Serum-free testosterone was significantly decreased 3 h post-exercise and remained significantly less than baseline 24 h post-exercise in both conditions (p < 0.05). In response to HL, AR-DNA binding significantly increased at 3 h post-exercise (p < 0.05), whereas no significant differences were observed at any time in response to LL (p > 0.05). Moreover, sarcoplasmic β-catenin was significantly greater in HL (p < 0.05) without significant changes in nucleoplasmic β-catenin (p > 0.05). In conclusion, increases in AR-DNA binding in response to HL RE indicate AR signaling may be load-dependent. Furthermore, despite the lack of increase in serum and muscle androgens or AR content following HL RE, elevations in AR-DNA binding with elevated sarcoplasmic β-catenin suggests β-catenin may be facilitating this response.The purpose of this study was (1) to determine the effect of single bouts of volume- and intensity-equated low- (LL) and high-load (HL) full-body resistance exercise (RE) on AR-DNA binding, serum/muscle testosterone and dihydrotestosterone, muscle androgen receptor (AR), and AR-DNA binding; and, (2) to determine the effect of RE on sarcoplasmic and nucleoplasmic β-catenin concentrations in order to determine their impact on mediating AR-DNA binding in the absence/presence of serum/muscle androgen and AR protein. In a cross-over design, 10 resistance-trained males completed volume- and intensity-equated LL and HL full-body RE. Blood and muscle samples were collected at pre-, 3 h-, and 24 h post-exercise. Separate 2 × 3 factorial analyses of variance (ANOVAs) with repeated measures and pairwise comparisons with a Bonferroni adjustment were used to analyze the main effects. No significant differences were observed in muscle AR, testosterone, dihydrotestosterone, or serum total testosterone in either condition ( Androgens, such as testosterone and dihydrotestosterone (DHT), play a pivotal role in muscle specific gene and protein expression, which can ultimately lead to skeletal muscle hypertrophy . PrimariIt is thought that the acute increase in serum/muscle androgen and/or AR content are responsible for the up-regulation in AR signaling. While this has been reported ,15,16,17Recent research suggests increases in AR signaling in response to mechanical loading may be due to up-regulation in specific AR-interacting proteins ,29,30. AIt is generally thought that higher volume and intensity RE are needed to elicit an appropriate stimulus for increasing AR signaling activity. While this may be the case, RE load may be the key variable responsible for increasing AR activity, provided it is accompanied with appropriate training volume and intensity. This is due to the overwhelming role load has on recruitment of motor units and the subsequent fiber type-specific metabolic and/or contractile stress placed on skeletal muscle. However, research investigating the load’s impact on muscular adaptations is commonly misinterpreted due to suboptimal methodological approaches and inconsistencies in terminology. For example, much of the literature does not clearly define and differentiate between intensity and load. In RE, intensity is commonly defined as a percent of 1 repetition maximum (1 RM) . HoweverParticipants visited the laboratory on 3 separate occasions in the following manner: visit 1 = entry/familiarization, medical/physical activity screening, and RE max testing; visit 2 = LL (50% 1 RM) RE; visit 3 = HL (80% 1 RM) RE session. Each visit was 7–10 days following the previous visit and participants were instructed to refrain from exercise for 48 h prior to RE max testing and RE protocols. In a crossover design and volume-equated manner, participants performed identical full-body RE consisting of barbell bench press, angled leg press, lat pulldown, and unilateral leg extension. During LL RE, participants performed 50% of their one repetition maximum to volitional failure for 4 sets in each exercise. Total exercise load volume (sets × repetitions × load) was calculated and equated in the HL RE during visit 3. During HL, sets were performed until participants reached the volume in order to match the total volume performed during LL. This allowed for volume and intensity to be equated between the two conditions. Each session involved the gathering of data for the analysis of biochemical and hormonal markers of blood and muscle metabolite changes.Ten apparently healthy, recreationally resistance-trained  for at least 1 year prior to the onset of the study) men between the ages of 18–35 volunteered for this study. Resistance training status was confirmed by a leg press 1-RM, which was compared to normal strength-to-body weight ratios. Participants were eligible for inclusion if their strength-to-body weight ratio was ≥2.82 times body weight . ParticiTo determine muscular strength and proper RE load prescription, participants performed 1-RM tests for bench press and angled leg press and 10-RM tests for unilateral leg extension and lat pulldown in accordance with the National Strength and Conditioning Association (NSCA) guidelines. Leg press foot placement and bench press/lat pulldown handgrip width were recorded and held constant over all testing conditions in order to maintain consistency. To ensure participants were moving through the full range of motion during each repetition, a goniometer was used to establish 90 degrees of knee flexion on the leg press. Moreover, during the bench press, participants were instructed to touch their chest with the barbell and extend their elbows to full extension to constitute a completed repetition. Similarly, participants were required to fully extend their elbows and pull the cable attachment until it made contact with their sternum/chest during the lat pulldown as well as fully extend their knees to constitute a completed repetition during the unilateral leg extension.Participants warmed up by completing 5 to 10 repetitions at approximately 50% of the estimated 1 RM/10 RM. Then participants rested for 1 min and then completed 3 to 5 repetitions at approximately 70% of the estimated 1 RM/10 RM. Load was then increased conservatively and the participant attempted to lift the load for 1/10 repetition(s). If the lift was successful, the participant rested for 5 min before attempting the next weight increment. As per NSCA guidelines, load was increased by 2.5–5% for upper body exercises and by 5–10% for lower body exercises. This procedure was continued until the participant failed to complete the lift. The 1 RM/10 RM was recorded as the maximum weight that the participant was able to lift for 1/10 repetition(s).®, Premier Nutrition Corporation, Kings Mountain, NC, USA, ). Additionally, as auxiliary measures of exercise intensity, muscle soreness and a rating of perceived of exertion (RPE) of each condition was gathered. Muscle soreness was assessed pre-exercise, 3 h post-, and 24 h post-exercise via a visual analogue scale in which participants were instructed to draw an intersecting mark across a 13-cm scale . The distance of each mark was measured from 0, and the measurement was used as the observed muscle soreness value. RPE was assessed immediately post-exercise via the modified Borg Scale (0–10). Lastly, in an attempt to control for variations in RE performance, skeletal muscle strength, and proper recovery, RE protocols were scheduled within 2 h of each other and separated by 7–10 days [During visits 2 and 3 participants performed full-body RE protocols consisting of barbell bench press, angled leg press, lat pulldown, and unilateral leg extension. As our main experimental variable, load varied between LL and HL RE while keeping all other controllable variables constant. During LL RE, participants performed 3 sets of each exercise at 50% 1 RM and were taken to volitional failure following the previously described warm-up protocol. During HL RE, participants performed each exercise at 80% 1 RM to volitional failure. Additionally, volume was equated between the two visits. Due to the greater amount of volume that can be accumulated with a lighter load, additional sets for each exercise were utilized (if necessary) in order to equate volume between LL and HL conditions. Moreover, when fatigue/failure occurred, study personnel provided assistance to help re-rack the weight safely. In all cases, 2–4 min rest occurred between all sets and exercises. Due to the diurnal nature and dietary influence of the biomarkers being investigated, participants reported to the laboratory upon waking and in a fasted state at 08:53 (±0:55) and 08:37 (±1:00) for LL and HL, respectively. Moreover, in order to minimize nutritional mediation of the markers we were investigating, participants received a standardized nutrition bar 30 min prior to RE  and height (cm) were determined on a standard dual beam balance scale . Percent bone mineral content, body fat, fat mass, and lean body mass were determined using dual-energy X-ray absorptiometry (DEXA) . Participants were required to record their dietary intake for 48 h prior to RE max testing and protocols. Participants’ diets were not be standardized but were asked to keep their dietary habits consistent throughout the study. The dietary recalls were evaluated with the MyFitnessPal mobile or desktop application  to determine the average daily macronutrient consumption of fat, carbohydrate, and protein in the diet for the duration of the study. Since previous research has demonstrated individuals who are dehydrated will have an attenuated testosterone response with RE [® 1 Automatic Biopsy Instrument  after subcutaneous administration of the local anesthetic (1 mL of 1% lidocaine/xylocaine). After the initial biopsy, the following biopsy attempts were made to extract tissue from approximately the same location as the initial biopsy by using the pre-biopsy scar, depth markings on the needle, and a successive incision that was made approximately 0.5 cm to the former from medial to lateral. After removal, adipose tissue was trimmed from the muscle specimens and was immediately frozen and stored at −80 °C for later analysis. Three muscle samples were obtained at visits 2 and 3 for a total of 6 muscle biopsies performed during the course of the study. Biopsies were taken pre-exercise and at 3 and 24 h post-exercise during visits 2 and 3.Percutaneous muscle biopsies (~30 mg) were obtained from the middle portion of the vastus lateralis muscle of the dominant leg (midpoint between the patella and the greater trochanter of the femur) at a depth between 1 and 2 cm using the fine needle aspiration technique. Muscle tissue was extraction using the TRU-COREVenous blood samples were obtained into 10 mL vacutainer tubes using a 21-gauge phlebotomy needle inserted into the antecubital vein. Blood samples stood at room temperature for 10 min and then centrifuged. The serum was then removed and frozen at −80 °C for later analysis. Six blood samples were obtained during the course of the study. The blood samples were collected pre-exercise and at 3 and 24 h post-exercise during visits 2 and 3.A portion of each muscle sample was weighed and homogenized using a commercial tissue extraction reagent  and a tissue homogenizer. Following total protein extraction, total sarcoplasmic and nucleoplasmic proteins were isolated separately using cytoplasmic and nuclear extraction buffers . All extracts were supplemented with phenylmethanesulfonyl fluoride and a protease inhibitor cocktail  with broad specificity for the inhibition of serine, cysteine, and metallo-proteases. Protein contents for total protein, sarcoplasm-, and nuclear-extracted samples were analyzed in duplicate and determined spectrophotometrically at a wavelength of 750 nm  and using bovine serum albumin as the standard .The concentrations of