Sarcopenia has become a leading cause of disability and mortality in the elderly. It has been reported that programmed cell death (PCD) is associated with the development of sarcopenia that is characterized by reduction of muscle fiber size and number. TNF-α is also validated to play a prominent role in sarcopenia through its complex signaling pathways including cell death signaling. However, it is still unclear whether TNF-α contributes to sarcopenia by mediating pyroptosis, one type of PCD. Here, we first established naturally aged mice with sarcopenia model and confirmed an inflammatory state represented by TNF-α in aged mice. Evidence of GSDME-mediated pyroptosis and activation of apoptotic caspase-8/-3 were also found in skeletal muscle cells of aged mice with sarcopenia. We demonstrated that TNF-α triggered GSDME-mediated pyroptosis in myotubes through activating caspase-8 and caspase-3 by using caspase-8 and caspase-3 inhibitors. Comparing the activation of caspase-8 and GSDME expression between TNF Complex IIa and TNF Complex IIb, TNF-α was found to be more inclined to assemble TNF Complex IIb in activating caspase-8 and triggering pyroptosis. Moreover, pyroptotic myotubes were validated to result in decreased expression of MHC1 and finally loss of myotubes by knockdown of GSDME. Our work reveals a novel mechanism that TNF-ɑ/caspase-8/caspase-3/GSDME signaling-mediated pyroptosis contributes to the development of sarcopenia. Caspase-3/GSDME signaling-mediated pyroptosis may be a promising therapeutic target for sarcopenia. Sarcopenia is a multifactorial disease characterized by age-related decline in skeletal muscle mass, strength, and physical performance . HistoloProgrammed cell death (PCD) refers to a type of strictly regulated cell death, including apoptosis, pyroptosis, necroptosis, ferroptosis, and autophagy . It has Morphologically, pyroptosis manifests as the formation of pores in the cell membrane, followed by cell swelling with membrane rupture and finally cell death . The exeInterestingly, interconnectivity exists between different types of PCD , 16. A tChronic low-grade inflammation was confirmed to develop with age in the elderly, with inflammatory cells infiltrating among the skeletal muscle cells and releasing pro-inflammatory cytokines such as TNF-α to cause muscle damage . TNF-α hIn this paper, we established naturally aged mice with sarcopenia model to observe the inflammatory state represented by TNF-α and to identify whether pyroptosis occurs in the skeletal muscle cells. TNF-α-stimulated myotube is a well-known in vitro study on sarcopenia . We thusThe gastrocnemius muscle index (GMI), absolute, and relative grip strength of aged mice were significantly lower than those of young mice, although there was no significant difference in the absolute mass of gastrocnemius muscle (GM) between the two groups. Meanwhile, the absolute mass of GM and GMI of aged mice were dramatically lower than those of middle-aged mice, whereas no significant differences in the absolute and relative grip strength were found between the two groups Fig. . The croThe expression of TNF-α was considerably higher in GM of aged mice than that in young and middle-aged mice Fig. . ConsistSince the common protein executors for pyroptosis are GSDMD and GSDME, we detected the expression of GSDMD and GSDME in GM of mice at different ages. Western blot showed that GSDMD was not cleaved in all of three groups, while GSDME was significantly cleaved in the aged mice compared with that in young and middle-aged mice Fig. . MeanwhiC2C12 myoblasts are mononuclear with spindle shape, whereas C2C12 myotubes are multi-nuclear with elongated morphology Fig. . The difNext, we investigated whether TNF-α induced GSDME-mediated pyroptosis in myotubes through caspase-3 and upstream caspase-8. Both caspase-8 and caspase-3 were activated with the concentration of TNF-α ranging from 0 to 100 ng/ml in myotubes Figs. A and 5A.TNF-α can trigger the activation of caspase-8 either by the assembly of TNF Complex IIa or by TNF Complex IIb . HoweverC2C12 cell has been recognized as a well-documented in vitro cell line for mimicking molecular mechanisms of sarcopenia , 29. DifThe expression of MHC1 declined with the concentration of TNF-α in myotubes Fig. , indicatTo further clarify the contribution of GSDME-mediated pyroptosis to TNF-α-induced sarcopenia, we transfected myoblasts either with lentivirus encoding shRNA targeting GSDME(LV-shGSDME) or empty lentiviral vector (LV-shNC) according to the optimal MOI for infection  and valiIn the current study, we identified that aged mice with sarcopenia presented a chronic low-grade inflammation with a high level of serum TNF-α and local TNF-α overexpression in GM, accompanied by evidence of GSDME-mediated pyroptosis and activation of apoptotic caspase-8/-3 in skeletal muscle cells. We demonstrated that TNF-α triggered GSDME-mediated pyroptosis in myotubes by activating caspase-8 and caspase-3 through TNF Complex IIb. Furthermore, pyroptotic myotubes resulted in decreased expression of MHC1 and loss of myotubes, finally leading to sarcopenia. Our study highlights the pathophysiological role of pyroptosis mediated by TNF-ɑ/caspase-8/caspae-3/GSDME signaling in the development of sarcopenia.According to the latest diagnostic criteria of the European Working Group on Sarcopenia in Older People in 2019 , low musWe further found a high level of serum TNF-α and local TNF-α overexpression in GM of aged mice, supporting the commonly recognized view that chronic low-grade inflammation is a hallmark feature of aging , as wellMuscle cell death may be the key factor for the loss of skeletal muscle fibers during aging . We obseWe observed activation of both caspase-8 and downstream caspase-3 in the GM of aged mice, consistent with the activation of caspase-8 and caspase-3 in TNF-α-treated myotubes, indicating that TNF-α could also activate the apoptosic caspase-8 and caspase-3 in aging skeletal muscle. As is well-known, TNF-α triggers apoptosis by activating caspase-8 through TNF Complex IIa or IIb, while activated caspase-8 blocks necroptosis by inhibiting RIPK1 and RIPK3 , 43. NotWe then investigated that knockdown of GSDME not only blocked the pyroptosis induced by TNF-α but also increased the expression of MHC1 reduced by TNF-α. These results further validated that TNF-α promotes sarcopenia by regulating GSDME-mediated pyroptosis. Notably, although knockdown of GSDME could reserve the expression of MHC1 reduced by TNF-α, it only partially reserved the expression of MHC1 as compared with untreated myotube, indicating that inhibition of pyroptosis may partially reserve the expression of MHC1 reduced by TNF-ɑ. One potential explanation is that although pyroptosis is inhibited, TNF-ɑ can still activate caspase-8/-3 and eventually stimulate apoptosis, which may also cause damage to the skeletal muscle. A recent study also found that knockdown of GSDME in macrophages reserved cell death from pyroptosis to apoptosis . Hence, Although our results suggested that inhibiting pyroptosis partially improves sarcopenia, we cannot completely negate the pathogenic role of GSDME-mediated pyroptosis in sarcopenia. Pyroptosis is a pro-inflammatory form of PCD that results in cell death and further tissue damage by inflammation cascades response . TherefoIn summary, our study reveals a novel mechanism that TNF-ɑ/caspase-8/caspase-3/GSDME signaling-mediated pyroptosis plays a pathogenic role in the development of sarcopenia, as illustrated in Fig. The following primary antibodies were used for immunoblotting, immunohistochemistry, and immunofluorescence: anti-GSDMD antibody (ab219800) and anti-GSDME antibody (ab215191) were purchased from Abcam . Anti-caspase-8 antibody (66093-1-Ig), anti-caspase-3 antibody (19677-1-AP), anti-TNF-α antibody(60291-1-Ig), anti-GAPDH antibody (60004-1-Ig), and anti-beta-Tubulin antibody (66240-1-Ig) were obtained from Proteintech . Cleaved-caspase-8 antibody (#8592) and anti-beta-actin antibody (#3700) was obtained from Cell Signaling Technology . Cleaved-caspase-3 antibody (GB11532) was purchased from Servicebio . Anti-myosin heavy chain 1 (MHC1) antibody was obtained from Developmental Studies Hybridoma Bank . Alexa Fluor 594-labeled secondary antibody (ZF-0516) and Alexa Fluor 488-labeled secondary antibody (ZF-051) were purchased from Zhongshan Golden Bridge . Mouse TNF-α ELISA Kit (EK282) was purchased from MULTISCIENCES . DeadEnd™ Fluorometric terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) System (G3250) was obtained from Promega . LDH cytotoxicity assay detection kit was obtained from Beyotime . Hoechst 33342/PI double stain kit was purchased from Solarbio . Recombinant murine TNF-α was purchased from PeproTech . Z-DEVD-FMK (A1920) and Z-IETD-FMK(B3232) were obtained from ApexBio Technology . Cycloheximide (CHX) and GSK’872 were obtained from MedChemExpress . TGF-β-activated kinase 1 inhibitor (TAK1i) 5Z-7-Oxozeaenol was purchased from Sigma-Aldrich .Healthy male C57/BL 6 J mice were purchased from Shanghai SLACCAS Laboratory Animal Co., Ltd , including 10 mice aged 12 weeks (weighting 24–26 g) as a young group, 10 mice aged 52 weeks (weighting 28–35 g) as a middle-aged group and 16 mice aged 80 weeks (weighing 30–40 g) as an old group. Mice were group-housed 4–5 per cage on a 12-hour light-dark cycle and bred under SPF conditions with suitable humidity and temperature for 56 days. Food and water were both available ad libitum. Body weight and grip strength were measured every 7 days. 1 mouse in the middle-aged group and 2 mice in the old group died during feeding. On day 56, there were 10 mice aged 20 weeks in the young group, 9 mice aged 60 weeks in the middle-aged group and 14 mice aged 88 weeks in the old group. All mice were fasted for 12 h overnight and euthanized for collecting blood and skeletal muscle samples at this time point. Blood samples were collected by retro-orbital venous plexus puncture for enzyme-linked immunosorbent assay (ELISA). Left and right GM were quickly isolated and weighted. The average mass of left and right GM was recorded as the absolute mass of GM. To exclude the influence of body weight on muscle mass, the absolute mass of GM was divided by body weight, which was defined as GMI. Right GM tissues were immediately frozen in liquid nitrogen and stored at −80 °C for western blot and gene expression studies. Left GM tissues were divided into two parts at the mid-belly. The proximal parts were fixed and stored in 4% parafomaldehyde for histological staining, whereas the distal parts were cut into pieces and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for TEM. The tubes containing the samples were digitally labeled to ensure examiners blinded. When establishment of sarcopenia model was confirmed,8 mice were randomly selected from the three groups respectively using random number table and used for the following experiments. All experiments were approved by the Animal Ethics Committee (Ethics Number: FJMU IACUC 2021-0385).The grip strength of the four limbs of mouse was measured using mice grip strength dynamometer . Mouse was gently placed on the mesh bar to allow its four limbs to hold the mesh. As the mouse grasped the mesh bar, its tail was gently pulled backward in a horizontal direction parallel to the mesh bar until the mouse released its grasp. The peak force applied by the mouse to grasp the mesh bar was recorded. Each mouse was tested repeatedly for six times. The average of six grip strength measurements was recorded as the absolute grip strength. To exclude the influence of body weight on muscle strength, the absolute grip strength was divided by body weight, which was defined as relative grip strength.Blood samples were centrifuged at 3000 g for 20 min in a refrigerated centrifuge. Then serum was separated and stored at -80 °C. The concentrations of serum TNF-α were measured using a high sensitivity ELISA Kit following the supplier’s protocol. Data were analyzed using Excel.Fixed GM tissues were dehydrated, embedded in paraffin, and sectioned at 10μm. Sections were dewaxed, and rehydrated for HE staining, immunofluorescence, immunohistochemistry and TUNEL staining. Sections were stained with hematoxylin and eosin for morphological evaluation and myofiber CSA analysis. Images were taken by Nikon Eclipse E200 microscope . At least five random fields at adequate magnification were acquired for each section. The mean CSA of fibers (at least 250) in five fields for each section was analyzed using Image J software. Representative images were presented at 40x magnification. Immunofluorescence was performed to assess the expression and location of GSDME. After antigen retrieval with Tris-EDTA by using a microwave oven, sections were permeabilized 0.5% TritonX-100 in PBS for 20 min, then blocked with 5% BSA in PBS for 1 h. Following blocking treatment, sections were incubated overnight at 4 °C with primary antibody against GSDME (1:50). Alexa Fluor 594-labeled secondary antibody (1:200) was used as a detection antibody. Nuclei were labeled with DAPI . Confocal images were acquired by using Zeiss confocal laser scanning microscope . Immunohistochemistry was performed to assess the expression of cleaved-caspase-8 and cleaved-caspase-3. After antigen retrieval, sections were incubated with 3% hydrogen peroxide to inactive endogenous peroxidases and blocked with 10% bovine serum albumin. Following blocking treatment, sections were incubated overnight at 4 °C with primary antibody . Sections were incubated with secondary antibodies, developed with DAB and counterstained with hematoxylin. Stained sections were photographed by using Nikon Eclipse E200 microscope . Images were analyzed by using Image-Pro Plus software. To analyze cell death, sections were stained with TUNEL according to the manufacturer’s instructions. TUNEL-positive cells were counted on five random fields of each section (20x magnification) using Image J software.3 in size and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer. For detecting ultrastructure of cells, cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer at room temperature for 5 min and then gently scraped, centrifuged and postfixed in 1%OsO4.Tissues and cells were embedded in resin for ultrathin sectioning. Ultrathin sections were then stained with uranyl acetate and lead citrate. Images were acquired by using Transmission electron microscopy .For detecting ultrastructure of GM tissues, tissues were cut into pieces approximately 1 mm2. To induce myoblasts differentiation into myotubes, the medium was replaced with DMEM supplemented with 2% horse serum  when the cells were >90% confluent. The differentiated medium was changed daily for 5 days. Differentiated myotubes were identified with immunofluorescence staining. Thereafter, Myotubes were either treated or not treated with drugs, depending on the experimental condition.Mouse C2C12 myoblast cell line (#CL-0044) was kindly provided by Procell Life Science&Technology Co., Ltd . Myoblasts were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium  supplemented 10% fetal bovine serum  and 1% penicillin-streptomycin  at 37 °C in a humidified atmosphere with 5% COC2C12 myoblasts were seeded in 6-well or 12-well plate, differentiated into myotubes and starved in serum-free medium for 24 h before the stimulation. For TNF-α treatments, the culture medium was switched to fresh medium containing different concentrations  of TNF-α for a period as indicated in the figures. For TNFα + CHX or TNF-α + TAK1i treatments, myotubes were treated with either 100 ng/ml TNF-α plus 10 μg/mL CHX to trigger TNF Complex IIa or 100 ng/ml TNF-α plus 1 μM TAK1 inhibitor 5z-7-oxozeaenol to trigger TNF Complex IIb for the indicated time points. To inhibit caspase-8 activity, myotubes were pre-incubated for 30 min with 1 μM GSK'872 and 1 h with 50 μM Z-IETD-FMK prior to treatment with 100 ng/ml TNF-α for 72 h.To inhibit caspase-3 activity, myotubes were pre-incubated for 1 h with 50 μM Z-DEVD-FMK prior to treatment with 100 ng/ml TNF-α for 72 h.Myoblasts were seeded on sterile coverslips and induced to differentiate into myotubes. Myotubes were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.25% TritonX-100 for 10 min at 4 °C and blocked with 5% BSA for 30 min at room temperature. Then myotubes were incubated with anti-MHC1 (1:100 dilution) antibody overnight at 4 °C. Following primary antibody incubation, myotubes were washed with PBS and incubated with an Alexa Fluor 488-labeled secondary antibody (1:200 dilution) for 1 h, and counterstained with DAPI . Fluorescence images were taken by using Nikon Eclipse TS2R microscope .For detecting the protein expression in GM tissues, approximately 25 mg of tissues were dissected and homogenized in 500 μl RIPA lysis buffer  with a Mill Cryogenic Grinder . For detecting the protein expression in cultured cells, cells and supernatants were both collected for protein extract after treatments and then extracted using lysis buffer  containing 1 mM PMSF  and 1% protease inhibitor cocktail . After protein concentrations were detected by BCA Protein Assay , western blot analysis was performed as described previously . Brieflyg for 5 min after various treatments. Aliquots of supernatants were transferred into 96-well plates, subjected to the LDH working liquids, and incubated for 30 min at room temperature protected from light. Thereafter, the absorbance was measured at 490 nm. The percentage of LDH release was calculated as follows: %release = 100 × / . Duplicate wells were run in each experiment, and each experiment was repeated four or five times.Myoblasts were seeded on 6- or 12-well plates, induced to differentiate into myotubes, and treated as indicated. Cell death was quantitated by assaying