To determine the factors affecting clinical decision-making about which patients should receive stroke rehabilitation.Data sources  were searched systematically from database inception to August 2018. Full-text English-language studies of data from stroke clinicians were included. Studies of patients were excluded. The included studies were any design focussed on clinical decision-making for referral or admission into stroke rehabilitation. Summary factors were compiled from each included study. The quality of the included studies was assessed using the Mixed Methods Appraisal Tool.n = 6 studies), presence of dementia (n = 6), patients’ social/family support (n = 6), organizational service pressures (n = 7) and the decision-making clinician’s own knowledge (n = 5) and emotions (n = 5).After removing duplicates, 1915 papers were identified, of which 13 met the inclusion criteria. Eight included studies were qualitative and one used mixed methods. A total of 292 clinicians were included in the studies. Quality of the included studies was mixed. Patient-level and organizational factors as well as characteristics of individual clinicians contributed to decisions about rehabilitation. The most often described factors were patients’ pre- and poststroke function (The results highlight a lack of clinical guidance to aid decision-making and reveal that a subjective approach to rehabilitation decision-making influenced by patient-level and organizational factors alongside clinicians’ characteristics occurs across services and countries. In addition, the Cochrane Central Register of Controlled Trials  and Cochrane Stroke Group Trials Register (August 2018) were also searched. No restrictions were placed on study design or publication date, with English language being the only restriction. The search terms were adapted to terminology used by each database (see 12 Studies that included a mixed diagnosis caseload (i.e. participants working in generic services) were excluded unless separate results for stroke were reported. Studies with patient participants were excluded. No restrictions were placed on the included study design.Studies were eligible for inclusion if they were full-text primary research published in peer-reviewed journals in which participants provided any type of stroke service . Studies were included that focussed on clinical decision-making for referral/admission to stroke rehabilitation, or that examined clinicians’ prioritization criteria for stroke rehabilitation or decision-making about rehabilitation potential, in essence any type of decision-making influencing subsequent access to stroke rehabilitation services. Studies focussed on decision-making between specific interventions or treatments were excluded, for example, decisions about which patients should receive a home visit.The first author (V.L.) reviewed studies against the inclusion criteria by title. All abstracts and full text were then reviewed for eligibility by V.L. and a reviewer independent of the research team in order to minimize selection bias. Two discrepancies in inclusion were resolved through discussion. Reference lists of the included studies and relevant review papers were hand-searched for studies not already identified in the searches. We extracted all factors from the included studies and organized them into patient-level (e.g. patient’s age), organizational (e.g. staffing levels) and clinician-level (e.g. experience) factors.13 was used. This allows for the appraisal of qualitative and quantitative study designs simultaneously and scores studies on design, sampling, appropriateness of outcome measures and analysis method, randomization (when appropriate) and completeness of data. Studies are scored in four domains and the total scores ranged from 0% to 100%. All studies were assessed by V.L. and an independent reviewer, and the scores were compared. Two discrepancies were resolved through discussion. Studies were included regardless of quality rating.In order to appraise the quality of studies, the Mixed Methods Appraisal ToolAfter removing duplicates, 1915 papers were identified, of which 13 met the inclusion criteria and were included see . Eight on = 5).1823 Two studies sampled from stroke units in more than one country.25 Three studies sampled clinicians involved in admitting patients to inpatient rehabilitation facilities and examined their admission criteria,25 and the others sampled referring clinicians. Two studies examined the factors influencing perceptions of rehabilitation potential after stroke, a complex concept sometimes used as a determinant for referring patients onto rehabilitation.17 A total of 292 clinicians were included in the studies. The study size varied from an ethnographic study of one multidisciplinary team16 to a survey of 77 discharge planners.24 And 671 patients were included across three of the studies,25 for example, in meetings observed about individual patients.Most included studies were conducted in Australia (25 how the findings may have related to researcher influence1720 and how the findings may have related to context of the research.15 Three of the four quantitative studies had poor response rates (under 60%);25 some had an unrepresentative sample of the study population, for example, some participants were sampled due to participation in previous research22 or reasons for non-participation from eligible individuals were not explained,24 and there was a lack of clarity about the measures used.25As shown in n = 8), organizational factors (n = 2) and the characteristics of individual clinicians (n = 4) were all found to influence clinicians’ decision-making for stroke rehabilitation. These key factors are described in Supplemental Table 1 and organized thematically below for clarity; however, categories were not mutually exclusive. Where possible, the positive or negative influences are described; however, some studies did not always specify the specific influence of factors. 24Patient-related factors , three of which described older age as negatively affecting rehabilitation services received by patients.22 Older age was identified as a barrier for referral into some rehabilitation services although the reasons why were unexplored.16 Patient age was also used as a proxy for associated disability, for example, older people being assumed to have a lower baseline functioning.18Five studies identified patient age as a factor perceived to influence decision-making  patients to rehabilitation. Putman et al.’s20 found that clinicians were less likely to refer patients for rehabilitation when they believed that the patient would not be accepted, for example, patients with a diagnosis of dementia. Similarly, Lam Wai Shun et al.17 identified that patients with severe memory problems  may be perceived to have low rehabilitation potential, as clinicians felt that they would be less likely to be accepted for rehabilitation. Neither study explored the reasons why staff believed this; however, in their other included study, Lynch et al.19 found that some participants considered that rehabilitation was not suitable for certain patients, particularly those with severe stroke or those with cognitive deficits/dementia. Clinicians perceived that these types of patients would never gain from rehabilitation, although the reasons why this was believed were not explored.19 Burton et al.14 found that some clinicians considered a premorbid diagnosis of dementia would indicate little rehabilitation potential and therefore limit the amount of rehabilitation that patients receive. Equally, clinicians in Longley et al.’s10 interview study found that clinicians perceived patients with dementia would lack rehabilitation potential or capacity to change unless they proved otherwise.A specific element of prestroke function, whether patients have preexisting dementia, was identified as an influential factor in decision-making not only affecting admission for rehabilitation, but also initial referrals. Lynch et al.25 found in four out of six stroke rehabilitation units studied that premorbid cognitive disability reduced the likelihood of a decision to admit. One Belgian unit specifically screened patients for advanced dementia, although the study does not detail how the result of screening would influence admission and the methodological quality of this study was rated poorly when appraised. In addition, Hakkennes et al.21 surveyed assessors from rehabilitation units and viewed premorbid cognition as the most important item to consider when accepting patients for rehabilitation. It is not clear, however, whether the premorbid cognition specifically refers to dementia. The National Institutes of Health Stroke Scale (NIHSS)26 score was used to gather information about cognition which has no way of indicating a premorbid diagnosis of dementia. Again, it was not specified whether cognition would positively or negatively influence a decision for rehabilitation, just that it would be taken into account.Putman et al.25 study when compared with other units in the study, although details remain unclear. Clinicians felt that families sometimes pressurized services into providing ongoing rehabilitation25 and influenced decisions about discharge destination.24 Home environment was also influential when deciding rehabilitation plans; patients from residential care were often not considered as candidates for rehabilitation unless families asked.20Social/family support was a factor that was considered to affect decisions about access to rehabilitation. A lack of social support, which may prevent patients from returning home, meant that patients were less likely to be admitted to one Swiss stroke unit in Putman et al.’sSupplemental Table 1). Occupational therapists in Daniëls et al.’s15 focus group study identified that patient motivation influenced their approach to rehabilitation and aided decisions on when to proceed with rehabilitation. While Daniëls et al.’s15 study focused on ongoing rather than access to rehabilitation, another study found that when participants were unmotivated to participate in therapy sessions, they were less likely to be referred for postacute rehabilitation in the first place.18 Two studies identified barriers to judging motivation, such as poststroke depression and attention,18 with some clinicians in Luker et al.’s18 acknowledging that low motivation would prevent access to rehabilitation but also feeling unable to influence it.Staff perceptions about patient-related factors also affected decision-making. Staff perception of patient motivation to engage in rehabilitation was found to affect decisions in five studies . Observed improvement in the acute phase was an important factor in clinicians’ decision-making about whether a patient possessed rehabilitation potential.17 Lynch et al.20 found that a lack of improvement within the first two weeks post stroke was linked to decisions about referral onto a residential care rather than an inpatient rehabilitation pathway.Patients were required to demonstrate progress with rehabilitation or have therapy-led goals in order to be referred for rehabilitation in five studies  were an important and sometimes overriding factor in decision-making. Predicting discharge destination determined clinical priority and the care patients would receive; patients for discharge into residential care would become low priority for rehabilitation.Supplementary Table 1). This predominantly related to bed shortages; some participants described having to discharge patients before they felt they reached the end of inpatient rehabilitation,16 and limited bed availability was identified as a barrier to referring patients for postacute rehabilitation.24 Participants in Lam Wai Shun et al.’s17 study described having limited time to assess patients, creating pressure for quick decision-making based on a single encounter with patients. Similarly, participants in Longley et al.’s10 study described the challenges of working with people with cognitive impairments within time-limited services, when they may require longer to progress with rehabilitation. Johnson et al.16 observed multidisciplinary team meetings and found that a barrier to decision-making about discharge destination was lack of time for some members of staff to actually attend meetings. Staffing shortages were also found to be barrier for admitting patients for rehabilitation23 and restricted the amount of time clinicians were