serum total and free testosterone were assessed via commercially-available enzyme-linked immunosorbent assay (ELISA) kits . The specificity of these kits are 100% with the sensitivity estimated to be 0.022 ng/mL and 0.018 pg/mL, respectively, for total and free testosterone. Samples were analyzed in duplicate and absorbances were read at a wavelength of 450 nm. Unknown concentrations were determined by linear regression against known standard curves using commercial software . The overall intra-assay percent coefficients of variation were 2.37% (±2.54) and 2.1% (±1.98) and, respectively, for total and free testosterone.The same ELISA kit employed for serum free testosterone was used to analyze intramuscular testosterone concentrations. Similarly, intramuscular DHT was assessed using a commercially available ELISA kit . These assays were performed using the total muscle protein cellular extracts ,28. The Total AR protein content was assessed in total muscle protein cellular extracts via commercially available ELISA kits . The assay is 100% specific and has a sensitivity of 0.1 ng/mL. All samples were analyzed in duplicate and absorbances were determined at a wavelength of 450 nm using a microplate reader  against known standard curves, and final concentrations expressed relative to total protein concentration. The overall intra-assay percent coefficient of variation was 7.91% (±6.64).AR-DNA binding was quantified in nucleoplasmic extracts by a commercially available ELISA kit . This particular immunoassay utilizes the qualitative technique of an indirect ELISA and the incorporation specific double-stranded (dsDNA) oligonucleotides (representing the ARE (5′-AGAACA-3′)). The assay is 100% specific and has a sensitivity of 0.3 μg of AR–DNA binding in nuclear-extracted MCF7 cells. All samples were analyzed in duplicate and absorbances were determined at a wavelength of 450 nm using a microplate reader . Absorbances were then expressed relative to total nucleoplasmic protein content. The overall intra-assay percent coefficient of variation was 9.89% (±7.59).Sarcoplasmic and nucleoplasmic β-catenin were assessed via commercially available ELISA kits  and performed using the sarcoplasmic and nucleoplasmic extracts. The assay is 100% specific and has a sensitivity of 0.156 ng/mL. All samples were analyzed in duplicate and absorbances were determined at a wavelength of 450 nm using a microplate reader  against known standard curves, and final concentrations expressed relative to sarcoplasmic and nucleoplasmic protein concentration, respectively. The overall intra-assay percent coefficient of variation was 9.79% (±6.86) and 9.04% (±7.70), respectively.2) are characterized 0.1–0.3 as small, 0.3–0.5 as medium, and ≥0.5 as large [Statistical analysis for serum and muscle hormone concentrations, protein metabolite concentrations, and AR-DNA binding activity were performed by utilizing separate 2 × 3  × Time ) factorial analyses of variance (ANOVAs) with repeated measures. Given significant baseline value differences, an ANCOVA was used to with the aforementioned baseline data as a covariate. If a significant interaction was found, simple effects analysis was conducted to determine where the interaction occurred. If a significant interaction was present, analysis of main effects was conducted using the simple effects, pairwise comparisons with a Bonferroni adjustment to compare dependent variables within each independent variable condition. If no interaction was present, then normal pairwise comparisons with a Bonferroni adjustment were used to test main effects. The magnitude of statistical significance was measured by effect size , which estimates the ratio of variance in the dependent variable that is explained by the independent variable. Partial Eta Squared effect sizes  or carbohydrate , protein , fat , or fiber intake  between the max testing, LL, or HL conditions. Similarly, total body water did not significantly change  between the max testing, LL, or HL conditions. For packed cell volume, no significant main effect for time  or condition  was observed between conditions.The results of the separate one way repeated measures ANOVAs indicated that there was no significant difference in total calories , barbell bench press , lat pulldown , single leg extension , and total testing session , or RPE . Additionally, no significant main effect for condition  or significant interaction for time and condition  for muscle soreness were observed. However, a significant main effect for time  was observed. Specifically, analysis revealed a significant increase in muscle soreness at 3 h post- (p = 0.003) and 24 h post-exercise (p = 0.001) compared to pre-exercise.The results of the separate pair samples p = 0.103, η2 = 0.525) or significant time and condition interaction  for serum total testosterone concentrations were observed. However, a significant main effect for time  was observed, which was revealed to be non-significant after adjusting for alpha level inflation via a Bonferroni adjustment (p = 0.091).The means (±SE) for serum total and free testosterone for each condition are indicated in p = 0.763, η2 = 0.011) or significant time and condition interaction  for serum free testosterone concentrations were observed. However, a significant main effect for time  was observed. Pairwise comparisons revealed that there was a significant decrease in serum free testosterone concentrations at 3 h post-exercise compared to pre-exercise (p < 0.001) in both conditions. Additionally, 24 h post-exercise was significantly greater than 3 h post-exercise (p = 0.041) but did not return to pre-exercise baseline values (p = 0.029) in either condition.Regarding serum free testosterone, no significant main effect of condition , condition , or significant time and condition interaction  for intramuscular testosterone concentrations were observed. For intramuscular DHT, no significant main effect of time , condition , or significant time and condition interaction  for intramuscular DHT concentrations were observed.The means (±SE) for intramuscular testosterone and DHT for each condition are indicated in p = 0.518, η2 = 0.062), condition  or significant time and condition interaction  were observed.The mean (±SE) for total intramuscular androgen receptor protein content relative to total muscle protein content (ng/mg) for each condition are indicated in p = 0.225, η2 = 0.141) or condition  for AR-DNA binding activity were observed. However, a significant interaction for time and condition  was observed. Analysis revealed that there was a significant increase in AR-DNA binding at 3 h post-exercise compared to pre-exercise in the HL condition (p = 0.030). The mean (±SE) for intramuscular androgen receptor-DNA binding relative to total nucleoplasmic muscle protein content (Abs/mg) for each condition are indicated in p = 0.002) of the data. Therefore, a Greenhouse–Geisser adjustment was used to meet the needed assumptions to run the appropriate statistical analysis. No significant main effect for time  or significant time and condition interaction  for sarcoplasmic β-catenin content were observed. However, a significant main effect for condition where HL was significantly greater compared to LL when collapsed for time . Regarding nucleoplasmic β-catenin, no significant main effect for time , condition , or significant time and condition interaction  for nucleoplasmic β-catenin content were observed.The means (±SE) for intramuscular sarcoplasmic and nucleoplasmic β-catenin for each condition are indicated in This appears to be the first study investigating the effects of RE load in an intensity- and volume-equated manner on AR-DNA binding activity, AR protein content, the influence of the androgenic hormones, as well as both sarcoplasmic and nucleoplasmic β-catenin concentrations on AR signaling. It is generally thought that LL (≤60% 1 RM) and HL (>60% 1 RM) RE have similar outcomes on skeletal muscle hypertrophy when performed to volitional muscular failure . TherefoFor its critical role in facilitating increases in AR signaling, the AR protein response may conceivably be mediating up-regulations in AR-DNA binding activity. However, we observed no significant changes in AR protein content across all time points in either condition. This suggests that our observed increases in AR signaling activity are not driven by changes in AR content. While more research is certainly warranted, this provides further evidence, along with others ,25,27,28The multifunctional Wnt-signaling protein, β-catenin, has been shown to robustly impact stability, activation, and transcriptional activity of the AR ,54,55. TThere is considerable evidence of crosstalk between Wnt/β-catenin and AR signaling in the literature ,56,57,58While in vitro and in vivo animal models provide valuable insight into the potential molecular mechanisms governing this response, human models are more comparable given our study design. In a recent study by Spillane et al. , they inPrevious research lends support to the notion that differential loading protocols do not dictate hypertrophy or increase AR signaling-related markers in skeletal muscle. A recent 12-week study by Morton et al. ,60. SpecIn RE research, rarely is the effect of load on muscular adaptations examined without being affected by confounding variables such as volume and intensity. Previous research has clearly demonstrated the overwhelming influence volume and intensity have on skeletal muscle hypertrophy and the molecular responses that regulate these adaptations ,60. At tThis appears to be the first study to date investigating the impacts of RE load, in a volume- and intensity-equated manner, on AR-DNA binding activity, serum and muscle androgen concentrations, AR protein content, and sarcoplasmic and nucleoplasmic β-catenin concentrations. In response to HL RE, we observed a significant ~74% increase in AR-DNA binding activity without significant elevations in serum or muscle androgen concentrations or AR protein content. However, sarcoplasmic β-catenin content was ~94% significantly greater when comparing the HL versus LL conditions regardless of time. Collectively, our findings provide evidence that when volume and intensity are equated, the acute AR signaling response to mechanical loading on skeletal muscle appears to be load-mediated. Moreover, the observed up-regulations in AR-DNA binding activity and greater sarcoplasmic β-catenin content suggest a preferential AR signaling response to HL RE. Our data further mechanistically support previous evidence of acute increases in AR signaling not being driven by changes in serum or muscle androgen concentrations nor AR protein content. Rather, AR co-activating proteins, such as β-catenin, may be largely responsible for mediating this response. Unfortunately, we cannot infer causality with the current dataset, considering the (time × condition) interaction effect observed in AR-DNA binding at 3 h was not mirrored by the observed condition-only effect in sarcoplasmic β-catenin. Furthermore, an inability to randomize study conditions to equate volume and intensity and a relatively small sample size are both clear limitations and must be considered when interpreting our findings. Many questions still remain about a variety of factors driving the acute AR signaling response to RE. Better understanding of molecular mechanisms in which AR co-activating proteins, such as β-catenin, interact with the AR may allow for more clarity and provide the necessary context to better explain these occurrences. Future research should investigate the impacts of other AR co-activating proteins and determine their differential impacts on the acute AR signaling response and, by extension, hypertrophic outcomes in skeletal muscle.


