the treatment of these patients.


Supplemental Digital Content is available in the text Patients with diabetes have a higher incidence of bladder cancer; however, the association between thiazolidinedione use and bladder cancer risk has been controversial. We aimed to investigate whether pioglitazone or rosiglitazone use is associated with bladder cancer risk in patients with type 2 diabetes mellitus.This nationwide nested case-control study used data set obtained from the Korean National Health Insurance Service National Sample Cohort 2002 to 2013. Among the 47,738 patients with incident diabetes, 85 cases of newly diagnosed bladder cancer and 850 controls  were recruited. Type 2 diabetes mellitus and bladder cancer were diagnosed using the International Statistical Classification of Diseases and Related Health Problems, 10th Revision code.More cases of bladder cancer were diagnosed in men (81.2%), and the stratified age peaked at 70 to 79 years old. Exclusive rosiglitazone use raised the incidence of bladder cancer . The risk of bladder cancer started to increase after less than 3 months use  and peaked at 3 to 12 months of rosiglitazone use . Patients were first exposed to exclusive rosiglitazone within 1 year  and those who had consistently used it for 1 year (OR = 4.48 95% CI = 1.51–13.31), had higher risks of bladder cancer compared with nonthiazolidinedione users. Neither pioglitazone use nor exclusive pioglitazone use were associated with an increased incidence of bladder cancer.Rosiglitazone use is associated with an increased risk of incident bladder cancer independent of age and sex in patients with type 2 diabetes mellitus. The highest odds of bladder cancer in rosiglitazone users was seen in those with <1 year of exposure. In the present study, we analyzed data from the Korean National Health Insurance Service (NHI), which is a nationwide cohort study with a nationally representative sample of the Korean population conducted by the Korea Centre for Disease Control and Prevention to regularly assess the health status of general civilians. Our aim was to investigate the association between TZD use and the incidence of bladder cancer in a nested case-control study of the T2DM population.Most studies have analyzed data derived from Caucasian subjects and mainly focused on pioglitazone.16 Under universal medical coverage, all medical claims data are collected by the NHI as a monopolistic health insurer in Korea. Therefore, all subjects in the NHIS-NSC were maintained until 2013 except for follow-up loss due to death or disqualification from the NHI . The ethics committee of the Yonsei University College of Medicine approved this study (4–2015–0579).We used 2002 to 2013 data from the South Korean National Health Insurance Service National Sample Cohort (NHIS-NSC). This is nationwide, representative 2%, stratified, random sample of a Korean population with a baseline of 1,025,340 subjects in 2002 to a final value of 1,014,730 subjects in 2013. It contains all inpatient and outpatient medical claims data, including personal information, prescription drugs, diagnostic and treatment codes, and primary and secondary diagnosis codes. Mortality data were provided by the National Statistical Office in Korea.7 We further excluded patients whose first antidiabetic agent was insulin and patients who were <40 years old at the time of first antidiabetic prescription. Finally, patients with type 1 diabetes mellitus or pre-existing T2DM patients were excluded. The date of incident diabetic cohort entry was defined as the date of first antidiabetic agent prescription. The last follow-up date was defined as the date of bladder cancer diagnosis, death, disqualification from NHI, or December 31, 2013, whichever occurred. The final incident diabetic cohort consisted of 47,738 patients.Within the NHIS-NSC, among subjects without any prescribed antidiabetic agent in 2002, we selected patients who were first prescribed oral antidiabetic agents from 2003 to 2013 so that all study subjects had at least 1 year, in which they were antidiabetic agent free.17 The index date was defined as the date 1 year before the date of bladder cancer diagnosis. Case patients were identified using the International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD-10).18In this nested case-control study with risk-set sampling (or incidence-density sampling), control patients were chosen from those in the incident diabetic cohort who were at risk of becoming a case at the time they were diagnosed.Case inclusion criteria were first-time diagnosed bladder cancer (ICD C67.0-C67.9); at least 1 year of a latent period after T2DM diagnosis; without prior diagnosis of urinary tract cancer  within 5 years before the date of bladder cancer diagnosis; and presence of clinical codes for treatment , codes for death due to bladder cancer, codes for diagnosis , or codes for death within 6 months of bladder cancer diagnosis. Exclusion criteria were a prior diagnosis of urinary tract cancer within 5 years before bladder cancer diagnosis. Control patients were randomly selected from the case risk set at a 1:10 ratio after they were matched on a 5-year interval age group at both 2002 and the time of T2DM diagnosis, sex, and date of T2DM diagnosis within 1 year. For random selection from the matched risk set, we applied the same exclusion criteria as that of the case group.For all analysis, prescription information before the index date was used for the identification of drug use. Rosiglitazone use was defined as at least one prescription between cohort entry and the index date. The same definition of drug use was applied to all types of drugs including nonantidiabetic drugs. Due to the absence of case patients who had used both rosiglitazone and pioglitazone, study patients were classified into 3 categories of TZD use never used