able to spend with each patient.18Organizational factors, such as service acceptance criteria and workforce capacity, were found to influence decisions about stroke rehabilitation. Service pressures were discussed in seven studies from four different countries using a mix of methods ; however, Lynch et al.19 found that lack of knowledge was a barrier for participants to refer patients to rehabilitation. They highlighted a belief from clinicians that rehabilitation was not suitable for patients with severe stroke, despite education sessions being provided demonstrating otherwise. Lack of knowledge about comorbid conditions  was found to influence decisions about ongoing stroke rehabilitation for patients in Longley et al.’s10 study, with participants highlighting a lack of availability of extra training. Clinicians’ awareness of rehabilitation services also influenced which services patients were referred to and when.18 Johnson et al.16 identified that clinicians’ lack of knowledge about rehabilitation services available in the community resulted in delays in discharge, or patients not being referred for rehabilitation at all. In addition, fear of damaging relationships with rehabilitation providers prevented some clinicians from referring patients when they considered them unlikely to be accepted.19Clinician’s knowledge and awareness influenced decisions. Lam Wai Shun et al.Supplemental Table 1). Participants in Longley et al.’s10 study described an element of “gut instinct” informed decision-making, particularly for less experienced clinicians. Luker et al.18 identified an “ethical strain” when attempting to provide equal levels of care; participants stated that they were aware that certain demographics of patients  had more difficulty in acquiring postacute rehabilitation and yet also acknowledged that they provided more rehabilitation to younger patients.18Finally, five qualitative studies identified an emotional element to decision-making for clinicians, with participants wanting to give all patients a chance with rehabilitation while not challenging limited resources  and level of social support. Service pressures and clinicians’ own knowledge also influenced whether patients would be referred or admitted for rehabilitation. Surprisingly, five studies described an emotional element to decision-making, which highlights the challenge faced by clinicians when formal guidance is lacking. This review reveals the complexity of decision-making and the delicate balance of factors that may lead to a patient receiving, or not receiving poststroke rehabilitation.27 and therefore consideration needs to be made when applying these results across services.The limitations of this review require consideration due to the mix of included studies. The low-quality appraisal scores of some included quantitative studies reflect a need for clearer reporting and more representative samples of participants in this area, for example, researchers could invite all people involved in discharge planning to participate from sampled services. Caution should be used when comparing their results to the highly appraised studies. There was heterogeneity in the organization of rehabilitation services and referral systems across the included studies which may limit the applicability of results, for example, some relied on external assessors selecting patients rather than patients being referred; some studies were carried out in generic acute or rehabilitation settings rather than stroke specific, and insurance was an influential factor in studies from countries requiring insurance to access healthcare. Organization of stroke services influences clinicians’ consideration about when to refer/admit patients for rehabilitation; service organization affects patient outcomes,8 which supports our findings. This review builds on the existing literature by summarizing research from clinicians’ perspectives and addressing the organizational and individual clinician-level factors as well.This review did find similarities across all nine countries covered by the included studies, which increases our confidence in the generalizability of our findings. The patient-related factors identified in this review are similar to those identified in a systematic review of patient-level studies that looked at prognostic factors influencing selection for rehabilitation,The review itself has a number of limitations. For one, search results were limited to English language. In addition, the search terms may have resulted in records being overlooked; the terms decision-making, clinical reasoning or clinical judgement were used based on previous studies and suggested search terms in databases, but despite this alternatives may still be used by some authors. There may have been ambiguity about what studies to include given the nature of the topic and the inclusion criteria, although all abstracts were reviewed by a researcher independent to the team in order to minimize this. One of the included studies was written by the authors of this review, which introduces elements of bias to the quality rating and importance given to certain factors. Again, quality was rated by an independent reviewer in order to minimize this bias.17 with the others being focussed on the perspective of rehabilitation assessors or discharge planners, that is, the clinician deciding whether to refer or accept patients for rehabilitation. These roles are reflective of different healthcare systems and therefore not necessarily generalizable to all countries, for example, the United Kingdom. Evidence suggests that decisions to accept patients to rehabilitation (including patients with stroke) are variable across clinical disciplines,28 which indicates a need for multidisciplinary guidance about rehabilitation potential. Stroke rehabilitation is multidisciplinary,1 and therefore the decision of whether a patient receives rehabilitation should be informed by all perspectives; future research needs to reflect this.Seven studies in this review were limited to single disciplines. Two were directly related to the experience of occupational therapists,19 specifically explored whether a nationally recommended assessment tool29 was being used in practice to guide assessment and referral for rehabilitation. They determined that only one out of eight sites studied used the recommended tool as the criteria to determine rehabilitation requirements, and four sites did not consistently use any type of assessment criteria. They recommended that more interdisciplinary guidance is required in order to ensure patients receive equal access to stroke rehabilitation.19 Our findings reveal that this subjective approach to rehabilitation decision-making occurs across services and countries, and more comprehensive methods of supporting decision-making are required.An important finding was that no service reported using formal criteria to aid decisions for rehabilitation. In fact, Lynch et al.30 and this review identified age as a barrier for acceptance into rehabilitation services.16 Similarly, prestroke dementia has been associated with poorer outcomes;7 however, it is unknown whether this is due to lack of opportunities for rehabilitation. While there is some recent evidence suggesting that prestroke dementia influences clinical decisions for stroke rehabilitation,10 this review has identified the need to further explore this in order to close gaps in inequality of access. There is no evidence to restrict access to stroke rehabilitation for certain patients,2 and therefore there is a need to challenge these barriers to stroke rehabilitation.Information about inconsistencies in access to rehabilitation has implications for clinical practice. Evidence suggests that older stroke patients are less likely to receive evidence-based stroke care processes than younger patients,This review highlights other barriers around access to stroke rehabilitation, particularly regarding clinicians’ own knowledge. Clinical decision-makers need to be aware that their perspective of patient-level and organizational factors, as well as their own individual characteristics, influences their decisions about stroke rehabilitation. Some of these barriers to rehabilitation are potentially modifiable by addressing staff knowledge deficits and attitudes to rehabilitation potential. Further studies on this topic require consideration of researcher influence, more representative samples of the study population and more specificity as to how factors positively or negatively influence decisions.Decisions about referring/accepting patients into stroke rehabilitation are influenced by not only patient factors, but also organizational factors and characteristics of the clinician.Clinical decisions appear to take a subjective approach due to lack of clinical guidance about which patients should receive stroke rehabilitation.Click here for additional data file.Supplemental material, Supplemental_Material for What factors affect clinical decision-making about access to stroke rehabilitation? A systematic review by Verity Longley, Sarah Peters, Caroline Swarbrick and Audrey Bowen in Clinical Rehabilitation


International Journal of Endocrinology has retracted the article titled “Vesicle-Associated Membrane Protein-Associated Protein A Is Involved in Androgen Receptor Trafficking in Mouse Sertoli Cells” [International Journal of Endocrinology and two in Cellular Physiology and Biochemistry. The details of the articles are as follows:International Journal of Endocrinology, vol. 2017, Article ID 3985916, 8 pages, 2017. https://doi.org/10.1155/2017/3985916. Published 31 May 2017, on AR localization to the membrane depending on caveolin [Q. Deng, Y. Wu, Z. Zhang, et al., “Androgen receptor localizes to plasma membrane by binding to Caveolin-1 in mouse sertoli cells,” Cellular Physiology and Biochemistry, vol. 43, pp. 223–236, 2017. https://doi.org/10.1159/000480343. Published 29 August 2017, on phosphorylation when AR localizes under testosterone treatment. Figure 1 is the same as Figure 1 in IJE/3985916. This does not cite IJE/3985916 [Q. Deng, Z. Zhang, Y. Wu, et al., “Non-genomic action of androgens is mediated by rapid phosphorylation and regulation of androgen receptor trafficking,” The retracted article. Published 4 March 2018, on the role of VAPA (a vesicle-associated protein) and OSBP in AR trafficking. In Figure 1(c), the top-left panels are the same as Figure 2(a) in IJE/3985916, though the western blots are intended to represent AR, VAPA, and pan cadherin versus AR, caveolin, and E-cadherin, respectively. This cites IJE/3985916 and CPB/480343 [Cellular Physiology and Biochemistry, vol. 47, pp. 2360–2368, 2018. https://doi.org/10.1159/000491608. Published 9 July 2018, on the role of CSN6 and Rab34 in AR trafficking. Figure 1 is the same as Figures 1(a) and 1(b) in IJE/4537214 and the text in the introduction and the beginning of the discussion is similar to IJE/4537214. This cites IJE/3985916 and IJE/4537214 [Q. Deng, C. He, Y. Wu, et al., “CSN6 and Rab34 are involved in androgen receptor trafficking in mouse testicular sertoli cells,” i Cells” . A readeThe negative controls of AR expression in IJE/3985916 ) and IJE/4537214 ) with or without testosterone look similar, but they are different from CPB/491608  and 4(c)); in the former two articles, the band without testosterone is not so faint as in the latter article.The authors apologized and said the articles were all prepared at around the same time, and for articles IJE/3985916 and CPB/480343, the data in Figure 1 in both of the articles are critical for the subsequent experiments. They explained that, in articles IJE/3985916 and IJE/4537214, they conducted two experiments at the same time, the mass spectrometry (MS) and the protein phosphorylation signaling pathway array, and that several candidate proteins involved in AR trafficking were screened by MS and they tried to verify the data by immunoprecipitation.Additionally, the authors clarified that they conducted each of the experiments at least three times. For the western blot assay, they kept the amount of protein and exposure time the same and clarified that the cell response to testosterone may be a little different though the trend was the same. They added that it might also be due to the negative control provided by the company indicated in the article.The authors provided duplicate figures, but the editorial board advised to retract the article.