The rate of Rehabilitation HPSSR published in rehabilitation journals did not change significantly over time (p = 0.47). The rates of publications with declared funding support increased significantly, but such growth did not differ significantly from that of the comparator fields. Finally, LMICs accounted for 9.3% of the country-focused rehabilitation HPSSR since 1990, but this percentage value increased significantly (p < 0.001) from 6% in 1990 to 13% in 2017. Conclusion: Rehabilitation HPSSR publications, i.e., those indexed in PubMed with related MeSH terms, have grown in both absolute and relative values. Rehabilitation HPSSR publications focused on LMICs also grew significantly since 1990, but still remained a tiny portion of the Rehabilitation HPSSR publications with country-specific MeSH terms.Background: Health policy, systems and services research (HPSSR) is increasingly needed to enable better access to, and value of, rehabilitation services worldwide. We aim to quantify the growth of Rehabilitation HPSSR publications since 1990, compared to that of overall rehabilitation research and overall HPSSR. Methods: Quantitative, comparative analysis of publication trends using the PubMed database and its indexation system. Comprehensive search filters, based on Medical Subject Headings (MeSH), were built and calibrated to locate research articles with content on HPSSR and rehabilitation of physical impairments. Additional filters were used for locating research publications declaring funding support, publications in rehabilitation journals, and finally publications focused on high-income (HICs) or low- and middle-income countries (LMICs). The same approach was used for retrieving data on comparator fields—overall HPSSR and overall rehabilitation research. Linear regressions, with ANOVA, were used for analyzing yearly publication growths over the 28-year time frame. Results: Rehabilitation HPSSR publications in PubMed have grown significantly from 1990 to 2017 in the percentage of all rehabilitation research (from 11% to 18%) and all HPSSR (from 2.8% to 3.9%; both  Rehabilitation services are increasingly needed around the globe, following the global ageing population and the increased rates of chronic conditions and disabilities ,2. HowevDiffering from clinical or basic research, health policy and systems research addresses the upstream aspects of health, organizations, and policies ,16,17. IIn the rehabilitation field, advancing HPSSR is an increasing priority. This has been evident through recent publications in scientific journals on the role for and value of advancing rehabilitation health-services research ,25,26,27Despite this growing interest, to our knowledge there is no systematic, quantitative analysis of the evolving research production on Rehabilitation HPSSR topics, even though its exists for other rehabilitation research areas ,37,38,39What is the total amount, absolute growth, and relative growth of Rehabilitation HPSSR publications ?Which portion of the Rehabilitation HPSSR has been published in rehabilitation journals, and has that changed over time?Which portion of the country-specific rehabilitation HPSSR publications focus on high-income countries (HICs) versus low- and middle-income countries (LMICs)?What are the yearly rates of the Rehabilitation HPSSR publications that declare funding support, and how does that compare to rehabilitation research overall and HPSSR overall.Hence, we aim to quantify the growth in the number of Rehabilitation HPSSR publications from 1990 and 2017, compared to that of overall rehabilitation research and overall HPSSR. This is operationalized through the following study questions:Answering these questions will provide information on crude trends in HPSSR articles in the rehabilitation literature from 1990 to 2017.Quantitative, comparative analysis of publication trends over time using data from the indexation system of a large health research database. http://www.ncbi.nlm.nih.gov/pubmed) and the indexation system for its largest sub-component  was used for this analysis. Although not exhaustive, PubMed is a comprehensive research database. For instance, there is evidence that PubMed contains about 85% of the studies included in health-based systematic reviews , which includes, for example, physical and occupational therapy. As a result of the search, 116 journals were identified and the respective MeSH terms were retrieved as alternative to one another.We use “Support of Research” [Publication Type] as the overarching MeSH term for articles declaring funding support from any venue. We used the World Bank’s classification to determine which countries and territories  are high- or low-income, then translated these to the respective MeSH terms, if existent . These terms were set as alternative to one another (using the operator “OR”) for the groups of HICs and LMICs. Of note, not all articles addressed specific countries , and articles could address no particular countries or HICs and LMICs at the same time. Also, when specific countries were addressed but this was not be explicit in the articles’ title or abstract, often the articles were not granted a country-specific MeSH term. Hence, the amount of country-specific rehabilitation HPSSR is not necessarily equal to that of overall rehabilitation HPSSR.For calibrating the search filters, we ran pilot searches with or without some of the MeSH terms the research team appraised as at the borderline for inclusion or exclusion, assessing the impact they had on the search results. For example, we tested the collective impact of introducing a set of MeSH terms focused on assistive devices  within the search filter for rehabilitation. Including this whole set of terms added less than 2% more Rehabilitation HPSSR articles retrieved. Hence, the inclusion of this set was not deemed to carry substantial risk to preciseness , with likely benefits for increased sensitivity . Theoretically, HPSSR cares about making medical products (including assistive technologies) available to those who need them . TherefoA final calibration exercise entailed a random selection of 30 articles from the authors’ personal resources, published before 2017, that were deemed relevant for the field of Rehabilitation HPSSR by the research team. These articles were searched for in PubMed, and their major MeSH term retrieved. This whole process enabled the identification of one more relevant MeSH term for the rehabilitation search filter  and one more relevant term for the filter on research publications. For instance, some research articles  were not indexed for any method/publication type in particular, but were indexed for “research support” [publication type]. Hence, we added this term to the research publications filter, assuming that all articles that declare “research support” are indeed research. This resulted in a substantial increase in the number of detected publications, sometimes over one-third more, with the added sensitivity coming with low risk to preciseness. The searches were conducted in late October 2019 but were narrowed down in publication dates from January 1, 1990 to December 31, 2017. This accounted for the fact that “publication types” have been systematically indexed in PubMed only since 1990 and for the typical 2-year delay in the PubMed indexation. Specifically, for the searches focused on funding support, the time span was narrowed down to end at December 2015. Indeed, in preliminary data visualizations, we observed outlier results  in every search, across the fields, i.e., focused or not on rehabilitation or HPSSR. This suggests a delay or relevant change in this indexation process in particular, rather than reflecting changes in publications data itself. Hence, data on 2016 and 2017 for declared funding support were excluded.r2 values and visual examination) were used to analyze any changes in the count data and computed percentages over time. We retained the linear models whenever the fit was similar .From each search in PubMed, the total and yearly volume of publications were exported to Excel (Microsoft Corporation). There, we computed relevant percentages . As this paper involved comparative analyses, the data were often analyzed and reported in those relative percentages. Run charts and then linear, exponential or logarithmic regression models, according to best fit  for this analysis. Below we present the results according to our initial research questions.p < 0.001; r2 = 0.96).From 1990 to 2017, a total of 23,105 Rehabilitation HPSSR publications were found, with a significant linear growth of 63.3 publications a year : 58.2–68.4; p < 0.001), albeit logarithmic in type (r2 (log model) = 0.83), i.e., more pronounced in the early years.For the relative growth, p < 0.001; r2 (linear model) = 0.9).p = 0.49), erratically moving around 24%. On the other hand, the percentage of the overall rehabilitation research  that was published in rehabilitation journals increased significant yet logarithmically over time  = 0.55), from 17% in 1990 to approaching 22% in 2017, the typical values of the Rehabilitation HPSSR. p < 0.001), from 6% in 1990 to 13% in 2017.From 1990 to 2017, a total of 9733 Rehabilitation HPSSR publications were indexed in PubMed with a focus on any HIC, and 986 on any LMIC. This means that LMICs account for up to 9.3% of the Rehabilitation HPSSR publications that had country-specific MeSH terms; however, p < 0.001; 95% CI: 58.2–68.4; r2 = 0.96). This growth was comparable to  rehabilitation research as a whole and HPSSR as a whole, with all three approaching 2015 with values within the 53–58% range.From 1990 to 2015, 11,037 Rehabilitation HPSSR publications declared funding support, i.e., 47.8% of them. Rehabilitation HPSSR publications, notably those indexed for rehabilitation and HPSSR topics in the PubMed database, have growth significantly from 1910 to 2017, in both absolute and relative values. Indeed, the growth of the rehabilitation HPSSR publications significantly outpaced that of both comparators, i.e., grew significantly in the percentage of all rehabilitation research, and in the percentage of all HPSSR. We also observed discrepancies in the Rehabilitation HPSSR publications focused on HICs versus LMICs, with the latter accounting for 9.3% of the Rehabilitation HPSSR publications that had at least a country-specific MeSH term. However, a significant, growing trend from 6% to 13% (1990–2017) was observed in that percentage value. Below we present five points of discussion related to our findings.First, the observed growth of publications on HPSSR topics within the overall rehabilitation research, i.e., from 11% to 18% (1990–2017), means that rehabilitation stakeholders are conducting, comparatively, more HPSSR. Nonetheless, HPSSR topics still represent less than one-fifth of all rehabilitation research publications and the observed relative growth was less pronounced  in more recent years. It is hard to know what is the desirable figure with regard to the proportion of rehabilitation research that should be devoted to HPSSR; however, what we do know is that Rehabilitation HPSSR helps to enhance the value or population impact of other forms of rehabilitation research . For example, HPSSR would help to translate the growing clinical knowledge into more common, more accessible, and more affordable rehabilitation practices, boost innovation and accountability for service organization and delivery, and make generalizable knowledge responsive or adaptive to identified local needs ,59,60.Second, Rehabilitation HPSSR represents a significantly increased proportion of publications within HPSSR, from 2.8% in 1990 to 3.9% in 2017. The growth over time has been significant and relatively linear, i.e., constant, over the 28-year period; nonetheless, rehabilitation represents a very small portion of all HPSSR. This may be concerning given the current high levels of physical rehabilitation needs worldwide ; hence, the effects of more recent pushes for advancing the Rehabilitation HPSSR ,11,13,34Moreover, we did not analyze the specific scope of Rehabilitation HPSSR by thematic areas ,44, but Finally, we focused only on research publications, while perspectives, commentaries, conceptual, narrative or other types of article, e.g., analyzing current situations  or descrHighlighting these limitations are critical in pinpointing the weaknesses of our methodology; however, given the limited availability of the publicly available information, we interpret this study as a starting point, and would invite the rehabilitation and/or HPSSR community to contribute to this discussion.This research uses the PubMed indexation capabilities to provide information on crude trends  on the evolution of the number of Rehabilitation HPSSR articles published from 1990 to 2017. With such a strategy, we have found that Rehabilitation HPSSR publications, i.e., those published and indexed in PubMed with rehabilitation- and HPSSR-related MeSH terms, have grown in both absolute and relative values. However, in 2017, Rehabilitation HPSSR still accounted for merely 18% of the rehabilitation research publications and 4% of the overall HPSSR publications in PubMed. We also found that the portion of Rehabilitation HPSSR publications focused on LMICs, among those that had country-specific MeSH terms, has been growing significantly since 1990, but is still minute, especially given the sizeable unmet needs for rehabilitation services in many LMICs.