TZDs, exclusive rosiglitazone use, and nonexclusive rosiglitazone use including exclusive pioglitazone users.7 Due to the absence of case patients who had used rosiglitazone >24 months, cumulative duration >12 months was the highest level in its category. The cumulative rosiglitazone dose was computed by summing the values of all prescriptions obtained by multiplying the daily dosage by prescribed days for each prescription. Then, the cumulative doses of rosiglitazone were categorized into tertiles. To consider the time of exposure, the time of first exposure to rosiglitazone was defined as the distance between the date of the first rosiglitazone prescription and the index date. We separated the above analyses into 2 distinct TZD uses: rosiglitazone and pioglitazone.As a secondary analysis, cumulative exposure and time of exposure among exclusive rosiglitazone users were examined. The cumulative duration was defined as the number of days between the first and last prescription plus the duration of last prescription up to the index date.19 No information on smoking habits was collected for the NHIS-NSC; therefore, we assumed that chronic lower respiratory disease indirectly represented cigarette smoking. A sensitivity analysis was conducted that excluded patients with a diagnostic code alone, those who died within 6 months of bladder cancer diagnosis, and those with any previous cancer history. All covariates were assessed by all information prior to the index date. All statistical analyses were conducted using SAS . A P value <0.05 was considered statistically significant.Characteristics of case and control patients are presented as numbers with percentages for categorical variables and as means with standard deviations for continuous variables. Conditional logistic regression was conducted to estimate odds ratios (ORs) and 95% CIs to assess the association between rosiglitazone use and the risk of bladder cancer. In addition to matching variables, we adjusted for urolithiasis and other urinary tract disease , renal disease , alcoholic liver disease and alcohol related metal and behavioral disorders , chronic lower respiratory disease except for asthma , congestive heart failure , Charlson comorbidity score calculated excluding diabetes and the comorbid diseases listed above, other antidiabetic drugs use , aspirin use, statin use, household income as a continuous variable , and residential area.From the original cohort of 47,738 subjects with incident T2DM, 120 patients with a first-time diagnostic code of bladder cancer were selected. We further excluded 35 patients who did not satisfy inclusion criteria. There were 85 final case patients, and the median follow-up time was 2880 days . Subjects who had taken rosiglitazone at any point had a higher incidence of bladder cancer  compared with patients who had never been exposed to rosiglitazone. The cumulative duration and doses of rosiglitazone use showed similar patterns to that observed for exclusive rosiglitazone use. Consistent use for rosiglitazone less than 12 months and a cumulative dose less than 2040 mg corresponded to 4 mg/day for 17 months and, were associated with higher risks of bladder cancer. However, a significant increase of bladder cancer incidence was not observed among subjects who had used pioglitazone for any time period (Supplementary Table 2).). Second, when we eliminated 17 case patients with any cancer history prior to the bladder cancer diagnosis, exclusive rosiglitazone use raised the incidence of bladder cancer by approximately 3-fold compared with no use of any TZD .The results of the sensitivity analysis did not show any differences from those of the main analysis. First, after excluding 13 case patients via diagnostic procedure or test  and 3 case patients due to early death within 6 months after the diagnosis of bladder cancer, exclusive rosiglitazone use raised the incidence of bladder cancer by approximately threefold compared with no use of any TZD  in T2DM patients. We used a nest-control model f, an incident diabetic cohort and found that rosiglitazone use increased bladder cancer risk. The risk was higher for subjects who had continuously using rosiglitazone for less than 1 year and recent users. However, we found that pioglitazone use was not related to the risk of bladder cancer, which is consistent with the above studies.Recent studies concluded that pioglitazone use was not associated with a significantly increased risk of bladder cancer.6 In our study of 47,738 T2DM patients, 120 incident bladder cancer patients were identified . This is a higher incidence than in general populations; the overall rate for bladder cancer in Korea was 4.6 per 100,000 person in 2011.21 The distribution of bladder cancer stratified by age peaked at 70 to 79 years old, which is the same as that of the general Korean populations.21 It was not known whether there was a difference between bladder cancer characteristics in T2DM and general population. In addition, the mechanism underlying bladder cancer in T2DM is not clearly understood. The incidence of bladder cancer is likely to be increased in a high-risk group such as T2DM.23 According to a Taiwanese study, older age, male sex, pre-existing nephropathy, and urolithiasis were associated with greater bladder cancer risk.22 Renal diseases did not increase the bladder cancer incidence in our study. However, we also found more urolithiasis, a well-established form of bladder tumorigenesis,24 significantly increased bladder cancer risk . Furthermore, we set latent period of bladder cancer for 1 year and 3 months to determine whether rosiglitazone acts as an initiator or promotor. Higher incidence of bladder cancer for patients with exposure ≤24 months and recent rosiglitazone use was observed in both settings. These results implied that rosiglitazone might act as a promoter, which