Background and Objective: Although triptolide was effective for prostate cancer (PCa), the mechanism is still unclear. Androgen receptor (AR) plays a large role in the development and progression of PCa, even after castration. The present study aimed at investigating the effects of triptolide on AR protein stability and the possible mechanism. Materials and Methods: By blocking protein synthesis with cycloheximide (CHX), the effect of triptolide on AR protein stability was investigated with western blot assay. The potential role of calpains in triptolide reduced AR protein stability was investigated with calpain inhibitor and Ca2+ chelator. Results: Triptolide down-regulated AR protein level when protein synthesis was blocked by CHX, demonstrating the decrease of AR protein stability. The AR protein level was restored when the cells were co-treated with triptolide and calpain inhibitor or Ca2+ chelator, indicating the important role of calpains. Conclusions: The results indicate that triptolide can activate calpain via promoting intracellular Ca2+ accumulation, and thus decrease the stability of AR protein, subsequently resulting in the breakdown of the AR protein in LNCaP cells. This work provides an experimental basis and evidence to elucidate the anti-PCa mechanisms of triptolide. Prostate cancer (PCa) is one of the most common malignancies in the male urinary system ,2. Both Tripterygium wilfordii Hook F (TWHF). Its prodrug, PG490-88, is experimentally effective for non-small cell lung carcinoma, fibrosarcoma, and colon carcinoma cells, and has been evaluated in phase I clinical trial for its anticancer activity [Triptolide is the primary active component of a traditional Chinese medicine, activity . We and activity . Our preactivity .2+ in some cells [Post-transcriptional regulation also plays an important role in the regulation of AR activity . AR has me cells ,19 promp2+ accumulation in the cells.In the present study, the effects of triptolide on AR protein stability and the possible mechanism were investigated. The results showed that triptolide decreased AR protein stability in LNCaP cells via calpain activation. Interestingly, the regulation of AR degradation by triptolide cannot be via altering the calpain protein levels, but via the promotion of CaTriptolide was purchased from Sichuan Weikeqi-Biotech Company ; Cycloheximide (CHX) was purchased from Dalian Meilun Biotechnology ; Probenecid was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. ; A23187 was purchased from BioVision, Inc. , BAPTA-AM was purchased from Selleck Chemicals ; ALLM was purchased from ApexBio Technology ; FLUO-3 AM was purchased from KeyGEN Biotech ; Phenylmethylsulfonyl fluoride (PMSF) was purchased Sangon Biotech Co., Ltd. .LNCaP cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences and cultured in RPMI-1640 media  with 10% fetal bovine serum .5/well) and treated with triptolide (100 nM). The viable cells were detected with SRB assay [To detect the effect of triptolide on cell growth, LNCaP were plated on 96-well plate  for 2 h and then triptolide (100 nM) was added to the media. After co-treatment with CHX and triptolide for 24 h, the cells were lysed with RIPA lysis buffer  and the protein concentration was detected with BCA kit .To detect the role of calpain in triptolide mediated AR degradation, calpain inhibitor ALLM and calcium chelator BAPTA-AM were used. After co-treatment with triptolide and ALLM (50 μM and 100 μM), PMSF (100 μM) or BAPTA-AM (10 μM) for 24 h, the cells were lysed with RIPA lysis buffer  and the protein concentration was detected with BCA kit .LNCaP cells were plated on 6-well plates and treated with triptolide (100 nM) for 24 h. Cells were harvested after trypsin digestion and then loaded with Fluo-3 AM (4 μM) for 30 min. Fluo-3 AM was solved in HBSS buffer containing pluronic (2.5 mM), probenecid (0.05%) and FBS (1%). The cells were washed with HBSS buffer (containing probenecid (0.05%) and FBS (1%)) twice and then incubated at 37 °C for 30 min. The intensity of fluorescence  was read with a microplate reader . The fluorescence intensity was normalized by cell protein concentration.Thirty μg proteins were subjected to SDS-PAGE, and the proteins on gels were transferred to PVDF membranes. The membranes were blocked with 5% skim milk, and incubated with anti-AR , anti-calpain-1 , anti-calpain-2 , anti-calpastatin  or GAPDH , then with a horseradish peroxidase-conjugated secondary antibody . The proteins were visualized using an ECL Western blot kit  and document with FluoChem E imaging systems .p < 0.05.All experiments were conducted in triplicate. Results were expressed as means ± SD. Statistical analyses were performed with Student’s t-test for comparison of differences between two groups, or ANOVA analysis for comparison of differences of three or more groups. Differences were considered statistically significant when To investigate whether triptolide can regulate the protein stability of AR, the protein synthesis inhibitor CHX was employed. The results showed that triptolide down-regulated AR protein level in parallel with the decrease of the cell viability after 24-h treatment . In addiProteases play important physiological role in regulatory mechanisms of other proteins by determining their lifetime. To determine whether the proteases involved in triptolide regulated AR down-regulation, protease inhibitor PMSF was first used. PMSF is typically an irreversible inhibitor of serine and cysteine proteases and reacts with common proteases. Although PMSF reacts with some cysteine proteases such as papain, it could not inhibit the activity of calpain effectively . We founThe influence on protein functions may be via the alteration of protein amounts or activity. In view of the essential role of calpain in the regulation of AR protein stability mediated by triptolide, the effects of triptolide on the expression of calpains and their endogenous inhibitory protein were determined first. After triptolide treatment, the protein levels of calpain-1, calpain-2 and their endogenous inhibitor, calpastatin, were determined by western blot assay. None of the three protein levels was significantly altered by triptolide . The res2+ levels within the cells, we further investigated the effects of triptolide on cytosolic Ca2+ levels. The Ca2+ level was determined using FLUO-3 AM. The data showed that, after triptolide treatment, the level of Ca2+ in the cells treated by triptolide was significantly higher than the control group, indicating that triptolide could promote Ca2+ accumulation in cells and elevate intracellular Ca2+  and calcium ionophore A23187 were used to explore the role of intracellular Ca2+ in AR protein down-regulation induced by triptolide. The data showed that calcium chelator BAPTA-AM recovered the protein level of AR that was decreased by triptolide (2+ level.To identify whether Caiptolide . Calciumiptolide . The resAlthough triptolide has been reported to be effective in PCa treatment, the mechanism of action is still unclear. Emerging studies have demonstrated that AR signaling plays a large role in the development and progression of PCa, even after castration ,4. AlterIn view of the important roles of protease in protein degradation, and calpains in AR protein hydrolyzation ,15,16,222+ in some cells [2+ concentration, triptolide can activate calpain and thus break down the AR protein. In the presence of triptolide, the elevation of Ca2+ level was observed, and chelating Ca2+ with BAPTA-AM restored AR expression. The result further supported our assumption. Additional investigation is still needed to discover whether the activity of calpains were changed and which isoform of calpains can be involved in AR degradation caused by triptolide.Several investigations suggested that calpains caused proteolytic elimination of AR and were beneficial for PCa treatment ,15,16. Hme cells ,19. Thes2+ indirectly independent. Though the current investigation did not rule out the role of cathepsins in triptolide induced AR degradation, taking together the information, we suggest that calpain activation plays an essential role in the down-regulation of AR protein by triptolide.Apart from calpains, there are other pathways that can modulate AR protein stability. The most common form of AR degradation is carried out by the ubiquitin proteasome system . However2+ concentration in the triptolide-treated group is lower compared with that of A23187 group, though the down-regulated AR level is similar in these two groups. AR mRNA is a significant contributor to AR protein level. Huang et al. [2+-dependent and Ca2+-independent pathways. However, quantitative investigation of the relative contribution of both pathways still needs further investigation. Moreover, though addition of A23187 following triptolide further down-regulated the level of AR protein, the concentration of A23187 employed in the current investigation also altered AR mRNA level [In addition, it is worth noting that the Cag et al.  and our g et al.  have shog et al. . Thus, iNA level . To furtAndrogen deprivation therapy is first-line treatment for PCa patients to impede the AR activity in metastasis PCa. However, gain-of-function mutations, in situ androgen production, androgen receptor gene amplification and AR splice variant have been reported to be responsible for the failure of androgen deprivation therapy . It woulIt is worth noting that the physiological functions of calpains are extensive, including regulation of apoptosis , cell mi2+ accumulation, and thus decrease the stability of AR protein, subsequently resulting in the breakdown of the AR protein in LNCaP cells. This work provides an experimental basis and evidence to elucidate the anti-PCa mechanisms of triptolide, a small-molecule natural product with great potential. However, further systematic studies are needed to acquire compelling support for triptolide as a potential drug for PCa.Together, these observations demonstrate that triptolide can activate calpain via promoting intracellular Ca