Ralaniten and analogs (EPI) bind androgen receptor N‐terminal domain (AR‐NTD) to block the transcriptional activities of full‐length AR (FL‐AR) and AR splice variants (AR‐Vs). Enzalutamide (ENZ) elevates levels of AR‐V7 leading to resistance and increased proliferation. Targeting AR‐NTD to block FL‐AR and AR‐Vs with EPI in combination with ENZ resulted in synergistic inhibition of proliferation of ENZ‐resistant prostate cancer cells. Resistance of castration‐resistant prostate cancer (CRPC) to enzalutamide and abiraterone involves the expression of constitutively active, truncated androgen receptor (AR) splice variants (AR‐Vs) that lack a C‐terminal ligand‐binding domain (LBD). Both full‐length AR and truncated AR‐Vs require a functional N‐terminal domain (NTD) for transcriptional activity thereby providing rationale for the development of ralaniten (EPI‐002) as a first‐in‐class antagonist of the AR‐NTD. Here, we evaluated the antitumor effect of a next‐generation analog of ralaniten (EPI‐7170) as a monotherapy or in combination with enzalutamide in prostate cancer cells that express AR‐V7 that were resistant to enzalutamide. EPI‐7170 had 8–9 times improved potency compared to ralaniten. Enzalutamide increased levels of AR‐V7 and expression of its target genes. Knockdown of AR‐V7 restored sensitivity to enzalutamide, indicating a role for AR‐V7 in the mechanism of resistance. EPI‐7170 inhibited expression of genes transcriptionally regulated by full‐length AR and AR‐V7. A combination of EPI‐7170 and enzalutamide resulted in synergistic inhibition of proliferation of enzalutamide‐resistant cells that was consistent with results from cell cycle and clonogenic assays. In addition, this drug enhanced the antitumor effect of enzalutamide in enzalutamide‐resistant CRPC preclinical models. Thus, a combination therapy targeting both the NTD and LBD of AR, and thereby blocking both full‐length AR and AR‐Vs, has potential for the treatment of enzalutamide‐resistant CRPC. ARandrogen receptorAR‐V7androgen receptor splice variant‐7CRPCcastration‐resistant prostate cancerENZenzalutamideFL‐ARfull‐length androgen receptorLBDligand‐binding domainNTDN‐terminal domain1Abiraterone and second‐generation antiandrogens such as enzalutamide (ENZ) target full‐length (FL) androgen receptor (AR) C‐terminal ligand‐binding domain (LBD). These AR‐LBD inhibitors have demonstrated significant responses for castration‐resistant prostate cancer (CRPC). Unfortunately the majority of these patients eventually develop adaptive resistance following an initial response , 2, 3, 4A key mechanism underlying lethal CRPC is the expression of constitutively active splice variants of androgen receptor (AR‐Vs) , 7, 8, 9Ralaniten and its analogs specifically inhibit the AR‐NTD to block the transcriptional activities of full‐length AR (AR‐FL) and AR‐Vs , 19, 20.22.1m) beginning in November, 2017 (media supplemented with ENZ was replenished every 3–4 days). VCaP‐C cells were developed by exposure to the corresponding concentration of DMSO as an appropriate control. VCaP cells were cultured in phenol red‐free DMEM supplemented with 10% FBS, LNCaP, and C4‐2B cells in phenol red‐free RPMI‐1640 supplemented with 10% FBS, VCaP‐ENZR cells in phenol red‐free DMEM supplemented with 10% FBS and 10 µm ENZ, C4‐2B‐ENZR cells in phenol red‐free RPMI‐1640 supplemented with 10% FBS and 20 µm ENZ. All cells were grown in a humidified incubator with 5% CO2 at 37 °C and maintained in culture for not more than 10 passages after resurrection. All cells were regularly tested to ensure they were mycoplasma‐free and were authenticated by short tandem repeat analysis . Cells were treated in phenol red‐free and charcoal stripped FBS (CSS) condition followed by androgen stimulation in assays to measure androgen‐induced signal.VCaP cells were purchased from the American Type Culture Collection . LNCaP cells were from L. Chung . C4‐2B and C4‐2B enzalutamide‐resistant (C4‐2B‐ENZR) cells were from A. Gao  . VCaP‐ENRalaniten was provided by NAEJA , and EPI‐7170 was synthesized by us. Enzalutamide was purchased from OmegaChem  and synthetic androgen (R1881) from AK Scientific Inc .2.2m), EPI‐7170 (2.5 or 3.5 µm), or its combination in phenol red‐free RPMI‐1640 plus 1.5% FBS for 48 h and harvested for protein isolation. Protein was extracted with RIPA buffer and protease inhibitor cocktails . The amount of protein was determined using a BCA protein assay kit . Equal amounts of denatured proteins were separated by SDS/PAGE gels and transferred to a PVDF membrane and blocked with 5% skim milk in PBS containing 0.1% Tween 20. The membranes were probed with the following primary antibodies: AR (N‐20) (ab108341) , AR‐V7 (RM7) , Cyclin D1 (#2978) diluted 1 : 1000, Cyclin A2 (#4656) diluted 1 : 1000, CDK4 (#12790)  diluted 1 : 1000, and Actin (A5441)  diluted 1 : 10 000.Cells  were plated in 6‐well plates in ENZ‐free media for 2 days prior to harvesting for protein isolation. For experiments for cell cycle protein, C4‐2B‐ENZR cells were plated in 6‐cm dishes and treated with vehicle, ENZ (20 µ2.35 cells) and C4‐2B‐ENZR (3 × 105 cells) cells were, respectively, plated and incubated for 24 h. siRNA/transfection reagent complexes were added into each wells. Forty‐eight hours post‐transfection, protein was harvested and analyzed by western blot analysis.siRNA against AR exon3b was obtained from Dharmacon . The target sequence for AR exon3b is 5′‐GUAGUUGUGAGUAUCAUGA‐3′. siRNA knockdown was performed using Lipofectamine RNAiMAX Transfection Reagent  according to the manufacturer's protocol. VCaP‐ENZR (5 × 104 cells/well) and C4‐2B‐ENZR (2 × 104 cells/well) were plated in 96‐well plates and transfected with siRNA 24 h prior to treatment with enzalutamide for 5 days (VCaP‐ENZR) or 3 days (C4‐2B‐ENZR). BrdU incorporation was measured using BrdU ELISA kit .For experiments combining drug treatment with siRNA transfection, VCaP‐ENZR  and PureLink RNA Mini Kit  according to the manufacturer's protocol. Reverse transcription reactions were conducted using cDNA with High Capacity RNA‐to‐cDNA kit . Quantitative real‐time RT–PCR was performed using Platinum SYBR Green qPCR SuperMix‐UDG with ROX (Invitrogen) and QuantStudio 6 Flex (Applied Biosystems). Data were normalized to RPL13a gene expression. Primers have been described previously .2.5m MgSO, 0.2 mm deoxynucleotide mix, and 2% DMSO with the AR‐NTD and LBD primers . The PCR fragment was gel purified and cloned into PCR 2.1 TOPO cloning vector using TOPO TA cloning kit from Invitrogen and transformed into chemically competent TOP10 cells. Plasmids were sequenced at the Sequencing and Bioinformatics Consortium at The University of British Columbia .Total RNA (1 μg) from VCaP‐ENZR cells was reverse transcribed using SuperScript IV VILO Master Mix (Invitrogen). The coding region of AR was PCR amplified with Platinum DNA Taq Polymerase High Fidelity in 20 µL with 1 U Taq polymerase, 2 m2.63 cells/well), VCaP‐C cells (8 × 103 cells/well), VCaP‐ENZR cells (1.2 × 104 cells/well), C4‐2B cells (3 × 103 cells/well), and C4‐2B‐ENZR (3 × 103 cells/well) cells were plated in 96‐well plates in their respective media . After incubating for 24 h (C4‐2B and C4‐2B‐ENZR) or 48 h (VCaP and VCaP‐ENZR), cells were treated with vehicle, EPI‐7170, ENZ, or a combination for 2 days (C4‐2B and C4‐2B‐ENZR) or 5 days (VCaP and VCaP‐ENZR). Cell proliferation assays were performed using the BrdU ELISA kit (Roche Diagnostics) according to the manufacturer's protocol. Absorbance was measured with a VersaMax ELISA microplate reader . The combination index (CI) values calculated by Chou‐Talalay method using compusyn software  where a CI value below 1 represents synergism.VCaP cells  in cell lysates were detected using Promega GloMax‐Multi Detection Luminometer . Protein concentrations of the cell lysates were determined by the Bradford method using Bio‐Rad Protein Assay Kit . Luciferase activities were normalized to protein concentration.LNCaP cells were plated in 24‐well plates and incubated for 24 h. Cells were cotransfected with PSA(6.1 kb)‐luciferase reporter or V7BS2.85 cells/plate) were plated in 6‐cm dishes and treated with vehicle, ENZ (20 µm), EPI‐7170 (2.5 or 3.5 µm), or its combination in RPMI‐1640 plus 1.5% FBS for 48 h. Cells were labeled with 10 μm BrdU for 2 h and fixed in 70% ethanol. BrdU‐labeled cells were probed with anti‐BrdU‐FITC antibody , and DNA was stained with 7‐aminoactinomycin D (Sigma‐Aldrich). Data were acquired using a FACS Calibur (BD Biosciences), and data were analyzed using flowjo software .C4‐2B‐ENZR cells (3.5 × 102.9m), ralaniten (25 µm), EPI‐7170 (2.5 µm), or a combination and then monitored for a colony formation for 2 weeks. Colonies were fixed with 4% paraformaldehyde and stained with 0.01% crystal violet. Colonies containing more than 50 cells were counted using imagej software version 1.45s .C4‐2B‐ENZR cells (400 cells/well) were plated in 6‐well culture plates, and after 24 h, cells were treated with ENZ (20 µ2.106 cells per injection) were suspended in 100 µL of PBS: Matrigel  (1 : 1) and subcutaneously injected into the flanks of mice. Mice were castrated when the tumors reached approximately 80 mm3 in volume. EPI‐7170 and ENZ were prepared in 5% DMSO, 10% Tween 80, and 1% carboxymethylcellulose. After 7 days, mice were divided into four groups and treated: control group (vehicle), ENZ group (20 mg·kg−1 body weight), EPI‐7170 group (30 mg·kg−1 body weight), and combination treatment group (concurrent treatment with ENZ and EPI‐7170). Body weight was measured every day, and tumor volume was measured twice a week. The tumor volume was calculated using the formula length × width × height × 0.52. After 31 days of treatment, the mice were euthanized and the tumors were processed for western blot analyses and IHC.The animal experiment protocols were approved by the University of British Columbia Animal Care Committee. Six‐ to eight‐week‐old male NOD/SCID mice were maintained in the Animal Care Facility at the British Columbia Cancer Research Centre. VCaP‐ENZR cells  sections. Sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked with 3% hydrogen peroxide in distilled water for 5 min. Incubation with anti‐Ki‐67  diluted in 1 : 50 was done at 4 °C overnight. Antigen was detected with 3,3‐diaminobenzidine and counterstaining with hematoxylin. For TUNEL staining, the ApopTag Fluorescein In Situ Apoptosis Detection Kit  was used according to the manufacturer's instructions.2.12graphpad prism 7 . P‐values < 0.05 were considered statistically significant.Differences in the quantitative data, which are expressed as mean ± SEM, between groups were compared by one‐way ANOVA Tukey's multiple comparisons test using 33.1m) over a period of 10 months. ENZ inhibited cell proliferation in parental cell lines but showed less effect on proliferation of VCaP‐ENZR and C4‐2B‐ENZR cells as determined with BrdU incorporation  cells to increasing doses of ENZ  revealed no new mutations compared to parental cells [Sequencing the FL‐AR from VCaP‐ENZ revealed no unique mutation compared to the sequence for FL‐AR in the parental VCaP cells (data not shown). The F876L mutation reported to confer resistance to ENZ , 30, 31 al cells .m)  Fig.  which ma3.250 of ENZ when combined with EPI‐7170. Combination index (CI) values were calculated from the fraction‐affected  value of each combination using the Chou‐Talalay method to determine potential synergism (CI < 1), additive effect (CI = 1), or antagonism (CI > 1). The CI values were lower than 1 at all the tested concentrations of EPI‐7170 and ENZ ‐luciferase reporter , whereaspromoter . Levels gen Fig. . These d3.4Androgen reduces, while ENZ increases, the expression of AR‐V7 in prostate cancer cells . Consist3.5m) monotherapy had little effect on cell cycle, whereas EPI‐7170 (3.5 µm) monotherapy led to an increase in G1 fraction and reduction of S phase. Combination therapy