LDH release into cell culture supernatants using LDH cytotoxicity assay detection kit according to the manufacturer’s protocol. Briefly, Culture supernatants were harvested and centrifuged at 400 × Myoblasts were seeded on 6- or 12-well plates, induced to differentiate into myotubes, and treated as indicated. Cell death was quantitated by assaying Hoechst 33342/PI double staining using Hoechst 33342/PI double stain kit according to the manufacturer’s protocol. The medium of each well was replaced with 1 ml of staining buffer containing 5 µl of Hoechst 33342 and 5 µl of PI, followed by incubation for 30 min at 4 °C without light. Thereafter, cells were washed with PBS and observed using Nikon Eclipse TS2R microscope . Four fields per well were randomly selected for counting the stained cells by Image J software.To construct cell lines with stable knockdown GSDME, a GSDME knockdown lentivirus (LV-shGSDME) was designed and constructed by Zolgene Biotech . For LV-shGSDME, the shRNA sequence of GSDME, 5’-GGA GCT GTT TGT GAA ACA AGA-3’, was cloned into the hU6-MCS-CMV-ZsGreen-PGK-Puromycin vector. The empty lentiviral vector was used as a negative control (LV-shNC). C2C12 myoblasts were transfected with LV-shGSDME at an optimal MOI determined in a preliminary experiment. After incubation for 72 h, myoblasts were selected with 4 μg/mL puromycin to construct stable knockdown myoblasts. Knockdown efficiency was confirmed by western blot. Thereafter, the stable GSDME knockdown myoblasts were induced to differentiate into myotubes by replacing DMEM supplemented with 2% horse serum. After 5 days for differentiation, the expression of MHC1 protein was detected by western blot to identify myotubes.q test. Pearson correlation analysis was used for simple linear correlation analysis. All the experiments were repeated at least four times, and a value of P < 0.05 was considered statistically significant.Data are presented as the mean ± standard deviation (SD) and analyzed with Graph Prism 9.0 software. Comparisons among groups were performed using one-way analysis of variance (ANOVA). Pairwise comparisons of multiple groups were performed by SNK-supplemental figure legendssupplemental figureOriginal western blots


This study describes the reporting of the preference-based health-related quality-of-life (HRQOL) instrument, the EQ-5D, and proposes strategies to improve reporting and reduce research waste. The EQ-5D is a validated instrument widely used for health economic evaluation and is useful for informing health policy.As part of a systematic review of papers reporting EQ-5D utility weights in patients with coronary artery disease, we noted the reasons data from some papers could not be reused in a meta-analysis, including whether health utility weights and sufficient statistical details were reported. Research waste was quantified using: (1) the percentage of papers and sample size excluded, and (2) researcher time and cost reviewing poorly reported papers.Our search strategy found 5942 papers. At title and abstract screening 93% were excluded. Of the 379 full text papers screened, 130 papers reported using EQ-5D. Only 46% (60/130) of those studies reported utility weights and/or statistical properties enabling meta-analysis. Only 67% of included papers had reported EQ-5D in the title or abstract. A total sample size of 133,298 was excluded because of poor reporting. The cost of researcher time wasted estimated to be between $3816 and $13,279 for our review.Poor reporting of EQ-5D data creates research waste where potentially useful data are excluded from meta-analyses and economic evaluations. Poor reporting of HRQOL instruments also creates waste due to additional time spent reviewing papers for systematic reviews that are subsequently excluded.Studies using the EQ-5D should report utility weights with appropriate summary statistics to enable reuse in meta-analysis and more robust evidence for health policy. We recommend authors report the HRQOL instrument in the title or abstract in line with current reporting guidelines (CONSORT-PRO and SPIRIT-PRO Extensions) to make it easier for other researchers to find. Validated instruments should also be listed in the Medical Subject Headings (MeSH) to improve cataloguing and retrieval of previous research. Being able to find and reuse published research is important for research progress, so time and money isn’t wasted repeating already completed experiments and so that new research can build on what is already known. Whilst researchers try to make their results available to others, sometimes the way scientific papers are written and catalogued makes it difficult to find and reuse previous research. This study highlights the difficulties researchers face when trying to find and reuse data from research on quality-of-life. We searched for research using the EQ-5D, a questionnaire about people’s health-related quality-of-life that is used in the evaluation of healthcare services. We wanted to use that previous research in an economic evaluation, rather than repeating a data collection. Because not all papers used the term EQ-5D, we searched for terms like “quality-of-life” to find relevant research. After reviewing thousands of papers, we found only about 2.2% [130/5942] had used EQ-5D, and less than half of those papers could be used for our research because of imperfect reporting. Our study shows that researchers could do better when reporting results of research using questionnaires like the EQ-5D. We suggest some simple ways to make it easier to find previous research including using the term “EQ-5D” in research summaries, and improving the way research papers are catalogued.Chalmers and Glasziou estimated 85% of health and medical research is avoidably wasted with a large part due to biased or poorly reported results , 2. ThisWhilst the focus on reducing waste has tended to be on biomedical research and the field of psychology, the same concepts apply to other areas, including quality-of-life research. Patient-reported outcome measures (PROMs) including quality-of-life measures are not routinely collected in most healthcare settings or registries, and given the resources required to follow-up and collect data, it is unrealistic for many researchers to undertake bespoke collection of these data. This means that health services researchers often rely on secondary use of data collected during clinical trials or observational studies to inform economic evaluations . SimilarCost-utility analysis, a type of cost-effectiveness analysis, uses health utility as the outcome of interest to assess the value of health services. Health utility is a measure of quality-of-life, between 0 (death) and 1 , elicited using preference-based methods, the standard gamble or time trade-off, which require participants to make choices between different states of health. The most commonly used utility instrument is the EQ-5D, a questionnaire which asks participants to rate their health across 5 dimensions; mobility, self-care, usual activities, pain/discomfort and anxiety/depression. The combination of responses to the 5 questions is used to generate a health utility weight. Health economic modelling and evaluation often make use of previously published health outcomes data because they are not often able to be measured directly from the study population. Collating and synthesising these data generally requires a systematic, or reproducible, review with meta-analysis to ensure the best estimates of quality-of-life are used .Systematic reviews rely on good reporting and cataloguing of studies by the original study teams for researchers to be able to find and reuse appropriate research. Quality-of-life and health utility measures are not often primary outcomes of trials and therefore may not be reported in abstracts of papers. This means it may not be evident from the title or abstract of a research paper that health utility was measured or what instruments were used. Whilst searches of clinical trial registries  may help identify trials with quality-of-life measures listed as secondary outcomes, additional work is required to find the associated papers, if indeed all trial outcomes have been reported . The CONFor researchers wanting to reuse health utility data, inadequate reporting of the instruments used makes the systematic review process more time-consuming because broad search criteria are needed to ensure potentially useful papers are captured, resulting in many papers not relevant to the meta-analysis topic also being captured and needing to be reviewed. Researcher time is thereby wasted, reviewing thousands of papers that end up being excluded from a review.One way avoidable waste occurs is when potentially useful papers do not report health utility results in a format compatible with reuse in meta-analyses.Research waste related to poor reporting of quality-of-life