Abnormal reactivation of androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) mainly results from overexpression and down-regulation of AR. Sumoylation of AR can influence its function. However, regulation of AR sumoylation by SUMO E3 ligases PIASs to modify AR distribution and stability are not well understood.We assessed the potential effect of SUMO3 modification on AR intracellular localization by immunostaining in AR-negative prostate cancer DU145 cells, and detected the effect of PIAS1/SUMO3 overexpression on AR sumoylation related degradation. Then we characterized AR sumoylation sites involved modified by SUMO3, and the key residue of PIAS1 involved in itself sumoylation and further mediated AR sumoylation (sumo3-conjugated), translocation and degradation. Finally we detected the recognition of PIAS1 (sumoylation ligase) to MDM2, a ubiquin ligase mediated AR degradation.We demonstrate that SUMO E3 ligase PIAS1, along with SUMO3, mediates AR cytosolic translocation and subsequent degradation via a ubiquitin-proteasome pathway. Although AR sumoylation occurs prior to ubiquitination, the SUMO-acceptor lysine 386 on AR, together with ubiquitin-acceptor lysine 845, contribute to PIAS1/SUMO3-induced AR nuclear export, ubiquitination and subsequent degradation. Moreover, PIAS1 itself is modified by SUMO3 overexpression, and mutation of SUMO-acceptor lysine 117 on PIAS1 can impair AR cytoplasmic distribution, demonstrating the essential role of sumoylated PIAS1 in AR translocation. We further determine that sumoylated PIAS1 interacts with AR lysine 386 and 845 to form a binary complex. Consistent with the effect on AR distribution, SUMO3 modification of PIAS1 is also required for AR ubiquitination and degradation by recruiting ubiquitin E3 ligase MDM2.Taken together, SUMO3 modification of PIAS1 modulates AR cellular distribution and stability. Our study provided the evidence the crosstalk between AR sumoylation and ubquitination mediated by PIAS1 and SUMO3. Androgen receptor (AR) signaling, activated by androgen, plays an essential role in the initiation and progression of prostate cancer (PCa) , 2. DespPost-translational protein modifications, such as ubiquitination or sumoylation, can regulate protein stability and affect protein levels in cells. Poly-ubiquitination of proteins with a K48-linked ubiquitin chain usually targets protein degradation via the 26S proteasome , 14. SimAR is a substrate for sumoylation, and PIAS family proteins act as E3 ligases to promote AR sumoylation . SUMO1 mIn this study, we discovered that AR is exported from the nucleus and degraded by PIAS1 together with SUMO3. Although increased sumoylation levels of AR are detected, only mutation of AR sumoylation site K386, but not K520, prevents cytoplasmic translocation and degradation of AR. This suggests that sumoylation site K386 plays a crucial role in nuclear export and subsequent degradation in a sumoylation-independent manner. PIAS1 itself, as a SUMO E3 ligase, is also modified by SUMO3, which results in cytoplasmic translocation of AR. Specific recruitment of the mouse homologue of double minute 2 protein (MDM2) ubiquitin E3 ligase of AR participates in the regulation of AR turnover. These findings reveal a novel role for self-sumoylation of SUMO E3 ligase PIAS1 in the regulation of AR cellular distribution and degradation, and also unveils a previously unknown crosstalk between components of the sumoylation (SUMO3 modified PIAS1) and ubiquitination machinery (MDM2) in the degradation of the AR protein.Sumoylation exerts diverse effects, ranging from regulation of transcription to intracellular trafficking . To asseBecause of the observation that partial AR translocation into the cytoplasm occured at 48 h post-transfection with PIAS1 and SUMO3, we followed the kinetics of localization and immunofluorescence intensity changes of AR induced by PIAS1 and SUMO3 at different time points  are commonly known to generate poly-ubiquitin chains on AR that target it for ubiquitin-proteasome mediated degradation , 15, 16.To confirm direct binding of MDM2 to Sumoylated-PIAS1, we performed immunoprecipitations using the myc antibody in cells expressing PIAS1 and SUMO3 together, with or without AR. As expected, MDM2 is equally co-precipitated in both myc-immunoprecipitations , a DNA-binding domain (DBD), a ligand-binding domain (LBD), and a hinge region that separates the LBD from the DBD . Access Many proteins that are exported from the nucleus, like P53, are degraded in the cytoplasm . MoreovePIAS proteins function widely as common co-transcriptional factors and SUMO E3 ligases. Although self-sumoylation of PIASs had yet to be addressed in vitro and in vivo , 28, 29,We explored a crosstalk mechanism between AR sumoylation and ubquitination, which is specifically mediated by PIAS1 and SUMO3. We discovered that SUMO3 modification by PIAS1 modulates AR cellular distribution and stability.We identified a PIAS1/SUMO3/AR complex, in which SUMO3 modified AR by PIAS1 (a SUMO E3 ligase), which functions for AR cytoplasmic translocation, and further degradation via recruitment of ubiquitin E3 ligase MDM2.Two AR sumoylation sites, K386 and K520, and two AR ubiquitination sites K845and K847R was reported in the previous study. Here we identified AR sumoylation site K386 and ubiquitination site K845, were necessary for the formation and function of PIAS1/SUMO3/AR cytoplasmic translocation complex.We demonstrated SUMO3 modification of PlAS1 K117 is necessary for PIAS1/SUMO3 complex formation and itself sumoylation further mediated AR sumoylation (sumo3-conjugated), which induced AR cytoplasmic translocation, and then SUMO3-modified PIAS1 recruited MDM2 to induce AR degradation.Our findings reveal a previously unknown crosstalk between the sumoylation and ubiquitination in castration-resistant PCa cells, and thus provide valuable insight into AR regulation in CRPC. Therefore, this study may prove useful to developing strategies for therapeutic intervention against AR overexpression in CRPC.SUMO1 has only around 45% identity with both SUMO2 and 3 . To acceProteasome inhibitors MG132 was purchased from Sigma. The following primary antibodies were used in this study: SUMO3, Ubiquitin and AR rabbit polyclonal antibodies (Santa Cruz), PIAS1 rabbit polyclonal antibody , MDM2 rabbit polyclonal antibody (Bioss), Flag tag and myc tag mouse monoclonal antibodies (TransGene Biotech). PIAS family genes were subcloned into pCMV-myc vector (Clontech). Full-length AR and various site mutations in pcDNA3-flag vector, including AR (K386R), AR (K520R), AR (K845R), and AR (K847R), were separately subcloned into VP16 vector by NdeI and XbaI. Full-length PIAS1 and a sumoylation site mutation PIAS1 (K117 L) were separately subcloned into GAL4 vector by TthIII and XbaI.Other plasmids have been described in Acknowledgments.Site-directed mutagenesis were used to engineer residues K386, K520, K845 and K847 site mutants of AR and residue K117 site mutant of PIAS1. Briefly, primers that included the residue mutation flanked by a wild-type sequence on either side, were generated. A PCR reaction produced a new complete copy of the plasmid containing the mutation coded for by the primers. The remaining parent plasmid pcDNA3-flag-AR or pCMV-myc-PIAS1 in PCR product was then digested by Dpn I and PCR product without parent plasmid was subsequently transformed into DH5αE.coli. DNA sequence containing the point mutation was verified by DNA sequencing.RNA extraction and reverser transcriptional PCR analysis were used to quantify the change of AR mRNA levels induced by PIAS1 and SUMO3 at different time points. We harvested cultured cells in TRIzol (Invitrogen) and extracted total RNA following the manufacturer’s instructions. Two μg total RNA were transcribed (RT) into 20 μl cDNA by SuperScript III kit (Invitrogen) with oligo (dT) primer. Two μl reverse transcribed cDNA were used for PCR. We designed AR primers for PCR to measure its gene expression level and used the β-actin expression level as control. The AR primers were 5′- CAGAAGACCTGCCTGATCT-3′ (forward) and 5′- CATCCCTGCTTCATAACAT-3′ (reverse). The β-actin primers were 5′- CATCCTGCGTCTGGACCTG-3′ (forward) and 5′- ATCTCCTTCTGCATCCTGTC-3′ (reverse).The human prostate cancer cell line DU145 was cultured in DMEM medium (GIBCO) plus 5% FBS. Transient transfections were carried out using Lipofectamine 2000™ (Invitrogen) following the manufacturer’s protocol.Mammalian two-hybrid assays and luciferase assays were employed to determine the protein-protein interactions between wild type AR or its ubiquitination site  mutants or its sumoylation site  mutants, and wildtype PIAS1 or its sumoylation site K117 L mutant, with or without SUMO3. Cells were transfected overnight at ~ 80% confluence in 48-well plates with 0.1 μg of 5 × GAL4Luc3 reporter and other expression vectors as indicated in the figures and legends. Renilla luciferase reporter was used as an internal control. After 48 h transfection, firefly luciferase activities were measured by using the Dual-Luciferase Reporter Assay System (Promega) and the ratio of firefly luciferase activity to Renilla luciferase activity was calculated as Relative luciferase activity. The results reflect the mean and standard deviation from triplicate samples.Immunofluorescent staining assays were performed to assess nuclear export of AR, sub-cellular localization of PIASs, SUMO1/3, MDM2 and molecular co-localizations. After transfection, cells were fixed with 3% formaldehyde for 30 min and then permeabilized with 0.1% Triton-100/PBS for 10 min. After being pre-blocked with 1% BSA/PBS, samples were incubated with indicated primary antibodies for 2 h, followed by incubation with fluorophore-conjugated secondary antibodies (Proteintech Group). Nuclei were counter-stained with 4′,6- diamidino-2-phenylindole (DAPI) after secondary antibody incubation. Cells were examined by fluorescence microscopy (Olympus).Immunoprecipitation and immunoblots were used to assess sumoylation of AR catalyzed by PIAS1, PIAS1 self-sumoylation, ubiquitination/degradation of AR and its protein levels, the inner complex formation of SUMO3 modified PIAS1 and AR, and also the binding of MDM2 to SUMO3 modified PIAS1. DU145 cells were transfected with various vectors for proper time and cross-linked with 1-2 mM dithio-bis succinimidyl propionate for 30 min. To prepare a total cell extract, transfected cells were lysed in lysis buffer  supplemented with the Complete Protease Inhibitor Mixture (Roche). Pre-cleared lysates were then incubated with pre-equilibrated protein-A or protein-G-Sepharose beads with either AR polyclonal antibody (Santa Cruz) or myc tag monoclonal antibody (TransGen Biotech) at 4 °C for 3 h. Eluted proteins were analyzed by immunoblots using SUMO3, ubiquitin, PIAS1 and AR antibodies at the appropriate dilutions.p < 0.05 or p < 0.01 using SPSS17.0.All results shown are reported as the mean ± standard deviation (SD). The One-way Anova was used to compare the means of 2 independent groups in more than two groups. Statistical significance was assumed for a tailed http://sumosp.biocuckoo.org/download.phpGPS-SUMO is an open software freely available for academic research at website:


In SH-SY5Y neuronal cells and primary human neuron cultures, H2O2 downregulated Lnc-EPIC1 and key MYC targets . Ectopic overexpression of Lnc-EPIC1 increased expression of MYC targets and significantly attenuated H2O2-induced neuronal cell death and apoptosis. Contrarily, Lnc-EPIC1 siRNA potentiated neuronal cell death by H2O2. MYC knockout by CRISPR/Cas9 method also facilitated H2O2-induced SH-SY5Y cell death. Significantly, MYC knockout abolished Lnc-EPIC1-induced actions in H2O2-stimulated neuronal cells. Together, these results suggest that Lnc-EPIC1 downregulation mediates H2O2-induced neuronal cell death.Excessive oxidative stress causes neuronal cell injury. Long non-coding RNA (LncRNA) EPIC1 (Lnc-EPIC1) is a MYC-interacting LncRNA. Its expression and potential functions in hydrogen peroxide (H It is possibly due to the high rate of oxygen consumption, enrichment of polyunsaturated fatty acids and defective Nrf2 cascade in neurons –5. Excese injury –8. H2O2 poptosis , 9, 10. Long non-coding RNAs (LncRNAs) are traditionally considered as transcriptional noise. Recently, there are growing literatures showing that LncRNAs are key regulators of almost all cellular and physiological processes , 12. LncCDC20, CDC45 and CCNA2) [2O2)-treated neuronal cells.Lnc-EPIC1 ENSG00000224271)’s binding to MYC occurs at Lnc-EPIC1’s 129-283 nucleotide region . Lnc-EPId CCNA2) , suppresd CCNA2) . Furtherd CCNA2) . Recentld CCNA2) , 22. Con4271’s bid CCNA2) , 16 and d CCNA2) –22, we h2O2. Expression of Lnc-EPIC1 was tested by qPCR assay using the described primers [U6 RNA. As demonstrated, H2O2 treatment (300 μM) time-dependently decreased Lnc-EPIC1 level in SH-SY5Y cells  in neuronal cells, differentiated SH-SY5Y neuronal cells were treated with H primers , 22. Its5Y cells . Its levectively . Further5Y cells . Lnc-EPIreatment . At the Cyclin A1, CDC20 and CDC45 [2O2 (100/300 μM) treatment in SH-SY5Y cells. Cyclin A1, CDC20 and CDC45 protein levels were decreased as well  significantly downregulated Lnc-EPIC1 expression  were established following