resulted in nearly complete inhibition of DNA synthesis in S phase  with ralaniten (25 µm) or low dose EPI‐7170 (2.5 µm) were more effective than monotherapies in C4‐2B‐ENZR cells , EPI‐7170 (30 mg·kg−1 body weight), or a combination (ENZ 20 mg·kg−1 body weight + EPI‐7170 30 mg·kg−1 body weight) for 31 days. EPI‐7170 alone significantly decreased the final tumor volume compared to the controls, whereas ENZ monotherapy did not. The combination of ENZ and EPI‐7170 significantly decreased tumor volume compared to ENZ alone and vehicle treatment  ; JQ1, a  synergy . To datey weight , whereasf 15.8 h . Thus, fClinical support for the AR‐NTD as a drug target can be drawn from the first in human clinical trial with ralaniten (NCT02606123) in heavily pretreated CRPC patients that had failed ENZ and/or abiraterone. This clinical trial revealed some signs of efficacy with ralaniten as indicated by reduction in serum prostate‐specific antigen (PSA) and stable disease in some patients in spite of having 50× lower Cmin blood levels than necessary for in vitro efficacy . This cl5In conclusion, this study revealed ENZ treatment leads to increased AR‐V7 expression that confers resistance to ENZ. By directly targeting AR‐NTD to block both FL‐AR and AR‐V7, EPI‐7170 showed synergistic effects in combination with ENZ. This study advances the current understanding of the role of AR‐V7 in the mechanism of resistance to antiandrogens and provides a new translatable therapeutic option targeting AR‐V7 to overcome CRPC.The authors declare the following competing interests: YH, MDS, KJ, and RJA are inventors of technology which was licensed by the BC Cancer to ESSA Pharma. MDS and RJA have equity and are Scientific Advisors for ESSA Pharma. Their interests were reviewed and are managed by the BC Cancer Agency and University of British Columbia in accordance with its research conflict of interest policies. TT has no competing interests.YH and MDS conceived the study, designed the experiments, interpreted the data, and wrote the manuscript. YH performed the biological experiments and analyzed the results. TT sequenced the FL‐AR in VCAP‐ENZR cells. KJ and RJA synthesized EPI‐7170. MDS supervised the study. All authors read and approved the final manuscript.Fig. S1. Relative protein levels of FL‐AR and AR‐V7 in cell lines. (A) Whole cell protein lysates from cells were run on a SDS‐PAGE gel and then analyzed for levels of FL‐AR (left) and AR‐V7 (right) using antibodies to the AR‐NTD (detects FL‐AR and AR‐Vs) and AR‐V7. Actin was used as a loading control. (B) Quantification of bands shown in “A” for FL‐AR and AR‐V7 normalized with levels of β‐actin that was used as a loading control. The numbers over the bars are the normalized values for AR and AR‐V7 in the two ENZR cell lines. n = 1.Click here for additional data file.Fig. S2. EPI‐7170 has improved potency compared to ralaniten (EPI‐002) to block androgen‐induced AR transcriptional activity and proliferation in C4‐2B‐ENZR cells. (A) Dose‐dependent inhibition of androgen‐induced transcriptional activity of FL‐AR in C4‐2B‐ENZR cells by EPI‐002 or EPI‐7170. C4‐2B‐ENZR cells transfected with the PSA‐luciferase reporter were treated with EPI‐002 or EPI‐7170 for 1hr prior to the addition of R1881 (1nM) for 24 hours. Values represent the mean ± SEM (n = 3). (B) Effects of EPI‐002 or EPI‐7170 on androgen‐induced proliferation of C4‐2B‐ENZR cells. IC50s of EPI‐002 or EPI‐7170 are shown in the table (right). Values represent the mean ± SEM (n = 3).Click here for additional data file.


Although recent studies have indicated that intestinal microbiota dweller are involved in the pathogenesis of allergy rhinitis (AR), the influence of gut microbiota on AR adult has not been fully elucidated yet. Hence, we carried out this study to uncover the distinctive bacterial taxa that differentiate allergy rhinitis patients from healthy individuals. Feces samples from thirty three AR patients and thirty one healthy individuals were analyzed by 16S rRNA gene sequencing.Prevotella_9, Phascolarctobacterium, Roseburia, Megamonas, Alistipes, Lachnoclostridium and Fusobacterium. The higher network complexity in AR group were dominated by taxa belonging to Firmicutes. The predicted function, alpha linolenic acid metabolism and bacterial invasion of epithelial cells pathway were higher in non-AR group while gonadotropin-releasing hormone (GnRH) signaling pathway, Fc γ-R mediated phagocytosis and endocytosis were higher in AR patients. Although the bacterial diversity between moderate and severe AR patients showed no significant difference, the significant correlation between featured genus and total nasal symptom score or rhinoconjunctivitis quality of life questionnaire, such as Butyricicoccus and Eisenbergiella, revealed the potential to intervene the AR status by means of gut microbiota.Results showed that the bacterial diversity in AR group was significantly higher than that of the non-AR group. Bacterial communities between AR and non-AR group were significantly differentiated as revealed by Principal coordinates analysis (PCoA) and the variation within non-AR were higher than that of the counterpart. Firmicutes, Fusobacteria, Actinobacteria, Cyanobacteria and Chloroflexi were the significantly differed phyla taxa and the top significantly distinguished bacterial genus included In conclusion, patients with allergy rhinitis had distinguished gut microbiota characteritics in comparison with healthy controls. The results suggest that gut microbiota might play crucial roles in influencing the course and different symptoms of AR.Trial registration ChiCTR, ChiCTR1900028613. Registered 29 December 2019, https://www.chictr.org.cn/showproj.aspx?proj=47650. Allergic rhinitis (AR) is one of the most globally common diseases and usually persists throughout life , 2. AR iThe mainstay conventional treatment for AR includes allergen avoidance, pharmacotherapy, immunotherapy and patient education , 4. AlthLactobacillus casei Shirota strain exerted effect on the immune system and allergic symptoms [Lactobacillus plantarum showed the ability to inhibit allergic cytokines and Bifidobacterium longum BB536 strain alleviated symptoms of seasonal AR [Recently, the role of probiotics in nutritional interventions have been investigated regarding to their beneficial effects on AR in improving patients’ quality of life and reducing medication use , 12. Thesymptoms , 19, whisonal AR –22. HencConsequently, in this study, we compared the intestinal microbiota characteristics between 31 non-AR and 33 AR patients through high-throughput 16S rRNA gene sequencing. The present study was to: (1) compare the composition and functional features of gut microbiota between AR and non-AR individuals; (2) identify the specific gut microbiota which was closely related to the severity of AR as well as the potential pathway of gut microbiota interacting with the host.The ethics committee of Xiamen hospital of Traditional Chinese Medicine approved this study (Permit Number ID: 2019-K020-01) and later it was registered in Chinese Clinical Trial Registry (ChiCTR1900028613). All healthy individuals and patients made agreement on the informed consent, and the study protocol complied with the ethical guidelines of the Declaration of Helsinki .Thirty one non-AR populations were enrolled from December 2017 to May 2018 at Zhongshan Hospital, affiliated with Xiamen University . The inclusion criteria were as follows: (1) no allergic history and no family history of allergic diseases; (2) sausage or snake shaped stool; and (3) voluntary participation in this study. The exclusion criteria of non-AR populations were based on previously reported  and indiThirty three AR patients were recruited at Xiamen hospital of Traditional Chinese Medicine  from December 2019 to March 2020. Patients were recruited with the following inclusion criteria: (1) age of 18–65 years; (2) meet the international diagnostic standard of AR  : (a) theAR patients who meet one of the following criteria were excluded: (1) upper respiratory tract infection, chronic obstructive pulmonary disease, emphysema, bronchiectasis, tuberculosis, pneumonia, bronchial asthma, secretory otitis media; (2) postural hypotension, organic heart disease or clinically significant ECG changes; (3) patients with thyroid disease, diabetes mellitus, hypoglycemia, cardiovascular and cerebrovascular disease, prostatic hypertrophy, glaucoma, epilepsy history, liver and kidney dysfunction, hematopoietic dysfunction, gastrointestinal diseases, malignant tumors; (4) patients had a history of mental and neurological diseases; (5) patients who had received the following treatment before the experiment: immunotherapy within 12 months, long-acting glucocorticoid injection therapy within 3 months, asmidazole within 6 weeks, glucocorticoids for systemic or local use within 2 weeks , sodium cromoglycerin or naidomide within 1 month, long-acting antihistamines within 2 weeks, short-acting antihistamines within 1 week, ketotifen, tricyclic antidepressants, macrolides and antifungal drugs within 2 weeks, systemic or local nasal decongestants within 24 h, using anti-histamine drugs, anticholinergics and antiarrhythmic drugs currently, using barbiturates, antispasmodics, phentolamine and digitalis drugs currently; or (6) abnormal physical examination results with clinical significance before admission, which may affect the achievement of the purpose of this study or hinder the realization of compliance with the research procedures according to the judgment of the researchers.TNSS and RQLQ were performed for AR patients to assess the severity of AR and the impact on patients’ quality of life caused by AR during the enrollment. Nasal symptoms were assessed using the TNSS score . RQLQ coThe procedure for DNA extraction of fecal samples (approximately 0.25 g) from non-AR healthy individuals and AR patients, the purity and concentration of the isolated DNAs, the 16S rRNA V3–V4 hypervariable region amplification for the library construction and paired-end sequencing were based on previous procedure . The bioBacterial alpha diversity indices, bacterial community ordination based on Bray-Curis distance, PERMANOVA and anosim analysis were based on the R package vegan (version 2.5.5) . The groIn this study, we used feces collected from 33 AR patients and 31 non-AR individuals  while not for J indices (p > 0.05) , average of 91,800 OTU sequence/sample and an average 316 OTUs were obtained from 64 samples  . In AR group, the average relative abundance of Bacteroidetes 49.27%) and Proteobacteria 3.61%) were lower than that in non-AR individuals  while Chloroflexi and Spirochaetes were the unique taxa which were absent in non-AR parts  value was 0.08 ± 0.02 for all samples  and severe 8–12) group to compare the bacterial diversity and community to probe into the role of bacterium in the progression of AR. Although moderate and severe AR patients showed significant difference in non-eye/nasal symptoms, practical problem, nasal symptom, eye symptoms, emotions and RQLQ  Observed species and (B) chao1. Figure S2. The bacterial community difference between AR and non-AR group. (A) The ANOSIM analysis are made to compare the intergroup and within group of samples. (B) The bacterial community variation comparison in AR and non-AR group. Figure S3. The predicted expression of distinguished pathway. The significantly distinguished pathway predicted by PICRUSt were screened by comparison between AR and non-AR individuals by Wilcox test and based on |logFC|>1. FC denotes fold change. Figure S4. Comparisons of bacterial diversity between moderate and severe AR patients. (A) The bacterial α diversity indexes comparison between moderate and severe AR, including Chao1, J and Shannon. Letters indicate the ANOVA groupings. (B) Differences in bacterial community structures between moderate and severe AR patients.