is in a line with the findings of an in vivo study.25 Given that T2DM patients might be prone to bladder cancer; rosiglitazone use might amplify the process in high-risk patients.Patients with diabetes are likely to have a modestly increased risk of bladder cancer.26 Long-term exposure to rosiglitazone from more than 2 to 5 years was associated with a higher risk of bladder cancer. However, the design and settings of previous studies were different from ours. Patients in the United Kingdom with pre-existing T2DM who were newly prescribed rosiglitazone were enrolled in a retrospective cohort study, which is dissimilar to nested case-control study approaches.23 In a Taiwanese nested case-control study, the index date for cases was defined as the date of first hospitalization for bladder cancer and included incident T2DM patients treated with rosiglitazone regardless of treatment duration.26 No latent period for bladder cancer was considered in either of those studies. As cancer would not appear in such a short period, the setting of the latent period for cancer would be very important and could influence the result. In addition, no dose–response relationship and the risk peak at less than 1 year of exposure stays support the hypothesis that rosiglitazone might act as a promoter and have an early effect of bladder cancer. It is also possible that rosiglitazone could promote bladder cancer in subjects with high sensitivity who develop bladder cancer during the early period of use.Our finding of a high incidence of bladder cancer in subjects who have used rosiglitazone for less than 1 year is inconsistent with previous studies that demonstrated a dose-response relationship of bladder cancer and rosiglitazone use.27 Proliferator-activated receptor alpha controls fatty acid catabolism, inflammatory response, and homeostasis in liver.30 Proliferator-activated receptor gamma regulates adipogenesis, insulin action, and plays roles in cell differentiation, and proliferation.32 This disparity could explain the different effects on lipid metabolism in previous studies.33 Moreover, PPAR gamma mediates the up-regulation of vascular endothelial growth factor, which plays a critical role in tumor angiogenesis.34 And oxidized low-density lipoprotein stimulates vascular endothelial growth factor expression via PPAR gamma pathway activation.35 Taken together, the potency of PPAR gamma agonism exerted by the 2 drugs might result in different effects regarding bladder cancer.The different effects of pioglitazone and rosiglitazone on bladder cancer are explained by their effects on PPAR gamma. Rosiglitazone is considered a pure PPAR gamma agonist, whereas pioglitazone is both a PPAR alpha and gamma agonist.36 we assumed that chronic lower respiratory disease indirectly represented smoking cigarettes and adjusted this in the final risk for bladder cancer. Likewise, no laboratory tests  of diabetic parameters were available, so we could not assess whether glycemic fluctuation or diabetes severity influenced bladder cancer risk. Thiazolidinedione is mostly prescribed as the second medication for patients whose T2DM is poorly controlled with metformin, so the population of current study would not be in the early disease stage. We matched cases and controls for age, sex, and date of T2DM diagnosis; therefore, the effect of those variables would be minimized. Second, as the data source was derived from a claim dataset, the actual adherence rates of medication are not reflected. This weak point was already noticed in a similar previous design.26 In this study, we assumed all medications prescribed were taken by the study patients and excluded those who failed to fill 2 or more prescriptions within 6 months. Third, the number of case patients was relatively small. We made an effort to improve on the retrospective cohort study by employing a nested case-control model. In addition, the study population was restricted to newly diagnosed T2DM patient; this allowed us to assess the pure effect of TZDs and let the case patients have a 1-year latent period of bladder cancer development. Because of these strict criteria, the final number of cases was smaller. Despite this, the results were statistically significant.There are several limitations of this study. First, several variables that could be associated with bladder cancer incidence, such as smoking, alcohol consumption, obesity, and exposure to environment and occupational chemicals are not measured in NHIS-NSC survey. For smoking, a well-known risk factor for bladder cancer,7 Second, the concept of the current study model is an aggregate of preceding analyses, so this nested case-control study is more sophisticated and provides higher accuracy. As described above, we determined the latent window period for bladder cancer after the diagnosis of T2DM and applied strict inclusion and exclusion criteria.There are major strengths of the current study. First, the nationwide database included a large population of incident T2DM patients and contained both outpatient and inpatient references, which were not included in a previous study.In conclusion, the present study shows that rosiglitazone but not pioglitazone increased bladder cancer risk in T2DM patients. In addition, bladder cancer incidence in rosiglitazone users was higher among those who had been taking the drug for less than 1 year. This suggests that rosiglitazone might promote the development of bladder cancer, but long-term studies are needed to assess the clear causality and mechanisms regarding rosiglitazone use and bladder cancer risk. Furthermore, it would be interesting to determine differences of bladder cancer in the general population and rosiglitazone users. If there is a clear relationship between rosiglitazone and bladder cancer, a rosiglitazone substitute might be prescribed for high-risk T2DM patients.