puromycin selection (see Methods). qPCR assay was performed to show that Lnc-EPIC1 levels were significantly elevated in the Lnc-EPIC1-OE cells, with/without H2O2 treatment  reduction  significantly increased Lnc-EPIC1 expression, even after Hmulation . H2O2-ineduction  and medieduction , was sigeduction –2K. Coll2O2 , with non-overlapping sequences [2O2-treated SH-SY5Y cells  reduction , showing completely depleted MYC mRNA . Consequ2O2, by showing significantly enhanced viability reduction and death versus control cells   and 4C. ol cells  and 4C. ol cells . Notablymulation .2O2-induced cytotoxicity , Lnc-EPIC1 overexpression (by “LV-Lnc-EPIC1”) or silencing (by “EPIC1-siRNA1”) failed to change Htoxicity  and 4F. al cells . Similar2O2-induced neuronal cell death.LncRNAs are involved in regulating almost all key cellular behaviors, by functioning as molecular signals, guides, scaffolds, enhancers or microRNA spongers, and altering gene transcription and/or functions , 12, 23.EPIC1 regulates MYC’s occupancy on several key MYC target genes, including Cyclin A1, CDC20 and CDC45 [EPIC1 functions as the “guide” RNA to promote expression of MYC targets [-EPIC1 . Here ou Lnc-EPIC1 is also vital for the survival of neuronal cells.LncRNA-mediated regulation on MYC transcriptional activity is largely unknown until recently . MYC by nd CDC45 . By dire targets . FurtherCyclin A1, CDC20 and CDC45. Contrarily, their levels were significantly reduced by siRNA-mediated silencing of Lnc-EPIC1. Importantly, Lnc-EPIC1 was decreased in H2O2-treated SH-SY5Y cells and primary neuronal cultures. Therefore, H2O2 downregulates Lnc-EPIC1 to inhibit MYC functions, which could be an important mechanism responsible for H2O2-induced neuronal cell death.We here reported a new mechanism of Lnc-EPIC1 in promoting neuronal cell survival via directly interacting with MYC, and Lnc-EPIC1 is important for MYC function in neuronal cells. In both SH-SY5Y cells and primary neuron cultures, forced overexpression of Lnc-EPIC1 significantly increased mRNA and protein expression of key MYC targets, including 2O2-induced neuronal cytotoxicity. Importantly, ectopic overexpression of Lnc-EPIC1 using a lentiviral construct significantly attenuated H2O2-induced neuronal cytotoxicity. On the contrary, siRNA-mediated silencing of Lnc-EPIC1 potentiated neuronal cell death and apoptosis by H2O2. Lnc-EPIC1 siRNA alone was cytotoxic to neuronal cells as well. Thus, targeting Lnc-EPIC1 could be a novel strategy for neuronal protection against oxidative injury.A number of recent data have implied that Lnc-EPIC1, by directly interacting with MYC, promotes survival of human cells –22. Howe2O2-induced neuronal cytotoxicity. Importantly, neither Lnc-EPIC1 overexpression nor Lnc-EPIC1 siRNA was functional in MYC-KO cells. These evidence indicated that MYC is the primary target of Lnc-EPIC1 in neuronal cells. Further insight studies are necessary to explore the interaction between Lnc-EPIC1 and MYC as well as their functions in neuronal cells. In summary, we conclude that Lnc-EPIC1 downregulation mediates H2O2-induced neuronal cell death.MYC is a transcription factor regulating expression of a wide variety of genes of cell survival. In the present study, we show that MYC KO by CRISPR/Cas9 method induced neuronal cell death and apoptosis. MYC KO also intensified H2O2, puromycin, polybrene, fetal bovine serum (FBS) and other cell culture reagents were provided by Sigma-Aldrich Co. . Antibodies were purchased from Cell Signaling Technology Co. . mRNA primers and sequences were synthesized by Genechem . All the transfection reagents were purchased from Invitrogen-Thermo Fisher Co. .H2O2 treatment, SH-SY5Y cells were cultured for 10 days in retinoic acid -containing complete DMEM medium, followed by another five days culture in serum-free DMEM with BDNF (50 ng/mL) and glutamine (2 mM).Human neuronal SH-SY5Y cells (from Dr. Gao ) were cuin vitro 8, DIV8) before any further experiments. The protocols of using human cells were approved by the Ethics Committee of authors’ institutions.The primary human neuron cultures were provided by Dr. Zhang . Briefly—ΔCt method using the following formula: 2—(Ct of target gene—Ct of reference gene). The data presented were normalized to GAPDH transcripts. mRNA primers for MYC, Cyclin A1,CDC20, and CDC45 [U6 RNA.Following the applied treatment, the TRIzol reagents (Promega) was applied to extract total cellular RNA. The Fast-Start Universal SYBR Green Master mix (Roche) was used for the qPCR. Melting curve analysis was performed to calculate the product melting temperature. qPCR quantification was through 2nd CDC45  and Lnc-nd CDC45  were synFor each treatment, 40 μg protein lysates were separated by 10% SDS-PAGE gels, transferred to PVDF blots. After blocking in 10% non-fat milk, the blots were probed with the appropriate primary antibodies, followed by incubation in the HRP-conjugated secondary antibodies. The blots were visualized by the ECL system (Sigma). Quantification of the band intensity was performed via using the Image J software.The pLenti6-GFP-puro expression vector with Lnc-EPIC1 sequence (“LV-EPIC1”) was provided by Dr. Zhang , transfe4 cells/cm2). Following treatment, cell viability was examined by the Cell Counting Kit-8 . CCK-8 optical density (OD) values at 550 nm were recorded.Neuronal cells were seeded in the 96-well plates  release to the conditional medium of dying cells was tested by a two-step LDH detection kit . Medium LDH was normalized to total LDH.4 cells/cm2), transfected with Lnc-EPIC1 siRNA or the scramble control siRNA (“scr-siRNA”) using the Lipofectamine RNAiMAX Reagent for 48h. Lnc-EPIC1 knockdown was verified by qPCR assay.Two Lnc-EPIC1 siRNAs, 5′-CCUUCAGACUGUCUUUGAA-3′ (“EPIC1-siRNA1”) and 5′-GCUUUCUCUCGGAAACGUG-3′ (““EPIC1-siRNA2”), were utilized , 22. SH-The caspase-3 activity assay was described in detail elsewhere .Single strand DNA (ssDNA) contents were measured as a characteristic marker of cell apoptosis. In brief, thirty μg cell lysates per treatment were analyzed by the ssDNA ELISA  to quantify DNA fragmentations. The ssDNA ELISA absorbance values were recorded at 405 nm.2O2 treatment, neuronal cells were first stained with JC-1 (10 μg/mL), washed and tested immediately via a fluorescence spectrofluorometer at 550 nm. The representative JC-1 images, integrating the green (at 550 nm) and red (at 625 nm) fluorescence wavelength were presented.JC-1, a fluorescence dye, can form green monomers and aggregate in mitochondria in stressed cells with mitochondrial depolarization . FollowiMYC knockoutMYC small guide RNA (sgRNA) . Statistical analyses among different groups were performed by the one-way analysis of variance (ANOVA) with Scheffe’s test using SPSS18.0 software . The two-tailed paired T tests (Excel 2007) were calculated to compare significance between two groups. Values of


Chronic exposure to environmental heavy metals is a worldwide health concern. It is acknowledged to be an important cause of lower respiratory tract damage in children. However, the molecular mechanisms underlying the heavy metal-induced cellular stress/toxicity are not completely understood. Small non-coding RNAs (sncRNAs), such as microRNAs (miRNA) and more recently identified tRNA-derived RNA fragments (tRFs), are critical to the posttranscriptional control of genes. We used deep sequencing to investigate whether cellular sncRNA profiles are changed by environmental heavy metals. We found that the treatment of arsenite, an important groundwater heavy metal, leads to abundant production of tRFs, that are ~30 nucleotides (nts) long and most of which correspond to the 5′-end of mature tRNAs. It is unlikely for these tRFs to be random degradation by-products, as the type of induced tRFs is heavy metal-dependent. Three most inducible tRFs and their roles in arsenite-induced cellular responses were then investigated. We identified that p65, an important transcription factor belonging to NF-κB family and also a key factor controlling inflammatory gene expression, is a regulated target of a tRF derived from 5′-end of mature tRNA encoding AlaCGC (tRF5-AlaCGC). tRF5-AlaCGC activates p65, subsequently leading to enhanced secretion of IL-8 in arsenite response. In this study, we also identified that endonuclease Dicer and angiogenin temporally control the induction of tRF5-AlaCGC, providing an insight into the control of tRF biogenesis and subsequently the prevention of cellular damage. There are an increasing number of reports showing that chronic exposure to arsenite, including the exposure to drinking-water arsenite, has been correlated with lower respiratory tract damage to cause both malignant and non-malignant lung diseases4. Despite strong associations between arsenite exposure and respiratory illness, the pathophysiological mechanisms by which arsenite acts on the lung remain largely unknown.Environmental exposure to arsenite is an ongoing worldwide health problem, with chronic ingestion of contaminated drinking water being the major source of exposure5, stress responses7, and host-virus interactions9. Continuous discoveries on sncRNAs have changed the landscape of human genetics and molecular biology, largely due to the fundamental role of miRNAs-the most extensively studied sncRNA class, as gene regulators10. Given their molecular nature and gene regulatory role in broad biological settings, sncRNAs are being recognized as regulatory objectives and innovative intervention tools to control many cellular responses by modifying the expression or gene silencing activity of sncRNAs11.Over the past decade, it has become clear that small non-coding RNAs (sncRNAs) play a significant role in many cellular processes, including cell proliferation12. There is increasing evidence supporting tRFs functional molecules, but not by-products from random degradation. First, tRFs are produced through specific cleavage of tRNA by endonucleases. Second, tRF expression is regulated by biological events including cellular stresses, cell proliferation, or viral infection. Third, some tRFs have gene trans-silencing activity, control protein translation, and/or carry out biological functions including virus replication control and cell division regulation18. tRF studies are fairly new and the molecular mechanisms underlying the functions of tRFs are largely unclear.tRNA-derived RNA Fragments (tRFs) are a recently identified class of sncRNAs. Their function and associated molecular mechanism(s) have not been well characterizedIn this study, we identified that arsenite-treated airway epithelial cells had a significantly higher abundance of tRFs, compared to the cells without the treatment. Three significantly induced tRFs derived from the 5′-end of tRNA-ProTGG (tRF5-ProTGG), tRNA-AlaCGC (tRF5-AlaCGC) and tRNA-GluCTC (tRF5-GluCTC) were selected for studying their roles in arsenite-induced cellular responses. We identified p65, an important transcription factor belonging to NF-κB family and also a key factor controlling inflammatory gene expression, as a regulatory target of tRF5-AlaCGC, but not of the other two tRFs. tRF5-AlaCGC activated p65, subsequently leading to enhanced secretion of an inflammatory cytokine IL-8. We also revealed that the biogenesis of tRF5-AlaCGC was temporarily regulated by endonuclease Dicer and angiogenin (ANG). Taken together with other facts that not all tRNAs were cleaved by heavy metals, and the cleavage of these tRNAs were at the 3′- side of the anticodon loop, we conclude that heavy metal-induced tRFs are functional molecules.20. SAE cells  were purchased from Lonza  and maintained as described21. Heavy metal compounds sodium