The benefit of inducing cellular senescence as a tumor suppressive strategy remains questionable due to the senescence-associated secretory phenotype. Hence, studies and development of senolytic compounds that induce cell death in senescent cells have recently emerged. Senescent cells are hypothesized to exhibit different upregulated pro-survival/anti-apoptotic networks depending on the senescent inducers. This might limit the effect of a particular senolytic compound that targets rather only a specific pathway. Interestingly, cellular senescence in prostate cancer (PCa) cells can be induced by either androgen receptor (AR) agonists at supraphysiological androgen level (SAL) used in bipolar androgen therapy or by AR antagonists. This challenges to define ligand-specific senolytic compounds.Here, we first induced cellular senescence by treating androgen-sensitive PCa LNCaP cells with either SAL or the AR antagonist Enzalutamide (ENZ). Subsequently, cells were incubated with the HSP90 inhibitor Ganetespib (GT), the Bcl-2 family inhibitor ABT263, or the Akt inhibitor MK2206 to analyze senolysis. GT and ABT263 are known senolytic compounds. We observed that GT exhibits senolytic activity specifically in SAL-pretreated PCa cells. Mechanistically, GT treatment results in reduction of AR, Akt, and phospho-S6 (p-S6) protein levels. Surprisingly, ABT263 lacks senolytic effect in both AR agonist- and antagonist-pretreated cells. ABT263 treatment does not affect AR, Akt, or S6 protein levels. Treatment with MK2206 does not reduce AR protein level and, as expected, potently inhibits Akt phosphorylation. However, ENZ-induced cellular senescent cells undergo apoptosis by MK2206, whereas SAL-treated cells are resistant. In line with this, we reveal that the pro-survival p-S6 level is higher in SAL-induced cellular senescent PCa cells compared to ENZ-treated cells. These data indicate a difference in the agonist- or antagonist-induced cellular senescence and suggest a novel role of MK2206 as a senolytic agent preferentially for AR antagonist-treated cells.Taken together, our data suggest that both AR agonist and antagonist induce cellular senescence but differentially upregulate a pro-survival signaling which preferentially sensitize androgen-sensitive PCa LNCaP cells to a specific senolytic compound. For decades, prostate cancer (PCa) ranks the most diagnosed cancer and the second leading cause of cancer-related deaths of men in Western countries , 2. It iWe have previously described that cellular senescence can be induced by AR-signaling in PCa. On the one hand, several AR antagonists such as atraric acid, compound C28, or aminosteroids have been shown to induce cellular senescence in PCa cells –11. On tCellular senescence is initially defined as an irreversible cell cycle arrest and has been proposed as one of the cancer inhibition strategies , 15. HowSenolytic agents are molecular compounds that induce cell death in senescent cells . SenesceIn this study, we first induced cellular senescence in androgen-sensitive PCa LNCaP cells by treating with either AR agonist at SAL or antagonist, and subsequently analyzed the senolytic effects of GT, ABT263, and the highly selective allosteric Akt inhibitor MK2206. Our data suggest that the pro-survival Akt-S6 signaling is more active in AR agonist-induced cellular senescent cells than antagonist. Interestingly, treatment with GT or MK2206 after AR ligand-induced cellular senescence exhibits distinct senolytic activities depending on the type of AR ligand, agonist or antagonist, whereas ABT263 lacks senolytic activity.In order to analyze the activity of senolytic agents in senescent LNCaP cells, first, cellular senescence was induced in the PCa cell line LNCaP cells by treating with AR agonist at SAL or antagonist for 72 h as described earlier , 13  mRNA was detected by ENZ treatment  were analyzed. The results show that specifically agonist treatment significantly downregulates BCL2 and upregulates BCL2L1 mRNA expression  were more sensitive to apoptotic inducers than wild-type MEFs [Next, the effects of MK2206, GT, and ABT263 were analyzed on the pro-survival Akt-S6 pathway. After induction of cellular senescence by AR ligands, as expected, MK2206 potently reduces p-Akt levels Fig. . Surprisype MEFs , 38. ThiInterestingly, GT treatment inhibits Akt-signaling as well Fig. . In lineUnlike MK2206 and GT, the inhibitor of the Bcl-2 family ABT263 does not interfere with Akt-S6 pathway Fig. . Both p-It is known that the AR is required for androgen-sensitive PCa cell survival. In line with this, AR knockdown mediates apoptosis in LNCaP cells . TherefoTaken together, our data suggest that the three inhibitors GT, ABT263, and MK2206 exhibit distinct mechanisms in AR ligand-induced cellular senescence. Mechanistically, GT treatment results in both reduction of AR protein level and inhibition of Akt-signaling. MK2206 does not reduce AR protein level but potently inhibits Akt phosphorylation, while ABT263 does neither affect AR protein nor Akt-signaling.Cellular senescence has been firstly introduced as an anti-cancer purpose, however, the effects of senescent cells on the tumor microenvironment via SASP factors have emerged –19, 43. BCL2 and BCL2L1). These data suggest that the pro-survival/anti-apoptotic pathways are distinct between AR agonist- and antagonist-induced cellular senescent LNCaP cells. Therefore, a suitable senolytic compound for particular survival pathway must be identified.In this study, we analyzed pro-survival pathways dependent on either AR agonist- or antagonist-induced cell senescence. First, we show that AR agonist R1881 at supraphysiological levels or the antagonist ENZ is capable of inducing cellular senescence but not apoptosis in the androgen-sensitive PCa LNCaP cells. We hypothesized that SAL or ENZ causes senescent cells to activate or upregulated distinct pro-survival/anti-apoptotic pathways. Our results indicate that the Akt-S6 pathway is more activated by AR agonist-induced cellular senescence compared to antagonist. Notably on the one hand, SAL induces phosphorylation of both Akt and S6. On the other hand, ENZ induces phosphorylation of Akt but not S6. Furthermore, in contrast to antagonist, AR agonist regulates the expression of Bcl-2 family members .For protein extraction, the assays were performed with 10 cm cell culture plates, and the cells were seeded at 500,000 cells per dish for MK2206 experiments and at 120,000 cells per dish for GT and ABT263 experiments. The whole-cell lysate preparation was adapted from Esmaeili et al. (2016) , 49. BriTo verify the detection of phosphorylated RIP3, the whole-cell lysate preparation was performed with NETN buffer with or without phosphatase inhibitors. The protein extracts were incubated at 37 °C for 3 h with or without alkaline phosphatase (FAST AP) (Thermo Scientific).TBP mRNA is served as the housekeeping gene for normalization. The primer sequences are listed as 5′ → 3′: TBP: fwd: GGCGTGTGAAGATAACCCAAGG, rev: CGCTGGAACTCGTCTCACT, BCL2: fwd: CATGTGTGTGGAGAGCGTCAA, rev: GCCGGTTCAGGTACTCAGTCA, BCL2L1: fwd: AACATCCCAGCTTCACATAACCCC, rev: GCGACCCCAGTTTACTCCATCC, BCL2L2: fwd: ACCCGTGAGATCCCTAACCTG, rev: CAGCTCCACAGACATAACCCT, CDKN2A (p14ARF): fwd: CCTGGAGGCGGCGAGAAC, rev: AGTAGCATCAGCACGAGGGC, CDKN2A (p16INK4a): fwd: CTTGCCTGGAAAGATACCG, rev: CCCTCCTCTTTCTTCCTCC, CDKN1A: fwd: TCGACTTTGTCACCGAGACACCAC, rev: CAGGTCCACATGGTCTTCCTCTG, CDKN1B: fwd: GGCCTCAGAAGACGTCAAAC, rev: ACAGGATGTCCATTCCATGA, KLK3: fwd: GAGGCTGGGAGTGCGAGAAG, rev: TTGTTCCTGATGCAGTGGGC.The assays were performed with RNA isolated from 10 cm cell culture dishes, and the cells were seeded at 500,000 cells per dish. The total RNA extraction was performed using peqGOLD TriFast (Peqlab) according to the manufacturer’s protocol. Two-step qRT-PCR was performed as described previously , 49 witht test was performed using the GraphPad Prism 8.0 software, which were calculated from the mean, standard deviation, standard error of mean (SEM), and number of replicates (n). A 95% confidence interval  < 0.05) was considered as statistically significant (*) between two subject groups. A 99% confidence interval (p < 0.01) and a 99.9% confidence interval (p < 0.001) were indicated by two (**) and three stars (***), respectively. Western blotting analysis was performed at least for biological duplicates to shown regulation at protein levels.For statistical analysis, a two-tailed unpaired Student’s Additional file 1: Fig. S1. Enzalutamide treatment significantly induces CDKN2A (p16INK4a) expression. Fig. S2. GT, ABT263, and MK2206 inhibit LNCaP cells proliferation through apoptosis induction in a concentration dependent manner. Fig. S3. p-RIP3 is dephosphorylated by alkaline phosphatase (FAST AP). Fig. S4. GT enhances detachment of cells after androgen-induced cellular senescence. Fig. S5. ABT263 and MK2206 induce LNCaP cell detachment. Fig. S6. Androgen regulates Bcl-2 family members transcription levels.