data, quantified as the proportion of papers reporting use of the instrument of interest, but not the statistical properties needed for reuse in meta-analysis, and,Research waste in the form of researcher time, related to reviewing papers that do not fit the review criteria, retrieved due to the need to use broad search terms.The aim for this paper is to examine two forms of avoidable research waste resulting from suboptimal reporting of quality-of-life data, relevant to systematic reviews and meta-analyses:The context is a systematic review of papers reporting EQ-5D utility weights for people with coronary artery disease. We present recommendations for improving reporting, to reduce waste both in terms of researcher time to find and review studies, and to enable reuse of results for new research.The results in this paper relate to a systematic review of papers using EQ-5D to assess quality-of-life and health utility of patients with coronary artery disease. Briefly, we undertook systematic searches of literature databases, for papers published from January 2003 to March 2020, assessing the quality-of-life of patients with coronary artery disease, using the EQ-5D. The review protocol was published prior to data collection , 14. TheWe quantified two types of research waste associated with poor reporting of EQ-5D data collection and results: waste due to inability to reuse reported data in a meta-analysis, and waste of researcher time reviewing poorly reported papers.We estimated the total time to review the title and abstract or full text of subsequently excluded papers using previous time estimates –17. BecaWe included the time wasted finding and cataloguing subsequently excluded full text documents for studies that could not be excluded at title and abstract screening. The rate at which full text documents were retrieved and screened for the “slow” scenario were taken from a cost-effectiveness study of systematic review methods . We doubWe calculated the costs of researcher time, using Queensland University of Technology salary scales for a mid-level professional staff  or an early to mid-career academic staff  in 2020 Australian dollars  of the total papers reviewed. Of the 60 included papers, only 40 (67%) included the words EQ-5D or EuroQoL in the title and/or abstract, meaning the full text needed to be screened to identify which if any quality-of-life instrument was used.Whilst title and abstract screening is relatively fast compared to full text screening, we estimated 36 to 93 h of researcher time was spent reviewing subsequently excluded papers, at a cost of between $1879 and $7366, Table We screened the full text of 379 papers. The largest group of papers excluded at full text review were those that did not use an instrument to measure quality-of-life or used an instrument other than the EQ-5D, 37% (118/319) of excluded papers. If the instrument had been listed in the title or abstract, then this would have avoided 10 to 20 h of reviewer time ($512 to $1562).We found a total of 130 papers reporting use of EQ-5D, but results reported in 70 (54%) were unable to be used in our meta-analysis, Table This case study of the process of systematically reviewing research highlights two ways in which research waste is generated due to poor reporting of methods and results. First, because the HRQOL instrument is often not reported in the title or abstract, systematic searches beyond the title and abstract are needed to retrieve potentially relevant papers, and their full texts need to be reviewed to correctly include or exclude them, creating waste in terms of researcher time. Secondly, waste is created when results are not reported in a way that enables reuse and/or meta-analysis.Because of the first problem, poor reporting of HRQOL instruments, we spent many hours reviewing papers that were eventually excluded. Whilst we recognise that not all researchers reporting HRQOL measurement would anticipate reuse of their data in a meta-analysis, new trials should be set in the context of existing research  to avoidThis first type of research waste could have been reduced and mostly avoided if other researchers had listed HRQOL instruments in their titles or abstracts, or if there were MeSH terms to capture them. Because one third (20/60) of included papers did not report the EQ-5D in the title or abstract, we needed to search for terms such as “quality-of-life”, meaning we captured many papers that used this term in a generic way but did not measure it. Additionally, whilst the EQ-5D measures preference-based health utility, that term is not often used outside of cost-utility analyses meaning potentially useful papers would be missed if we did not search for the term quality-of-life. Further, when it was clear from the abstract that HRQOL had been measured, for many papers, we needed to review the full text to know which instrument had been used. We reviewed the full text of 118 papers that used an instrument other than the EQ-5D, which we would not have had to review had the instrument been stated in the abstract. Whilst our research examined only use of the EQ-5D, we expect similar problems to arise when searching for research using other HRQOL instruments. The waste created here was researcher time spent reviewing papers that could have been more quickly included or excluded had reporting been better.As a result of the broad search strategy needed to capture papers that did not report the HRQOL instrument in the title or abstract, we found that only 1% (60/5942) of papers we reviewed could be included in our meta-analysis and estimated the cost of researcher time wasted to be between $3816 and $13,279 for our review. Had we used a more limited search strategy, for example restricting the search only to papers which reported the EQ-5D in the title and/or abstract, we would have missed one third of the eventually included papers.Whilst there will always be some time spent reviewing papers that are eventually excluded, excessive waste of researcher time can be avoided. Guidelines exist for reporting patient-reported outcome measures , 11, howMore disappointing and wasteful than reviewing papers not fitting inclusion criteria, was the number of studies that had used EQ-5D but could not be included in our meta-analysis. We found that only 60 from 130 papers reporting use of the EQ-5D could be used in the meta-analysis. More than half of the papers we found that had collected potentially useful data could not be included, which was over 130,000 participants. Adding to this, we may also have missed papers that did include useful data for our meta-analysis, by inadvertently excluding them at title and abstract screening, if it appeared that HRQOL had not been measured.This second form of waste, due to inability to reuse results, is most problematic because as Chan et al.  point ouSimilarly, a potential criticism is that we only searched for studies which had measured EQ-5D directly from participants, when it is possible to map EQ-5D outcomes from other instruments. Whilst we will have excluded studies which took that approach, there is debate about the reliability of mapping algorithms particularly for lower health utility values . TherefoQuality-of-life, catalogued under both Epidemiological measurements and Philosophy. There is a MeSH term for Patient-Reported Outcome Measures (PROMs), but the only instrument listed is a sino-nasal outcome test for rhinosinusitis. A solution would be to include a more complete list of validated PROMs in the MeSH terms that would enable papers to be classified and more easily retrieved by researchers. The Australian Commission on Safety and Quality in Health Care has collated lists of all validated PROMs [Despite substantial resources being invested to catalogue papers using Medical Subject Headings (MeSH)  terms aned PROMs , which cIn summary, the problem of poor reporting of HRQOL data can create research waste both from the perspective of researcher time spent retrieving and screening papers, and for the reliability meta-analysis results. The solutions we recommend are simple to implement and should improve both our ability to find previous research and our understanding of the value of treatments to patients through more reliable meta-analyses.New MeSH terms for all validated PROMs to be proposed to the National Library of Medicine.Improve use of reporting guidance such as CONSORT-PRO and SPIRIT-PRO Extensions , 11 to eFurther extension of the guidelines to include improved reporting of EQ-5D data, to enable reuse: mean health utility weights, with standard deviation or standard error, number of people in the sample, and the tariff used (which country/region) to generate utility weights.Encourage researchers to use and report EQ-5D questionnaire results and avoid reporting only the Visual Analogue Scale (EQ VAS), which is not a preference-based measure.