arsenite, nickel (II) chloride, and cobalt were purchased from Sigma-Aldrich . Primary antibodies against P65 and lamin B were purchased from Millipore  and Sigma-Aldrich respectively. Primary antibodies against ANG, Dicer and Drosha were from Santa Cruz Biotechnology . Horseradish-coupled secondary antibodies were also purchased from Santa Cruz.A549 cells  were from ATCC, Manassas, VA, and maintained as described22  or available upon request.Total RNAs from A549 cells, treated w/wo heavy metal solution, were extracted by TRIzol Reagents . The RNAs were delivered to the Genomics Core of the University of Texas Medical Branch (UTMB) for small RNAs sequencing. In brief, small RNA libraries were created using the New England Biolabs small RNA library protocol (New England Biolabs). Library construction used a two-step ligation process to create templates compatible with Illumina based next generation sequence (NGS) analysis. Where appropriate, RNA samples were quantified using a Qubit™ fluorometric assay (Thermo Fisher Scientific). RNA quality was assessed using a 2100 Bioanalyzer and the RNA 6000 Pico LabChip® (Agilent Technologies). Library creation uses a sequential addition of first a 3′ adapter sequence followed by a 5′ adapter sequence. A cDNA copy was then synthesized using ProtoScript® II reverse transcriptase (New England Biolabs) and a primer complementary to a segment of the 3′ adapter. Amplification of the template population was performed in 15 cycles . The libraries were not size selected. All NGS libraries were indexed. The final concentration of all NGS libraries was determined using a Qubit™ fluorometric assay and the DNA fragment size of each library was assessed using a DNA 1000 high sensitivity chip and an Agilent 2100 Bioanalyzer. Sequence analysis, 2 × 50 bases, was performed on an Illumina Hi-Seq. 1500 using the TruSeq SBS kit v3. The sequence reads ≥ 10 after adaptor sequence removal were used for further classification and small RNAs were mapped using Novoalign software as we previously described in22. Briefly, RNA was separated in 8% or10% denaturing polyacrylamide gel with 7 mol/l urea and then transferred to a positively charged nylon membrane. The membrane was hybridized with 32P-labeled probes in ULTRAhyb®-Oligo solution, followed image development.Northern hybridization for sncRNAs induction was performed, as we previously describedConfluent A549 cells were transfected with an antisense oligo (100 nM) against a specific tRF or a control α-oligo using Lipofectamine® 2000 DNA transfection reagent according to manufacturer’s protocol . At 2 hours (h) post-transfection, cells were treated with/without (w/wo) arsenite (10 μM) for 15 h at 37 °C, followed by sample harvesting.24. Membranes were blocked with 5% milk in TBS-Tween 20 and incubated with the proper primary antibody to detect p65. Membranes were then stripped and re-probed for lamin B detection.Nuclear extracts of cells were prepared, as we previously described, and quantified with a protein quantification kit from Bio-Rad . Proteins were fractionated by 10% SDS-PAGE denaturing gels, and transferred to polyvinylidene difluoride membranes, as we previously describedThe level of IL-8 was quantified using human IL-8 Quantikine ELISA Kit from R&D Systems , according to the manufacturer’s instructions.Total cellular RNAs were extracted using TRIzol reagents (Invitrogen). First strand cDNA was synthesized by using TaqMan RT reagents . The RT reaction was performed under the following conditions: 25 °C, 10 minutes; 48 °C, 30 minutes; 95 °C, 5 minutes. Quantitative RT-PCR amplification was performed by using SYBR. Sequence information on green-labeled primers for target genes and internal control 18 S rRNA is available upon request. Quantitative PCR reactions were performed with the FastStart Universal SYBR Green Master (ROX)  in the ABI 7500 Sequence Detection System using following conditions: initial steps: 50 °C, 2 minutes and 95 °C, 10 minutes; PCR steps: 95 °C, 15 seconds and 60 °C, 1 minute for 40 cycles. The information on RT primers to quantify ANG, Dicer, and Drosha is available upon request.t test for paired or unpaired data, or one-way ANOVA, as appropriate.Data are presented as means ± SEM, and statistically significant differences were assessed by Student’s 25, a chemical compound which can be inhaled or absorbed through the skin, leading to various types of disease symptoms including death  &26. Although the induction of tRFs was suggested to contribute to arsenite-induced cellular stress, there was no direct evidence. In addition, the study was done in a microarray format, which might leave some tRFs with significance in heavy metal-induced diseases unrevealed. Since the chronic exposure to arsenite has been correlated with lower respiratory tract damage4, A549 cells, a lower respiratory epithelial cell model, were used and the change of a global sncRNA expression profile was obtained in response to arsenite treatment by Illumina ultra-high-throughput sequencing. These raw data were analyzed as depicted in Fig. http://www.mirbase.org), the rRNA database , the tRNA database , and the Exon-Intron database . In high-throughput sequencing, the cloning frequency provides a digital expression of sncRNAs. Among sncRNAs, no significant changes in miRNAs or rRNA-derived sncRNAs were found in response to arsenite (data not shown). At 6 h post treatment with arsenite, the percentile of tRNA-derived sncRNAs was increased by the fold of 1.6 in A549 cells. The majority of affected tRFs was derived from the 5′-end and for those with induction larger than two by arsenite were listed in Table Heavy metal-induced tRFs were first reported for arseniteHere, we selected three tRFs with different fold induction to validate the deep sequencing data, using probes demonstrated in Fig. 18, further supporting stimulus-dependent tRF induction.It is interesting that tRF5-GluCTC-S, but not tRF5-GluCTC-L, is also inducible by RSV, a leading cause of the lower respiratory tract infection in the pediatric population29, it is physiologically needed to confirm that primary airway epithelial cells can also produce these tRFs upon the treatment of arsenite. To investigate that, primary cultured human small airway epithelial (SAE) cells were treated with 10 µM arsenite for 15 h, followed by total RNA extraction and Northern blot described in Fig. Although A549 cells are a common cell model for respiratory infection and stress studies31. Herein, we investigated whether and how arsenite-induced tRFs contribute to cellular responses to arsenite. Since NF-ĸB, a transcriptional factor, has been shown previously to be responsible for carcinogenesis, apoptosis, and inflammation in arsenite responses33, we first studied whether tRFs affect the activation of NF-ĸB. As shown in Fig. 34. It is also known that IL-8 is tightly regulated by NF-ĸB in airway epithelial cells35. We, therefore, investigated whether arsenite-induced IL-8 can be inhibited by the treatment of anti-tRF5-AlaCGC oligo. As shown in Fig. Although arsenite is a significant environmental contaminant found in soil and water, and besides its neurobehavioral and carcinogenetic effects, it is also highly associated with lower respiratory tract damage, the molecular mechanism contributing to its impact on cellular responses is largely unknown), supporting tRFs are not randomly generated, but produced via a specific pathway. Previously, we and others have shown that both prokaryotes and eukaryotes use ribonucleases to target the tRNA anticodon loop38. In particular, a ribonuclease called ANG is responsible for stress- or virus-induced cleavage of tRNA at the anticodon loop to produce tRFs derived from the 5′-end of tRNAs (tRF-5 series)25. Dicer, a ribonuclease commonly recognized to be responsible for miRNA biogenesis, was also reported to cleave tRNAs to produce tRFs from the 3′-end of tRNAs (tRF-3 series)39, although a recent report demonstrated that some tRF-3 series to be Dicer-independent40. Dicer is also responsible for tRNA cleavage on D arms of mature tRNAs in cancer cells to produce tRF541. Whether Dicer contributes to heavy metal-induced tRFs is currently unknown. Since tRF5-AlaCGC is involved in NF-kB-mediated inflammatory response to arsenite, studying the biogenesis mechanism(s) of tRF5-AlaCGC may provide an insight into arsenite-induced inflammation control. We, therefore, suppressed several types of ribonucleases, including ANG and those responsible for miRNA production, by their respective siRNA, and measured tRF5-AlaCGC in arsenite-treated A549 cells. As shown Fig. 22. Although ANG has been demonstrated in the previous study in mediating arsenite-induced tRFs, their role was focused on the early time point post treatment14. This is the first report demonstrating a temporal role of ANG in tRF biogenesis in the context of heavy metal treatment. We also discovered that Dicer can produce tRF-5 series by cleaving tRNA at the anti-codon loop, in addition to its previously described role in generating tRF-3 series and tRF-5 series ending at D arms of mature tRNAs41. Collectively, our data demonstrated that ANG and Dicer play a role in inducing tRF5-AlaCGC by arsenite. However, ANG only contributes to its early induction.Since not all tRNAs produced tRFs in response to arsenite treatment, we do not think that arsenite-induced tRFs are random degradation products. Close inspection of tRFs in the high-throughput data revealed the 3′-ends of major tRF5 isoforms were located at the anti-codon loop see Fig. , supportIn this study, we demonstrated that tRF induction is a common biological event in response to heavy metal exposures. Depending upon the type of heavy metal, the profile of induced tRFs varied. However, induced tRFs were always derived from a limited population of mature tRNAs, regardless of which heavy metal was used for the induction. Structurally, the 3′-ends of induced tRFs ended nearly by the anticodon loop. More importantly, a tRF from arsenite-treated cells was proved to be functional in arsenite-induced p65 activation and IL-8 production. All of these supported that tRFs induced by heavy metals were not random degraded byproducts, but functional molecules with biological importance. This discovery may launch a new angle to study the cellular/stress responses to the exposures of heavy metals.41. In sex hormone-dependent cancers, ANG seems important to generate tRF5 via targeting the anticodon loop42. In response to RSV infection, we found that ANG is responsible for tRNA cleavage on the anticodon loop22. In prostate cancer, ELAC2 is responsible for generating tRFs derived from 3′ end of tRNA precursor transcript43. Overall, these studies seemed to support specific cleavage of tRNA by a specific nuclease. Interestingly, our study revealed that both ANG and Dicer are responsible for generating a tRF5 with its 3′-end in the anticodon loop. Why cells need two enzymes to temporally regulate the tRF biogenesis in response to arsenite needs to be examined further. It is well known that Drosha is in the nuclear compartment and also a key enzyme to cleave a long RNA primary transcript known as a pri-miRNA to produce a stem-loop structure of about 70 base pairs long, known as a pre-miRNA. Cytosolic Dicer then cleaves pre-miRNAs to produce miRNAs44. Therefore, our study clearly demonstrated that the biogenesis of tRF is different from that of miRNA, as Drosha was not involved in the tRF biogenesis. The independence of tRF on Drosha also suggested that tRF cleavage did not occur in the nuclear compartment, which is consistent to what we reported for tRFs’ cytosolic presence22. Other than the mechanism