Background: Multidrug-resistant pathogens are a major health problem in many countries. In Germany, in accordance with the German Antibiotic Resistance Strategy (DART), the surveillance of antibiotic consumption in acute care hospitals and rehabilitation facilities was made mandatory by the Infection Protection Act in 2011 and 2017.Whereas comparable reference data for acute care hospitals are available, such data is lacking for rehabilitation facilities. Therefore, the Rhine-Main network on MDRO (Multi Drug Resistant Organisms) has offered to evaluate the antibiotic consumption of the network's rehabilitation facilities. Antimicrobial consumption  and patient days from 2016–2018 were requested.Materials and methods: By October 31, 2019, nine clinics, including a facility for early neurological rehabilitation, reported their consumption of antibiotics  and patient days from 2016–2018. The information from the clinics was entered in an Excel table and the DDD calculated if necessary. In order to compare the facilities, the DDD/100 patient days (pd) were calculated for the individual active substances.Results: Antibiotic consumption in general rehabilitation facilities decreased slightly from 4.8 DDD/100 pd to 4.4 DDD/100 pd from 2016 to 2018. In early neurological rehabilitation, antibiotic consumption increased from 10.2 DDD/100 pd to 13.1 DDD/100 pd in the same period. Among the most commonly used antibiotics, cefuroxime came first, followed by ciprofloxacin and amoxicillin in third place.Discussion: To our knowledge, this is the first antimicrobial consumption data from rehabilitation facilities in Germany. Antibiotic consumption in general rehabilitation facilities is less than 10% of the consumption in normal/regular wards of acute care clinics, and the consumption in neurological early rehabilitation was approximately 10% of the consumption in intensive care wards within acute care clinics. Reserve or broad-spectrum antibiotics were rarely or not used at all in the rehabilitation facilities. Despite this overall positive situation, antibiotic stewardship should also be introduced in rehabilitation facilities, possibly with the support of the regional MDRO networks. Staphylococcus aureus (MRSA) strains are declining, an increase in vancomycin-resistant enterococci (VRE) and multidrug-resistant Gram-negative pathogens (MDRGN) is particularly evident , th, th8], tAntibiotic consumption in general rehabilitation facilities (GR) slightly decreased from 4.8 DDD/100 pd to 4.4 DDD/100 pd  from 2016 to 2018. In the facility for early neurological rehabilitation (ENR), antibiotic consumption was significantly higher  and increased from 10.2 DDD/ 100 pd to 13.1 DDD/100 pd from 2016 to 2018. The decrease in GR is due in particular to the decrease in cephalosporin consumption from 1.31 DDD/100 pd in 2016 to 0.9 DDD/100 pd in 2018 (particularly due to the decreasing consumption of cefuroxime from 1.17 DDD/100 pd in 2016 to 0.7 DDD/100 pd). The consumption of the other antibiotic groups remained largely stable. In contrast, the increase in antibiotic consumption in the ENR is mostly due to the increasing consumption of cephalosporins  and quinolones (from 2.0 to 3.3 DDD/100 pd) and to a lesser extent also penicillins (increase from 3.43 DDD/100 pd to 4.08 DDD/100 pd) Table 1 .Table 1 Antimicrobial consumption in the individual rehabilitation facilities differed significantly in some cases. Over all years and all GRs, the total consumption ranged from 1.7 DDD/100 pd to 9 DDD/100 pd, the consumption of penicillins from 0.4 DDD/100 pd to 1.5 DDD/100 pd, that of cephalosporins from 0.2 DDD/100 pd to 3.8 DDD/ 100 pd and quinolone consumption from 0.2 DDD/ 100 pd to 1.9 DDD/100 pd. Apparently, some clinics had already started antibiotic stewardship. For example, two GRs had significantly reduced their total consumption and especially their consumption of cephalosporins.Figure 1 Before discussing the antibiotic consumption data presented here, we would like to point out the study’s limitations. The data originate from a small number of rehabilitation facilities with different rehabilitation indications and only one facility for early neurological rehabilitation (ENR) in a single region in Germany. The comparability of the data to other rehabilitation facilities in other regions in Germany can only be confirmed (or excluded) by further surveys. However, there is no evidence of bias regarding the therapeutic indications of participating rehabilitation facilities.On the other hand, to our knowledge, this is the first paper on antibiotic consumption data from rehabilitation facilities in Germany – including facilities with various rehabilitation indications. In addition, this is current data, from the past 3 years up to 2018. As long as there are no further publications on antibiotic consumption in rehabilitation facilities available – neither in national nor in international literature –, this data can be used as comparison data for other rehabilitation facilities.In addition, we suggest that the Robert Koch Institute also enables rehabilitation facilities to participate in its antibiotic consumption monitoring system  and offeBecause hospital antimicrobial consumption data published by the European Centre for Disease Prevention and Control (ECDC) use another denominator (per 1000 inhabitants), our data are not comparable to the European data for methodological reasons . Hence, When considering individual active substances, cefuroxime was the most frequently used antibiotic, followed by ciprofloxacin and amoxicillin. This means that one penicillin compound was already the third most often consumed antibiotic and a total of 4 penicillins (2 of which had an enzyme inhibitor) were among the top 10. Cefuroxime is also the most frequently used antibiotic active ingredient in acute hospitals – and in the outpatient sector – in Germany , [10], . This acIn the general rehabilitation facilities presented here, cefuroxime consumption decreased by more than a third as well, from 1.17 DDD/100 pd to 0.7 DDD/100 pd from 2016 to 2018, despite the fact that the data was collected retrospectively without previous systematic intervention in the facilities. However, since colleagues from rehabilitation facilities have been attending the Antibiotic Stewardship working group of the MDRO network Rhine-Main , [16], . H. H24]. HNevertheless, further efforts should be made to improve (reduce) the use of antibiotics in rehabilitation facilities. For acute care hospitals, numerous studies have shown that improved, careful antibiotic use not only reduced colonization and infections with multi-resistant pathogens, but also reduced severe Clostridium difficile infections , 31], , . Given tThe MDRO networks in Germany not only advise institutions – including rehabilitation facilities – on questions about multi-resistant pathogens. Most MDRO networks have also started projects on the responsible use of antibiotics and offer information flyers on this. The MDRO network Rhine-Main for example, offers flyers on the responsible use of antibiotics for respiratory infections, urinary tract infections, and ear infections. For instance, with these flyers, patients or relatives can be informed that 80% of respiratory infections are caused by viruses and antibiotics do not help, or that 70% of uncomplicated urinary tract infections heal even without antibiotics. The flyers also provide information on suitable “home remedies”. They were created in cooperation with the relevant professional medical associations, based on current guidelines, and are available free of charge on the homepage or can be ordered from the network .In Germany, rehabilitation facilities have been obliged to monitor their antibiotic consumption since 2017. In this paper, antibiotic consumption data from the years 2016–2018 from rehabilitation facilities in Germany are presented for the first time.Antibiotic consumption in the rehabilitation facilities was generally less than 5 DDD/100 pd, but in a facility for early neurological rehabilitation between 10 and 13 DDD/100 pd. This is about 10% of the consumption in acute care hospitals . Broad-spectrum or reserve antibiotics are prescribed very rarely or not at all.Even if antibiotic consumption in the rehabilitation facilities was low compared to that in acute care clinics, antibiotic stewardship should also aim to reduce and improve the use of antibiotics in rehabilitation facilities. This article offers suggestions to this end, e.g., cooperation with the regional MDRO networks.The authors declare that they have no competing interests.


Dysregulation of long noncoding RNAs (lncRNAs) is associated with drug resistance in multiple cancers. We explored the roles of lncRNA p38 inhibited cutaneous squamous cell carcinoma-associated lincRNA (PICSAR) in cisplatin (DDP) resistance of cutaneous squamous cell carcinoma (CSCC).in vivo. The morphology of exosomes was analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA).Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure the expression of lnc-PICSAR, miR-485-5p and reversionless 3-like (REV3L) mRNA. The cell counting kit-8 (CCK-8) assay was conducted to evaluate DDP resistance and cell viability. The transwell assay was performed to determine cell migration and invasion. Western blot assay and immunohistochemistry (IHC) staining assay were carried out to measure protein levels. The dual-luciferase reporter assay was used to investigate the association between miR-485-5p and lnc-PICSAR or REV3L. Murine xenograft model was constructed to explore the function of lnc-PICSAR in vivo. In addition, lnc-PICSAR was increased in the exosomes derived from CSCC patients’ serum and CSCC cells.Lnc-PICSAR was elevated in DDP-resistant CSCC cells. Lnc-PICSAR silencing suppressed cell viability, DDP resistance, migration and invasion in DDP-resistant CSCC cells. MiR-485-5p acted as a target of lnc-PICSAR, and miR-485-5p inhibition reversed the impacts of lnc-PICSAR silencing on DDP resistance and cell progression in DDP-resistant CSCC cells. Lnc-PICSAR promoted REV3L expression via sponging miR-485-5p. Moreover, REV3L overexpression overturned the effects of lnc-PICSAR on cell progression and DDP resistance. Lnc-PICSAR knockdown suppressed DDP resistance Lnc-PICSAR enhanced DDP resistance via miR-485-5p/REV3L axis in DDP-resistant CSCC cells. Besides, exosome-mediated lnc-PICSAR might be involved in the regulation of drug resistance in CSCC. BecauseLong noncoding RNAs (lncRNAs), a series of noncoding RNAs (ncRNAs) with >200 nucleotides (nts), play crucial roles in cellular processes . AberranMicroRNAs (miRNAs), ncRNAs with about 22 nts in length, modulate gene expression via recognizing the 3′-untranslated region (3′UTR) of target mRNAs . MultiplReversionless 3-like (REV3L), the catalytic subunit of DNA polymerase ζ, alters the development and chemoresistance of diverse cancers . Wang etIn this research, we measured the expression of PICSAR, miR-485-5p and REV3L in DDP-resistant CSCC cells. Moreover, their roles and mechanisms in tumor progression and DDP resistance were explored.22.1Serum samples were obtained from 30 CSCC patients and 30 healthy volunteers at the Affiliated Hospital of Jiangnan University. The samples were preserved at −80°C until use.Informed consent: Informed consent has been obtained from all individuals included in this study.Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration and has been approved by the Ethics Committee of Affiliated Hospital of Jiangnan University.2.22 at 37°C.Normal human epidermal keratinocytes (NHEKs) and CSCC cells (A431) were bought from the American Type Culture Collection . CSCC cells (HSC-5) were bought from Japanese Collection of Research Bioresources . DDP-resistant CSCC cells (A431/DDP and HSC-5/DDP) were established by exposing A431 and HSC-5 cells to gradually increased concentrations of DDP . All cells were grown in Dulbecco’s modified Eagle’s medium  including 10% fetal bovine serum  and 1% penicillin–streptomycin (HyClone) in an incubator containing 5% CO2.3Small-interfering RNA against lnc-PICSAR (si-lnc-PICSAR) and its control (si-NC); the overexpression vector of lnc-PICSAR (lnc-PICSAR) and the overexpression vector of REV3L and their control (pcDNA); miR-485-5p mimic (miR-485-5p) and its control (miR-NC); miR-485-5p inhibitor (anti-miR-485-5p) and its control (anti-miR-NC); and lentiviral vectors (sh-lnc-PICSAR and sh-NC) were purchased from RIBOBIO . Lipofectamine 2000  was utilized for cell transfection.2.4−ΔΔCt method. The primers used were as follows: lnc-PICSAR: (F: 5′-GTGAGCAGAGGGACCTGAAG-3′ and R: 5′-ACATGTGCTCCCCACCTAAG-3′); miR-485-5p: (F: 5′-CCAAGCTTCACCCATTCCTAACAGGAC-3′ and R: 5′-CGGGATCCGTAGGTCAGTTACATGCATC-3′); REV3L: (F: 5′-TGATGTCTTCAGCTGGTATCATGA-3′ and R: 5′-CCGCCCTTCAGGTTCACTT-3′); GAPDH: (F: 5′-GGAGCGAGATCCCTCCAAAAT-3′ and R: 5′-GGCTGTTGTCATACTTCTCATGG-3′); and U6: (F: 5′-TGCGGGTGCTCGCTTCGGCAGC-3′ and R: 5′-CCAGTGCAGGGTCCGAGGT-3′).Total RNA in serums, cells and exosomes was extracted using Rneasy Mini Kit . RNAs were reversely transcribed into cDNAs using PrimeScript™ RT reagent Kit  or miScript II Reverse Transcriptase Kit (Qiagen). Then, qRT-PCR was conducted with BeyoFast™ SYBR Green qPCR Mix . The expression was examined with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or small nuclear RNA U6 as an internal control using the 22.550) was analyzed.DDP resistance and cell viability were evaluated by the CCK-8 assay. To determine DDP resistance, A431, A431/DDP, HSC-5 and HSC-5/DDP cells were seeded into 96-well plates and maintained overnight. Then, cells were treated with different doses of DDP (Solarbio) for 48 h. Next, 10 μL CCK-8  was added and incubated for another 2 h. The absorbance at 450 nm was measured using a microplate reader . The 50% maximal inhibitory concentration  was added at indicated time points and incubated for another 2 h. Finally, the absorbance at 450 nm was examined.2.6For the analysis of cell migration, A431/DDP and HSC-5/DDP cells in serum-free DMEM (HyClone) were seeded into the upper chamber of a transwell insert , and DMEM (HyClone) containing 10% FBS (HyClone) was added into the lower chamber. Forty-eight hours later, migrated cells were treated with methanol, stained with crystal violet (Solarbio) and then counted under a light microscope . For the analysis of cell invasion, the transwell chamber was precoated with Matrigel (Solarbio), and the other steps were consistent with the cell migration assay.2.7Total protein was isolated from serums, cells and exosomes using RIPA buffer (Beyotime) and quantified using BCA protein assay kit . Then, equal amount of proteins was separated by sodium dodecyl sulfonate–polyacrylamide gel (Solarbio) and transferred onto polyvinylidene difluoride (PVDF) membranes . After being blocked with skim milk for 2 h, membranes were incubated with primary antibodies against multidrug resistance-associated protein 1 , multidrug resistance protein 1 , REV3L  or GAPDH  overnight followed by incubation with corresponding secondary antibody  for 2 h. The proteins were visualized using the enhanced chemiluminescence detection kit .2.8The sequences of lnc-PICSAR and 3′UTR of REV3L, which contained the binding sites of wild-type or mutant miR-485-5p, were inserted into pmirGLO vector  to establish luciferase reporter plasmids lnc-PICSAR WT, lnc-PICSAR MUT, REV3L 3′UTR-WT and REV3L 3′UTR-MUT, respectively. A431/DDP and HSC-5/DDP cells were transfected with indicated plasmid and miR-485-5p or miR-NC using Lipofectamine 2000 (Invitrogen). Dual-luciferase reporter assay kit (Promega) was adopted to examine the luciferase activity after 48 h of co-transfection.2.9n = 7). Sh-lnc-PICSAR or sh-NC transfected HSC-5/DDP cells were injected into the nude mice. After 7 days, the mice were treated with 6 mg/kg of DDP (Solarbio) or equivalent phosphate-buffered saline  every 3 days. Tumor weight was examined every 3 days and calculated with the formula: (length × width2)/2. On day 25, mice were sacrificed, and tumor samples were weighed and harvested for subsequent experiments.Six-week-old nude mice were bought from Shanghai SLAC Laboratory animal Co., Ltd  and divided into four groups  and incubated with normal goat serum for 20 min. Next, the samples were maintained with anti-REV3L  at 4°C overnight and HRP-conjugated secondary antibody  for 30 min at room temperature. After diaminobenzidine  staining and hematoxylin counterstaining, the sections were photographed using a digital microscope camera .The mice tumors were fixed in 4% paraformaldehyde (Beyotime) for 48 h, embedded in paraffin and sectioned into 4 µm thick. Then, the slides were deparaffinized, hydrated with a graded ethanol series and treated with H2.11g. Then, the supernatant was mixed with ExoQuick and incubated for 30 min at 4°C and then centrifuged for 30 min at 1,500 × g. Next, the supernatant was removed, followed by centrifugation for 5 min at 1,500 × g to remove the residual liquid. Exosome pellets were resuspended in PBS (Solarbio). Exosomes from cultured cells were isolated and purified via differential centrifugation as previously described [Exosomes were isolated from serums using ExoQuick precipitation kit  based on the instructions of the manufacturer. In brief, serums were centrifuged for 15 min at 3,000 × escribed .2.12Exosomes were placed on carbon-coated copper grids and stained with the phosphotungstic acid solution. The morphology of exosomes was observed by TEM .2.13The size distribution of exosomes was analyzed using Delsa Nano Analyzer  based on the protocols of the manufacturer.2.14t-test or one-way analysis of variance (ANOVA). It was considered statistically significant if the P value is less than 0.05.Data were collected from three independent experiments and presented as mean ± standard deviation (SD). Data analysis was conducted using GraphPad Prism 7 software . The difference was analyzed via Student’s 33.1To begin with, we measured the expression level of lnc-PICSAR in the serum of CSCC patients and healthy volunteers by qRT-PCR. The results showed that lnc-PICSAR was conspicuously elevated in CSCC patients’ serum compared to normal serum . Afterwa3.250 of cisplatin in A431 cells, HSC-5 cells and corresponding DDP-resistant cells was assessed via the CCK-8 assay. The data manifested that IC50 of cisplatin was increased in A431/DDP and HSC-5/DDP cells in reference to A431 and HSC-5 cells . CCK-8 assay indicated that the viability of A431/DDP and HSC-5/DDP cells was enhanced compared to the viability of A431 and HSC-5 cells . Besides-5 cells , suggest3.350 of cisplatin was reduced in A431/DDP and HSC-5/DDP cells transfected with si-lnc-PICSAR, indicating that lnc-PICSAR knockdown enhanced the sensitivity of A431/DDP and HSC-5/DDP cells to DDP (To explore the exact role of lnc-PICSAR in the resistance of DDP-resistant CSCC cells to DDP, si-lnc-PICSAR was transfected into A431/DDP and HSC-5/DDP cells to downregulate the expression of lnc-PICSAR. As shown in s to DDP . As suggs to DDP . Besidess to DDP . To sum 3.450 of cisplatin was decreased in A431/DDP and HSC-5/DDP cells by lnc-PICSAR deficiency, while inhibition of miR-485-5p partly overturned this effect (To reveal the underlying mechanisms of lnc-PICSAR in the regulation of DDP resistance in DDP-resistant CSCC cells, online website Starbase was used to search the potential target of lnc-PICSAR. As shown in s effect . The CCKs effect . Knockdos effect . Westerns effect . All the3.5By using software Starbase, we found that REV3L might be a target gene of miR-485-5p, and their potential binding sites were shown in 3.6To further determine the relationship among lnc-PICSAR, miR-485-5p and REV3L in DDP-resistant CSCC cells, A431/DDP and HSC-5/DDP cells were transfected with si-lnc-PICSAR, si-NC, si-lnc-PICSAR + anti-miR-NC or si-lnc-PICSAR + anti-miR-485-5p, and then, the protein level of REV3L was measured. The data displayed that lnc-PICSAR silencing drastically decreased REV3L expression in A431/DDP and HSC-5/DDP cells, but miR-485-5p inhibition restored the effect . The out3.7To further explore the relationship between lnc-PICSAR and REV3L in DDP-resistant CSCC cells, we first determined REV3L level in A431/DDP and HSC-5/DDP cells transfected with pcDNA or REV3L. The data demonstrated that REV3L transfection led to obvious elevation of REV3L mRNA and protein levels in A431/DDP and HSC-5/DDP cells . Afterwa3.8in vivo, we established a xenograft tumor mouse model by injecting sh-NC or sh-PICSAR transfected HSC-5/DDP cells into nude mice. Seven days later, the mice were treated with 6 mg/kg DDP or equivalent PBS every 3 days. We observed that tumor volume and tumor weight were markedly blocked by the DDP treatment or lnc-PICSAR knockdown (in vivo.To reveal the effect of lnc-PICSAR on DDP resistance nockdown . Moreovenockdown . We conc3.9Subsequently, CSCC patients’ serum-derived exosomes and HSC-5 cells–derived exosomes were isolated. The morphology of exosomes was observed by TEM, and the results showed that vesicles had a round or oval membrane . The NTA4Chemoresistance is an enormous obstacle for the therapy of human cancers. LncRNAs have been demonstrated to act as vital regulators in tumor progression and drug resistance in cancers. However, the effects of lncRNAs in chemoresistance of CSCC have been limited to date. Herein, we explored the roles of lnc-PICSAR in DDP resistance in CSCC. We found that lnc-PICSAR was drastically increased in DDP-resistant CSCC cells. Silencing of lnc-PICSAR enhanced the DDP sensitivity via suppressing cell viability, migration and invasion in DDP-resistant CSCC cells. Moreover, we established a novel network of lnc-PICSAR/miR-485-5p/REV3L in the drug resistance of CSCC.in vitro and blocked the tumor growth in vivo [in vivo. All these observations suggested that the level of lnc-PICSAR might be a prognostic marker, and lnc-PICSAR enhanced DDP resistance in DDP-resistant CSCC cells.Piipponen et al. validated that lnc-PICSAR was elevated in CSCC cells, and its deficiency hampered CSCC cell growth and migration  in vivo ,23. Mult in vivo . Li et a in vivo . We inveLncRNAs can function as miRNA sponges to alter the biological function of miRNAs . We wondExosomes are tiny vesicles about 40–150 nm in diameter secreted by cells . It has In conclusion, lnc-PICSAR was highly expressed in DDP-resistant CSCC patients’ serum and cells. Lnc-PICSAR/miR-485-5p/REV3L axis participated in the regulation of DDP resistance via altering cell viability and metastasis. These findings suggested that lnc-PICSAR might be a diagnostic biomarker for CSCC and provide a new treatment strategy for CSCC patients.