Here, we show that TNF-α promotes a reduction and expansion in the frequency of both T cell subsets producing IFN-γ and IL-17, respectively. Selective blockade of TNFR1 or TNFR2 on Th1 and Th17 cells revealed that TNFR2 mediates the decrease in IFN-γ production, while signaling through both receptors augments IL-17 production. We also demonstrate that Th1, but not Th17 cells from RA patients present lower levels of TNFR1 compared to healthy controls, whereas TNFR2 expression on both T cell types is similar between patients and controls. Since TNF-α receptors levels in RA patients are not significantly changed by the therapeutic blockade of TNF-α, we propose that targeting TNFR2 may represent an alternative strategy to normalize the levels of key cytokines that contribute to RA pathogenesis.Tumor necrosis factor (TNF)-α is a pleiotropic cytokine implicated in the etiology of several autoimmune diseases, including rheumatoid arthritis (RA). TNF-α regulates diverse effector functions through the activation of TNF-α receptor (TNFR)1 and TNFR2. Although the detrimental role of this cytokine has been addressed in distinct disease settings, the effects of TNF-α on cytokine production by isolated CD4 Based oTNF-α is a pro-inflammatory cytokine that is abundantly present in the inflamed synovium, where it is produced by macrophages, fibroblasts, lymphocytes, and endothelial cells . In RA, TNF-α binds to two transmembrane receptor molecules, TNF-α receptor (TNFR)1 (p55) and TNFR2 (p75). While TNFR1 is expressed at low levels in a wide variety of cell types and tissues, TNFR2 expression is inducible on endothelial and immune cells, such as monocytes, macrophages, natural killer cells, and T and B lymphocytes . TNFR2 iIn the synovium of active RA patients, both TNF-α receptors are upregulated, at the protein and mRNA levels . In addi+ T cell subpopulations remained to be evaluated. Here, we sought to determine the production of IFN-γ and IL-17 by TNF-α-stimulated peripheral Th1 and Th17 cells upon selective blockade of TNF-α, TNFR1, or TNFR2. Our results show evidence for a differential effect of TNF-α on TNFR1 and TNFR2-mediated cytokine production by both T cell subtypes, which may have an impact on RA outcome.Although the effector functions of TNF-α and TNF-α blockade have been extensively approached in several disease settings, the effect of TNF-α neutralization or TNF-α receptors blockade on cytokine production by circulating CD4+ T cells using a RosettSep™ kit (gating strategy shown in + T cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin plus anti-CD28 and anti-CD49d monoclonal antibodies (mAbs), we purified Th1 and Th17 lymphocytes by cell sorting based on their signature cytokines, IFN-γ and IL-17, respectively, applying a cytokine capture assay. Purification of CD4+IFN-γ+ (Th1) and CD4+IL-17+ (Th17) cells yielded >95% enrichment  for 4 days, followed by stimulation with PMA and ionomycin plus brefeldin A to induce cytokine production. Increasing concentrations of TNF-α  were used to stimulate Th1 and Th17 cells. As TNF-α at 1000 ng/mL consistently induced a response on both T cell subsets , we deci+, while the baseline levels of IFN-γ in both T cell subsets did not change in the presence of TNF-α plus infliximab  [Not only did TNF-α reduce IFN-γ production by Th1 and Th17 cells; the cytokine was also able to stimulate both T cell subpopulations to produce IL-17. Although several reports indicate that TNF-α does not participate directly in the differentiation of the Th17 cell profile ,39,40, o7 cells) , suggest7 cells) , thus emTNF-α-mediated induction of IL-17 production by Th17 cells may play a role in the pathogenesis of RA, given that patients with active disease and high levels of TNF-α in the blood present increased levels of IL-17A in the joints and augmented frequency of peripheral and tissue-infiltrating pathogenic Th17 lymphocytes ,44. It i+ T cells producing IFN-γ and IL-17 in the lymph nodes of CIA-stimulated mice lacking TNFR1 was significantly higher than in TNFR2-knockout or wild-type mice, indicating that inhibition of Th1 and Th17 responses is mediated by TNFR1. Conversely, in a mouse model that overexpresses TNF-α and develops spontaneous RA-like lesions in the joints, deficiency of TNFR2, but not TNFR1, led to a more aggressive disease, with exacerbated synovial hyperplasia and enhanced destruction of bone and cartilage [The biological effects of the TNF-α/TNF-α receptors axis on autoimmune diseases, including RA, have been intensively explored. Of importance to our current study is the work of Notley et al. , who havartilage . Moreoveartilage . Accordiartilage . These aartilage  thus proIn this sense, here we observed that, constitutively, Th1 and Th17 cells presented similar levels of TNFR1, whereas TNFR2 expression was higher on Th1 cells compared to Th17 lymphocytes. Interestingly, although TNF-α did not exert a significant effect on the levels of both receptors on the membrane of T cell subsets compared to their baseline expression, this apparent lack of effect does not exclude the possible impact of this cytokine on its cognate receptors’ activation thresholds and/or the triggering of specific intracellular signaling pathways on distinct T cell subpopulations.+ Th1 and IFN-γ+ Th17 cells observed in the absence of TNF-α treatment, suggesting that TNFR2 signaling regulates the TNF-α-induced decrease in the frequency of Th1 and Th17 cells producing IFN-γ. It is important to mention that, even though TNFR2 full activation is mediated by membrane-bound TNF-α [Indeed, our results demonstrate that, under TNF-α stimulation, selective blockade of TNFR2, but not TNFR1, reestablished the baseline proportions of IFN-γnd TNF-α , we cannnd TNF-α , and whind TNF-α . As LT-αnd TNF-α , it woulAs for IL-17, inhibition of either TNF-α receptor individually, in the presence of exogenous TNF-α, restored the baseline proportions of Th1 and Th17 cells producing IL-17, indicating that engagement of TNFR1 and TNFR2 is necessary to achieve the TNF-α-mediated increase in IL-17 production by these T cell subsets. Since neutralization of TNF-α was effective at re-establishing the baseline frequencies of Th1 and Th17 cells that produce IFN-γ and IL-17, blockade of either TNF-α or TNFR2 may be sufficient to revert the TNF-α-induced alterations in cytokine production by these T cell lineages.+ T cells expressing TNFR1 [+ T cells did not differ between RA patients and healthy donors [+ Th1 and TNFR1+ Th17 cells were found to be significantly increased in RA patients compared to healthy controls. In contrast, the percentages of Th1 and Th17 cells expressing TNFR2 were decreased in RA patients compared to controls, although the levels of TNFR2 on these T cell subsets were similar between both groups of individuals.Previous reports have shown that total PBMCs from patients with RA present higher levels of TNFR1 mRNA , as wellng TNFR1 . In turnng TNFR1 ; accordiy donors . Partial+ Th1 and TNFR2+ Th17 cells may be explained by the well-known mechanism of receptor internalization, since stimulation with TNF-α can lead to TNFR1 and TNFR2 endocytosis and further inhibition of their long-term functions [The decreased expression of TNFR1 on Th1 cells and the lower frequency of TNFR2unctions ,53. Howeunctions .