by which ribonucleases use to generate specific tRFs in certain biological settings, it is also possible that the enzymes actually cleave all tRNAs. However, some tRFs are not detectable due to their instability. Currently we do not know whether some tRFs are regulated at the post-cleavage steps. However, for tRF5-AlaCGC, it is inducible by arsenite but not by cobalt. Since both treatments were done in A549 cells, it is less likely for tRF5-AlaCGC to be induced by cobalt but unstable in A549 cells. As discussed, arsenite is an important health risk factor and its contribution to respiratory illness is significant, with mechanisms largely unknown45. The investigated response to arsenite treatment is mainly oxidative stress and its regulation at the protein level47. It has been suggested that cells deficient in eIF2α phosphorylation have a higher accumulation of tRNA fragments derived from initiator-tRNAMet in response to arsenite, which may affect the rates of protein synthesis16. The accumulation of tRF5-MetCTA by arsenite was also observed in our study (Table 48. In this study, we investigated whether there are any other mechanisms than tRF-controlled protein synthesis contributing to heavy metal-induced pathogenesis, we discovered that a tRF actually was involved in the regulation of the activation of transcription factor p65 and subsequently enhanced the IL-8 secretion in response to arsenite treatment. In the future, we will investigate the molecular mechanism underlying the interaction of tRF5-AlaCGC and p65. Overall, this study generated a new study angle to use tRF to examine mechanisms underlying pathogenic diseases by heavy metals, including arsenite, cobalt and nickel52.The induction of tRFs is more or less mediated by ribonucleases. As mentioned, Dicer is responsible for the biogenesis of some tRF3s and D-arm cleaved tRF5sSupplementary figure and tables


To determine if female football players who had longer durations of rehabilitation, measured in months, after anterior cruciate ligament reconstruction would have lower tuck jump scores (fewer technique flaws) and smaller asymmetries during drop vertical jump landing.One-hundred-and-seventeen female football players, aged 16–25 years, after primary unilateral ACL reconstruction  were included. Athletes reported the duration of rehabilitation they performed after anterior cruciate ligament reconstruction. Athletes also performed the tuck jump and drop vertical jump tests. Outcome variables were: tuck jump score, frontal plane knee motion and probability of peak knee abduction moment during drop vertical jump landing.P = 0.01). Athletes with > 9 months of rehabilitation had more frontal plane knee motion  than athletes with < 6 months (P = 0.01) or 6–9 months (P = 0.03).There was no difference in tuck jump score based on duration of rehabilitation (n.s.). No interaction (n.s.), difference between limbs (n.s.), or duration of rehabilitation (n.s.) was found for peak knee abduction moment during drop vertical jump landing. No interaction (n.s.) or difference between limbs (n.s.) was found for frontal plane knee motion, but there was a difference based on duration of rehabilitation (As there was no difference in tuck jump score or peak knee abduction moment based on duration of rehabilitation, the results of this study press upon clinicians the importance of using objective measures to progress rehabilitation and clear athletes for return to sport, rather than time alone.II. Time from surgery or duration of rehabilitation is an easy measure for athletes, clinicians, and surgeons to track, but time alone may not give the most complete information on an athlete’s recovery . Time isMany athletes expect to return to sport in 6–12 months after ACL reconstruction . HoweverThe tuck jump test aims to identify technique flaws during plyometric activity, with the goal of identifying athletes that could be at risk for injury , 20. OriThe purpose of this study was to explore if there were differences in tuck jump score and asymmetry during DVJ landing based on duration of rehabilitation in female football players. Expanding on the previous findings relating longer durations of rehabilitation to more symmetrical performance , the autThis study was a secondary aim and analysis of baseline data collected as part of a prospective cohort study. The primary analysis, and its methodology, exploring knee function and return to sport outcomes has been published , and proAthletes were identified through the Swedish National Knee Ligament Register, and via advertisement on the websites of three regional football districts near Linköping University, Sweden. Inclusion criteria were female football players, 16–25 years old, who had a primary ACL reconstruction in the past 6–36 months at a clinic in one of the three regional football districts. Exclusion criteria were a concomitant posterior cruciate ligament injury, medial or lateral collateral ligament knee injury that was surgically treated , 10, nevFive hundred and thirty-five athletes were identified through the Swedish National Knee Ligament Register as meeting inclusion criteria Fig. . These aThe web-based questionnaire asked athletes about their age, level of football, and duration of supervised rehabilitation after ACL reconstruction . DuratioAthletes performed one trial of the tuck jump test. The tuck jump involves the athlete jumping continuously for 10 s. The standardized instructions were to lift the knees to hip height and attempt to land in the same place. Athletes were unaware of the grading criteria for the tuck jump test. Two video cameras (Panasonic HC-V500M), one in the frontal and one in the sagittal plane, 5 m and 3.5 m from the athlete, respectively, were used. Video was recorded at 50 Hz with advanced video coding high-definition format at 1080/50p. The tuck jump was analyzed by one researcher (IM), blinded to duration of rehabilitation, according to a clinician-friendly grading tool . PreviouTo perform the DVJ test athletes stood on a box (31 cm high) with their feet on marks 35 cm apart. Athletes were given standardized instructions to drop down off the box and immediately jump as high as possible reaching with both arms towards a ball suspended at a height of 260 cm. Data were captured with the same two video cameras, 70 cm high, one 3.5 m from the expected landing position in the frontal plane and one 2.5 m in sagittal plane . The thrThe study was approved by the Regional Ethical Review Board (Dnr 2012/24-31 and 2013/75-32) and the Swedish National Knee Ligament Register Board.Variables of interest were the duration of rehabilitation each athlete had , the total tuck jump score, the frontal plane knee motion and the pKAM during the DVJ. Presence of a concomitant meniscal or cartilage pathology was used as a covariate because such pathologies, particularly if surgically treated or repaired, can prolong rehabilitation.P < 0.10. The presence of concomitant meniscal or cartilage pathology was included in all models as a covariate.One-way ANOVAs and chi-squared tests were used to assess if there were differences in demographic and surgical variables between each duration of rehabilitation. An ANCOVA with planned least squares comparisons was used to determine if there were differences between each duration of rehabilitation and total tuck jump score. A chi-square test was used to determine if there were differences in the proportion of athletes who had an abnormal tuck jump score  based on duration of rehabilitation. Repeated measures (limb × duration of rehabilitation) ANCOVAs with planned least squares comparisons were used to examine differences in frontal plane knee motion and pKAM between the surgical and non-surgical limbs and between each level of rehabilitation duration. Planned comparisons were the duration of rehabilitation and explored if the main effect was P ≤ 0.05 and effect size was reported as partial eta-squared (np2). The sample size was set by the wider prospective cohort study. Sensitivity analysis indicated that given this sample size (N = 117), with a power of 0.80, three groups (duration of rehabilitation) and two measures (limb), an effect size of np2 = 0.08 could be detected in frontal plane knee motion. Effect sizes were considered small (np2 = 0.01), medium (np2 = 0.06), and large (np2 = 0.14) , indicating that regardless of limb there was a difference in frontal plane knee motion based on duration of rehabilitation  or 6–9 months (P = 0.03) of rehabilitation had less frontal plane knee motion during the DVJ than an athlete who had > 9 months of rehabilitation. There was neither limb x duration of rehabilitation interaction effect (n.s.) for pKAM during the DVJ, nor main effects of limb (n.s.) or duration of rehabilitation (n.s.) , nor was there a main effect of limb (n.s.) indicating no significant asymmetry during the DVJ. However, there was a significant main effect of duration of rehabilitation [F,113 = 4.The most important finding of this study was that tuck jump score did not differ based on duration of rehabilitation after ACL reconstruction. The women had no difference between limbs or in probability of a high peak knee abduction moment during DVJ landing based on duration of rehabilitation. There was also no difference between limbs in frontal plane knee motion. However, regardless of limb, athletes with > 9 months rehabilitation had more frontal plane knee motion than those with < 9 months. Although increased medial/valgus knee motion has been associated with a higher risk of both primary and secondary ACL injuries , 15, 26,A systematic review by Barber-Westin et al.  showed tA community-level study found that athletes who had ≥ 6 months of rehabilitation with structured agility and plyometric training achieved ≥ 90% quadriceps strength and single-legged hop test limb symmetry , whereasFuture studies are needed to examine rehabilitation prior to ACL reconstruction and the interaction between rehabilitation before and after surgery. Details of rehabilitation protocols, interventions, rehabilitation progression guidelines, dosage, compliance, and return to sport criteria were not collected in this study. Rehabilitation details would have enabled the authors to understand the results in further depth, however support future work on the outcomes of specific interventions and guidelines for progression during ACL reconstruction rehabilitation.There are some limitations to this study. Although three-dimensional motion analysis allows for more precise measurements and force plates facilitate the calculation of peak knee abduction moment, the two-dimensional video used in this study is clinically accessible and feasible, and both the DVJ and tuck jump are tests used frequently to assess jump landing performance , 19, 20.The primary clinical take-home from the current study is the importance of using objective criteria to assess athletes as they progress through their rehabilitation after ACL reconstruction. Objective criteria should include functional measures such as strength and patient-reported outcome measures , 11, butThis study found no differences in tuck jump score or probability of high peak knee abduction moment during DVJ landing. Regardless of limb, female football players who had > 9 months of rehabilitation had more frontal plane knee motion during DVJ landing than athletes with < 9 months of rehabilitation after ACL reconstruction. In conclusion, these results suggest that return to sport should not be determined purely based on time or duration of rehabilitation, but rather reinforce the need to use objective measures to guide rehabilitation progression and discharge.