+ T lymphocytes that migrate to rheumatoid synovium [Alternatively, the proteolytic shedding of membrane-bound TNF-α receptors ectodomains by the metalloprotease TNF-α converting enzyme  ,55, resusynovium , it woulIn this sense, our study is subject to some limitations. First, we only evaluated the frequency of Th1 and Th17 cells producing IFN-γ and IL-17 by flow cytometry. Although such an approach does not reveal the capacity of these cells to secrete the cytokines analyzed, which would adequately reflect their ability to affect other cells, intracellular cytokine staining provides the possibility to identify which specific cells are producing the cytokine of interest. Here, we aimed at assessing IFN-γ and IL-17-only producers within each isolated T cell subtype, and this can only be achieved on a single-cell basis . Second, although we found no change in the expression of TNF-α receptors on Th1 and Th17 cells in RA patients before and after adalimumab treatment, we did not evaluate whether this conserved phenotype relates to T cell effector functions in these patients. Moreover, our study only focused on the effects of TNF-α treatment and TNF-α receptors blockade on cytokine production by peripheral T lymphocytes derived from healthy donors. Since RA patients present T cells with altered functionality, which contributes to the pathogenesis of RA , we beliAlthough the intervention of RA patients with adalimumab induces rapid and sustained improvements in disease activity and ameliorates physical function , a signi+ T cell subpopulations and on the levels of cytokines they produce to potentially uncover biological mechanisms that may help develop novel therapeutic strategies to treat RA or other autoimmune diseases.Nevertheless, it is important to bear in mind that stimulation of TNFR2 with agonist antibodies has been shown to promote proliferation and activation of regulatory T cells (Tregs) with immunosuppressive function . In addiIn conclusion, the data presented herein suggest that, on one hand, TNF-α affects the phenotype of Th1 and Th17 lymphocytes by decreasing their production of IFN-γ through TNFR2 activation and by stimulating IL-17 synthesis through TNFR1 and TNFR2 signaling, thus proposing that TNFR2 could represent an interesting target to normalize the levels of IFN-γ and IL-17 produced by Th1 and Th17 cells. Hence, TNFR2 neutralization may be taken into consideration when designing TNFR-based biologicals to antagonize the harmful effects of TNF-α in RA. Not only can TNF-α participate in the processes of perpetuation and chronicity of this disease, but also in its upstream pathogenic mechanisms, actively modulating the effector functions of key subsets of T cells through differential actions on its receptors.Nine healthy donors  aged 48 ± 7 years  and ten female RA patients aged 52 ± 11 years  were included in this study. Patients were recruited at the Hospital Clínico, Universidad de Chile. At the time of study entry, patients presented with RA duration of 15 ± 7 years , and none of them had received biologic disease-modifying anti-rheumatic drugs. RA characterization was defined according to the American College of Rheumatology (ACR) criteria . All patBuffy coats from healthy volunteers were provided by the Hospital Clínico, Universidad de Chile. Peripheral blood samples from RA patients and controls were used for PBMCs isolation by density gradient centrifugation using Ficoll-Hypaque . PBMCs were cryopreserved in liquid nitrogen in fetal bovine serum (FBS)  and 10% dimethyl sulphoxide, until use.+ T cells by negative selection using the RosetteSep™ Human CD4+ T cell enrichment Kit , according to the manufacturer’s instructions. Enrichment of CD4+ cells was assessed by flow cytometry using a PECy7-conjugated anti-CD4 mAb (clone OKT4), and antibody specificity was validated with a PECy7-conjugated mouse IgG2b, κ isotype control . For the isolation of specific cytokine-producing T cells, cytokine capture assays were performed [+ T cells were stimulated with soluble anti-CD28 (0.5 mg/mL) and anti-CD49d (1 mg/mL) mAbs  plus 15 ng/mL of PMA and 1 µg/mL of ionomycin  for 3 h and 30 min at 37 °C and 5% CO2 to induce cytokine production. Cells were washed with 1% FBS in phosphate-buffered saline (PBS) and incubated in the dark, for 12 h at 4 °C, with anti-CD45-IFN-γ-biotin-avidin or anti-CD45-IL-17-biotin-avidin antibody conjugates (both from Thermo Fisher Scientific) to obtain IFN-γ and IL-17-secreting CD4+CD45+ T cells, respectively. Cells were then stained with anti-IFN-γ-FITC and anti-IL-17-PE mAbs (both from Thermo Fisher Scientific) for 30 min at 4 °C in the dark for further Th1 and Th17 cell sorting, respectively, using a BD FACSAria III cell sorter .Buffy coats from healthy donors were used to enrich CD4erformed . For thiThe purity of sorted Th1 and Th17 lymphocytes was evaluated by flow cytometry following cell permeabilization with the BD Cytofix/Cytoperm™ kit (BD Biosciences) for 20 min plus intracellular staining with anti-T-bet-PerCP/Cy5.5 and anti-RORγt-APC mAbs (both from Thermo Fisher Scientific) for 30 min at 4 °C in the dark. Cells were then analyzed by flow cytometry with a FACScalibur flow cytometer (BD Biosciences).4 cells/well in 200 µL of X-Vivo 20 medium , in flat-bottomed 96-well plates and cultured for 4 days, at 37 °C and 5% CO2, with recombinant human TNF-α (1 µg/mL) (Thermo Fisher Scientific) in the presence or absence of an anti-TNF-α blocking mAb (infliximab) (10 µg/mL) , Schering-Plough, Kenilworth, NJ, USA). Next, cells were stimulated with 15 ng/mL of PMA and 1 µg/mL of ionomycin plus 10 µg/mL of brefeldin A (Thermo Fisher Scientific) for 5 h, after which they were harvested, washed, permeabilized with a fixation and permeabilization kit (Thermo Fisher Scientific), and incubated with anti-IFN-γ-FITC and anti-IL-17-PE mAbs for 30 min at 4 °C in the dark. Cells were further analyzed by flow cytometry using a FACScalibur flow cytometer. Unstained cells were acquired to control for the absence of carryover fluorescence from the cell sorting procedure.To evaluate the effect of TNF-α on intracellular cytokine production, Th1 and Th17-sorted cells were resuspended and distributed, at a density of 7.5 × 10® staining kit (ThermoFisher Scientific).All data from cell sorting and flow cytometry were acquired with the CellQuest program (BD Biosciences) and evaluated with the Weasel Software . The lymphoid population was defined according to forward and side scatter patterns. In all experimental settings, dead cells were excluded from analysis using the LIVE/DEADTo determine TNF-α-mediated apoptosis in cell cultures, Th1 and Th17-sorted lymphocytes were resuspended and cultured, for 4 days, with TNF-α (1 µg/mL) in the presence or absence of infliximab (10 µg/mL). Cells were then labeled with PE-conjugated annexin-V and 7-AAD (both from Thermo Fisher Scientific), and cell viability was immediately determined by flow cytometry.To evaluate the levels of TNFR1 and TNFR2 on the cell membrane, in the presence of TNF-α, purified Th1 and Th17 lymphocytes were maintained for 24 h in culture medium prior to a 24 h stimulus with 1 µg/mL of TNF-α. Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803)  and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark. A mouse IgG1κ mAb (clone P3.6.2.8.1) and a rat IgG2bκ mAb (clone eB149/10H5) (both from ThermoFisher Scientific) were used to test anti-TNFR1 and anti-TNFR2 mAbs specificity, respectively. Cells were further analyzed by flow cytometry, as previously described.For TNF-α receptors blockade assays, purified Th1 and Th17 cells were incubated with 5 ng/mL of either an unconjugated mouse anti-human TNFR1 mAb (clone 16803) or an unconjugated mouse anti-human-TNFR2 (clone 22210) (both from R&D Systems) for 1 h. An IgG1κ isotype control (clone 11711) (R&D Systems) was used to rule out non-specific effects of the neutralizing antibodies. Cells were then stimulated with 1 µg/mL of TNF-α for 4 days, after which intracellular staining of IFN-γ and IL-17, followed by analysis by flow cytometry, were performed.+CCR6-CXCR3+CD161- Th1 cells and CD4+CCR6+CXCR3-CD161+ Th17 lymphocytes.To analyze the levels of TNFR1 and TNFR2 on the membrane of Th1 and Th17 cells present in the blood of RA patients treated with adalimumab, PBMCs from RA patients and healthy donors were incubated with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems), a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences), a mouse anti-human CD161-FITC mAb (clone HP-3G10), a mouse anti-human CCR6-PerCP/Cy5.5 mAb (clone G034E3), and a mouse anti-human CXCR3-APC/Cy7 mAb (clone G025H7)  for 30 min at 4 °C in the dark. TNF-α receptors levels were evaluated, by flow cytometry, on CD4P-values lower than 0.05 were considered statistically significant. For all analysis, GraphPad Prism v7.04  was employed.Normality was evaluated by the Kolmogorov–Smirnov test. Significance was determined using either the Mann–Whitney test, Kruskal–Wallis test with Dunn’s multiple comparison post hoc test, or one-way ANOVA with Tukey’s multiple comparison test, all with a confidence interval of 95%.


However, to date, approved drugs targeting TNF‐α are restricted to biological macromolecules, largely due to the difficulties in using small molecules for pharmaceutical intervention of protein–protein interactions. Herein the power of a natural product‐enriched DNA‐encoded library (nDEL) is exploited to identify small molecules that interfere with the protein–protein interaction between TNF‐α and the cognate receptor. Initially, to select molecules capable of binding to TNF‐α , “late‐stage” DNA modification method is applied to construct an nDEL library consisted of 400 sterically diverse natural products and pharmaceutically active chemicals. Several natural products, including kaempferol, identified not only show direct interaction with TNF‐α, but also lead to the blockage of TNF‐α/TNFR1 interaction. Significantly, kaempferol attenuates the TNF‐α signaling in cells and reduces the 12‐O‐tetradecanoylphorbol‐13‐acetateinduced ear inflammation in mice. Structure‐activity‐relationship analyses demonstrate the importance of substitution groups at C‐3, C‐7, and C‐4' of kaempferol. The nDEL hit, kaempferol, represents a novel chemical scaffold capable of specifically recognizing TNF‐α and blocking its signal transduction, a promising starting point for the development of a small molecule TNF‐α inhibitor for use in the clinical setting.Tumor necrosis factor  nDEL) approach, merging natural products with DEL, is applied in a reverse pharmacology study targeting tumor necrosis factor α (TNF‐α), a key cytokine in inflammation. Several natural products, particularly kaempferol, a flavonoid, has been identified to recognize and attenuate the TNF‐α signaling in cells as that of adalimumab, and reduce 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced ear inflammation in mice.The natural product‐enriched DNA‐encoded library ( However, this major advancement does itself bring new challenges in terms of library expansion and DEL selection. The ability to select functional molecules using DEL technology is undoubtedly dependent upon both the total number and the scaffold diversity of members comprising the initial DEL library. Academic laboratories and pharmaceutical corporations are continually developing new DNA‐compatible chemical reactions, leading to a significant increase in the number of chemical classes that can now be included in DELs. At the same time, new selection strategies, especially for membrane‐bound protein targets, are being developed. Consequently, more and more DEL‐derived new drug candidates are entering clinical development.DNA‐encoded chemical libraries (DELs) link the power of chemistry and genetics and have revolutionized drug discovery.1 DELsα (TNF‐α), a crucial regulator in the immune response, has been the target of multiple approved antibody drugs such as adalimumab, golimumab, and certolizumab. TNF‐α antagonism has shown therapeutic efficacy for several autoimmune diseases such as rheumatoid arthritis (RA), psoriatic arthritis, multiple sclerosis (MS), and Crohn's disease. Nevertheless, only 60–70% of patients achieve a long‐term clinical response. In the remaining patients, the poor response to antibody therapies is thought to be at least partially due to the immunogenicity of these biological molecules. This, in combination with other shortcomings of bio‐macromolecular drugs, such as high cost (for chronic usage), poor stability, and the requirement for parenteral administration, has prompted increasing interest in the development of orally bioavailable small molecule inhibitors targeting TNF‐α.Tumor necrosis factor olizumab.6 TNF‐ Many highly effective drugs are, in fact, derived from natural products, that is, Taxol and artemisinin. However, due to their polypharmacology, the mechanisms of action for most therapeutic natural products remain largely unclear. Although the polypharmacology of natural products hinders their further pharmaceutical development, it makes them ideal tools for chemogenomic studies. By merging natural products with DEL technology, one is able to fully leverage the power of natural selection in evolution. We developed a late‐stage DNA‐coding method that allowed us to generate a natural product‐enriched DEL library (nDEL), in which molecules with biological activities are DNA‐labeled and can be profiled via affinity panning.Over the course of evolution, natural products, including the active ingredients in Traditional Chinese Medicines (TCMs), have developed with highly diverse and complicated chemical scaffolds. These molecules have been extensively studied and used in the clinical treatment of disease for thousands of years.11 MannDEL, an approach that combines the advantages of both structural diversity and polypharmacological function, to probe small molecule binding pockets on TNF‐α. A natural product, kaempferol, was selected and shown to not only bind TNF‐α but also disrupt the interaction between TNF‐α and its cognate receptor TNFR1. It was capable of potent inhibition of both TNF‐α‐induced cell death and the corresponding signaling