Mortality at six months was lower for patients receiving rehabilitation compared with those who had not (adjusted odds ratio (OR): 0.49; 95% confidence interval . Sub-analysis showed the benefit of cardiac , but not geriatric rehabilitation . A program of rehabilitation after TAVI has the potential to reduce mortality. Future studies should focus on health-orientated behavior and identifying risk factors for declining rehabilitation programs.The benefit of rehabilitation in elderly patients undergoing transcatheter aortic valve implantation (TAVI) for treatment of severe aortic stenosis is unknown. The impact of declining rehabilitation programs on mortality has also not been described. In a longitudinal cohort study of 1056 patients undergoing elective TAVI between 2008 and 2016, logistic regression analysis was used to assess the relationship between treatment modality and outcome according to whether or not patients participated in a three-week rehabilitation program after TAVI. Subgroup analyses included patient outcome separated according to cardiac, geriatric, or no rehabilitation. A total of 1017 patients survived until hospital discharge (96.3%) and were offered rehabilitation, 366 patients (36.0%) declined to undergo rehabilitation, with the remaining patients undergoing either cardiac ( Cardiac rehabilitation programs improve functional capacity and quality of life in patients with aortic valve replacement . The devTo date, approaches to encourage post-TAVI recovery have included early mobilization, exercise training, patient education, and psychological support ,4,5,6. AWe hypothesized a rehabilitation program would produce reduced cardiovascular mortality.All patients undergoing elective TAVI at the Brandenburg Heart Centre in Bernau, Germany between 2008 and 2016 were enrolled and prospectively followed by telephone calls or letters. Subjects were excluded if they died during initial hospital stay. Therefore, hospital discharge was considered as baseline for further analyses.The study was performed in accordance with the Declaration of Helsinki. The Strengthening the Reporting of Observational Studies in Epidemiology guidelines for reporting observational studies was used .The ethics committee advised that no formal vote and no written informed consent beyond the agreement at hospital admission was necessary because data collection and assessment was part of hospital-wide measure of quality management.2 increase, glomerular filtration rate [GFR] was considered as less than vs. 60 or more mL/min, LVEF by an increase of 1%, the EuroScore by an increase of 1%, and the remaining variables as yes vs. no.Patient characteristics were recorded at baseline, including demographics and comorbidities. A full cardiac history was taken and the logistic EuroScore I was calculated. Echocardiographic parameters were documented, including left ventricular ejection fraction (LVEF), and New York Heart Association (NYHA) class was determined. Levels of N-terminal brain natriuretic peptide (NT-proBNP) were measured. Age was analyzed by one year increments, body mass index (BMI) by a 1 kg/mWe chose 6-month mortality as the follow-up period to discriminate potential intermediate effects of a rehabilitation program from comorbidities becoming more relevant for long-term patient outcome, such as for 1-year mortality.During the TAVI procedure, complications were documented. Prior to discharge, a full cardiac assessment was performed including aortic valve function, LVEF, and NT-proBNP levels.Patients were subsequently evaluated at an outpatient appointment 6 months after TAVI. Also, the patients’ vital status was assessed by telephone or letter. If no reply was received, the family doctor was contacted by telephone. Mortality and rehospitalization since discharge were documented and cardiac function was again assessed.n = 4) were representative for post-interventional rehabilitation programs. The components of the rehabilitation included patient health education, advice on cardiovascular risk reduction, including lifestyle and dietary advice, psychological support, and physical activity.In accordance with standard clinical practice in Germany, all patients were offered rehabilitation after the TAVI procedure in preparation for hospital discharge. Those considering post-interventional rehabilitation programs either underwent cardiac or geriatric rehabilitation on an inpatient basis for approximately 3 weeks. The rehabilitation centers  but present with a Barthel index of at least 70, and thus with a high degree of independence. There are various training methods, such as ergometer and treadmill training, Thera Band ™  use, strength and muscle building, and swimming.Often cardiac rehabilitation is divided into three phases with phase I initiated during the index hospital stay focusing on early progressive mobilization of the patient to enabling self-care by discharge, and counseling about the illness, the treatment, risk factors management, and follow-up planning. The next phase is a 3–4-week in-patient rehabilitation program or a supervised ambulatory outpatient program of 3 to 6 months’ duration, which both consist of monitored exercise and further risk factor reduction. Phase III is a lifetime maintenance phase in which physical fitness and additional risk-factor reduction are emphasized.p-value < 0.05 was considered statistically significant. Unadjusted odds ratios (OR) with 95% confidence intervals (95% CI) are provided for mortality and rehospitalization at 6 months. Kaplan-Meier analysis was performed to assess survival during the 6 months after hospital discharge, with a Breslow test used to compare the rehabilitation and no rehabilitation groups. Multivariable analysis of the variables independently associated with mortality at 6 months was performed considering the following variables recorded prior to baseline: age, male sex, body mass index, glomerular filtration rate <60 mL/min, diabetes mellitus, left ventricular ejection fraction, logistic EuroScore I, the use of balloon expandable valves, and the attendance at rehabilitation. The covariates for the regression model were based on statistically significant differences at baseline (threshold p ≤ 0.1) as well as on clinical judgement. Statistical analysis was performed using SPSS version 23.0 .Data are provided as patient numbers and percentages, normally distributed data are presented as means and SD, and non-normally distributed data as medians with 25–75th percentiles. Data distribution was evaluated using histograms. For comparison of linear variables, one way analysis of variance (ANOVA) or the Mann-Whitney U-test were used. Fisher’s exact test or the chi-square test was used for categorical values as appropriate. A two-sided A total of 1056 patients underwent elective TAVI during the study period. Of these, 1017 survived until hospital discharge and were included in the subsequent analysis . Althougp = 0.965) and chronic kidney disease . Diabetes mellitus was less common in patients who underwent rehabilitation than for those who did not , whereas NT-proBNP levels were lower .The baseline characteristics of the patients with and without rehabilitation were similar . In accop = 0.467) (p = 0.198), whereas pacemaker implantation was more frequently required in those patients who underwent rehabilitation . The median length of hospital stay after TAVI was longer for the group of patients that went on to undergo rehabilitation : 6.0, 9.0) than those that declined it .Few patients required conversion to open surgery during the TAVI procedure  . Similarp = 0.334) (p = 0.031), whereas the peak valve gradient and levels of aortic insufficiency did not vary significantly.Patient evaluation prior to hospital discharge identified lower NT-proBNP levels than those recorded prior to TAVI, with little variation between the rehabilitation and no rehabilitation groups  . The meap = 0.003)  .p = 0.032). All other variables were not independent risk factors for the endpoint assessed   .Assessment of cardiac function at six months identified little variation depending on whether or not patients had undergone rehabilitation . Few patThe finding of a reduced mortality being associated with rehabilitation prompted the question of whether a cardiac and geriatric rehabilitation program would potentially differ with respect to outcomes based on their distinct profile.Comparing the survival rate for patients with cardiac, geriatric, or no rehabilitation showed the highest survival rate during the six months after TAVI for the group of patients who underwent cardiac rehabilitation .p = 0.003) with more patients that underwent prior CABG  but with a lower NYHA class  and logistic EuroScore I . Cardiac rehabilitation patients had a shorter median hospitalization after TAVI  and lower levels of NT-proBNP .p = 0.006), whereas GFR <60 mL/min was associated with an increased risk  (p = 0.65). There was a non-significant effect of the GFR  and BMI .Cardiac rehabilitation was associated with reduced mortality  . GeriatrIn this population of 1017 consecutive patients receiving TAVI, we found six-month survival to be higher in patients that underwent a rehabilitation program after their procedure compared to those that voluntarily declined participation. A cardiac rehabilitation program enforcing physical exercise, in addition to psychosocial training after TAVI, was associated with a higher survival at six months than a geriatric rehabilitation program. The reduced lower all-cause mortality at six months in patients receiving rehabilitation was mainly an effect of a decrease in non-cardiovascular mortality.The benefits of rehabilitation for patients with CAD, including those undergoing percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), are well-established. Not only have improvements in exercise capacity been demonstrated ,11 but dIn the present study, superior mortality over the six months after TAVI was observed for the patients that underwent rehabilitation compared to those that declined it. This advantage appeared to be predominantly a result of a decreased likelihood of non-cardiovascular mortality, with the observed reduction in cardiovascular death being less significant. This suggests that improvements in cardiovascular health were not the main driving force behind the lower mortality for the patients that received rehabilitation. This is in agreement with the data regarding cardiac function at six months after TAVI, which showed no significant differences between the two groups. In broad populations of patients with cardiac disease, patients who choose to not participate in cardiac rehabilitation are more likely to be depressed, of low socio-economic status, and physically inactive, predicting poorer clinical outcomes compared to patients receiving rehabilitation ,17.Diabetes is an important risk factor for one-year all-cause mortality in patients scheduled for TAVI . In our A recent meta-analysis summarizing data from 292 TAVI patients undergoing cardiac rehabilitation program demonstrated improvements in six 6-minute walk distance (6 MWD) and exercise capacity for TAVI patients after rehabilitation . Most ofA further notable finding of this analysis was the high proportion of patients that voluntarily declined rehabilitation. Poor uptake of such programs after cardiac surgery has been previously demonstrated ,25. FurtThe study was designed as a longitudinal observational cohort study with prospective follow-up focusing on six-month mortality. Thus, the results enabled us to generate hypotheses for future research questions when preparing a study with a randomized controlled design. Insufficient information regarding exercise capacity and six MWD was available, in particular, for the patients that did not attend rehabilitation. This prevented comparisons with previous studies that evaluated these outcomes. Also, evaluation of variables, such as frailty, nutritional status, depression, or musculoskeletal status, may have provided further insight into the benefits of the different components of the rehabilitation programs. Patients declining rehabilitation may have been behaviorally and socio-economically different from those who chose to perform rehabilitation, which should be focus in future prospective studies. Health-oriented behavior should be assessed in a controlled design as it was not the primary question of this study.Patients that underwent rehabilitation after TAVI had superior overall survival compared to those that voluntarily declined to participate in a program. Tailoring of the components of the rehabilitation to the needs of this unique population, as well as increasing patient participation, could further foster the benefits of TAVI. Future studies should focus on health-oriented behavior as well as identifying risk factors for declining participation in rehabilitation programs.