pathways in L929 cells. Furthermore, studies using a murine ear model demonstrated that kaempferol reduced inflammation in vivo.In this study, we used the 22.1α (hTNF‐α) was purified with Ni‐NTA and size‐exclusion chromatography (SEC)  with an SEC retention volume close to that of BSA (MW: 66.4 kDa)  was used to measure the affinity of the interaction. A CM5 chip was used with a running buffer consisting of 1% DMSO. Recombinant hTNF‐α first flowed through and immobilized on the CM5 chip via amine coupling to form a uniform single molecular layer of hTNF‐α on the chip surface. Of the eight nDEL hits, all showed some degree of interaction with the hTNF‐α coating surface  and kumatakenin B (KB), showed similar KD values of 45 ± 5.5 and 15 ± 5.8 µm, respectively; whereas, the natural product, gancaonin I (GCN), and the antibiotic, moxifloxacin hydrochloride (MHCl), showed more potent binding with KD values of 1.9 ± 0.2 and 7.8 ± 1.5 µm, respectively , galangin (GLG), kaempferide (KMF), and 4',5‐dihydroxyflavone (DHF) all interacted with the recombinant TNF‐α. Compared to Kae, removing any of the —OH group at the C3 , C7 , or C4’  position resulted in improved binding to hTNF‐α. In contrast, methylation at C3 and C7  or at C4’  had little effect on the binding affinity , a competitive affinity assay was performed using ELISA. First, the cognate ligand‐receptor interaction was established in a 96‐well microtiter plate using recombinant hTNF‐α and HRP‐tagged hTNFR1 extracellular domain (hTNFR1‐ECD‐HRP)  with test compounds at various concentrations in a 100 µL assay solution, followed by incubation in a 96‐well microtiter plate coated with hTNF‐α . In the presence of TNFR1, the non‐flavonoid compounds, MHCl and GCN, showed either no or greatly reduced (>5000‐fold less) binding potency, respectively  did not affect the potency of competitive binding, whereas removal of either the –OH at C3  or at C7  resulted in a potency decrease of greater than two orders of magnitude  or removal  of the –OH substitution at C4’ both resulted in a modest 2–5 fold reduction in binding potency  stimulation of cell survival and expression of pro‐inflammatory genes or b) apoptosis and cell death. TNF‐α‐induced activation of the caspase cascade represents a hallmark of TNF‐α signaling leading to programmed cell death (apoptosis). We further investigated if KB and Kae rescued cells from TNF‐α‐induced cell death by inhibiting the caspase cascade activation in L929 cells. The intrinsic effects of the tested compounds on the caspase cascade were first ruled out  of caspase cascade activation at 100 μm.TNF‐optosis).20 We α signaling and is exerted by the activation of key transcription factors such as NF‐κB and AP1. NF‐κB activation results in the degradation of IκBα protein, a negative regulator of NF‐κB, in cells. We examined the ability of Kae and KB to inhibit TNF‐α‐induced NF‐κB activation by monitoring IκBα degradation in L929 cells. The intrinsic effects of the tested compounds on IκBα degradation were first ruled out . Moreover, when compared to the background control, MHCl showed no inhibition of TNF‐α‐induced IκBα degradation at 100 μm.Induction of mitogenesis is another hallmark of TNF‐in cells.21 We 2.6Kae and KB, an in vivo, phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced skin edema model was established in C57BL/6 mice. TNF‐α is a key factor involved in TPA‐induced dermatitis, especially at the stage where dermal edema is significant. Topical application of TPA has been shown to induce TNF‐α expression at the application site. Exposure of a mouse ear to 2.4 µg TPA (in neat DMSO) for 30 min led to a significant increase in ear thickness and tissue weight , a COX inhibitor, was used as a positive control for anti‐inflammation. In a typical experiment, Indo (0.5 mg in neat DMSO) or the test compound, Kae , was applied topically to the TPA‐treated area at 30 min and 3.5 h after TPA exposure. 3 h following the second application of Indo or Kae, tissue samples were collected for analysis. In experimental mice (four mice/cohort group), the contralateral ear served as an internal background control and was not treated with either TPA or test compounds. As shown in Figure Indo or 0.5 mg Kae  were measured, respectively. TPA‐treated mice had an increased level of neutrophils in their peripheral blood, as well as increased mRNA expression of TNF‐α, IL‐1α, and CXCL2 in the tissues of the treated ear. Application of Kae following TPA exposure completely suppressed the increase in peripheral neutrophils and the increase in mRNA expression of all three cytokines , and cancer.14a Naα before. In the current study, we took advantage of the polypharmacological characteristic of an nDEL library consisting of 400 biologically active compounds  mouse model. In addition, it has a high safety profile in terms of both liver and renal side effects. Our current study established that kaempferol can exert its anti‐inflammatory effect via interruption of TNF‐α cell signaling similar to Humira .DEL approach was successfully applied in an attempt to identify novel small molecule inhibitors of TNF‐α before.24 In MHCl, a quinolone antibiotic, is known to potently inhibit the enzymatic activities of bacterial DNA gyrase and topoisomerase IV leading to strong antimicrobial activity. Its off‐target inhibition of mammalian topoisomerase II at high concentration (>100 μm), on the other hand, is believed as one of the important factors causing various adverse events of MHCl such as cardiac, hepatic, and cutaneous toxicities. Our observation that MHCl is a potent TNF‐α binder (apparent KD = 7.8 μm) provides new insight into the inflammatory nature of this molecule. Interestingly, our study showed that the binding of MHCl to TNF‐α did not interfere with the TNF‐α‐TNFR1 signaling. MHCl showed only partial inhibition of TNF‐α induced caspase cascade with no inhibition of TNF‐α induced NFκB activation , could serve as an invaluable polypharmacological probe in chemogenomic research. As a pilot study for proof‐of‐concept, TNF‐α, a key cytokine in inflammatory responses, was used as the target protein. A natural‐product small molecule binder of TNF‐α, Kae, was identified by nDEL panning and showed impressive inhibitory efficacies on TNF‐α induced cell signaling both in vitro and in vivo similar to that of the known anti‐inflammatory antibody drug, adalimumab (Humira). Preliminary SAR analysis of kaempferol analogs showed potential to develop an “Oral Humira” by further improving the potency and efficacy of Kae upon structural modification. Moreover, the identification of TNF‐α as one of the cellular targets for MHCl, a quinolone antibiotic, provides a plausible mechanism accounting for some of its adverse effects. The nDEL approach, thus, is undoubtedly a powerful tool for next‐generation pharmaceutical discovery and development.In this study, we demonstrated that DEL technology, in combination with natural products and functional small molecules , or human TNF‐α gene  was PCR amplified from Hela cell cDNA and cloned into a pET‐28a expression vector (Novagen) with an N‐terminal 6 × His tag. The plasmid was transformed into BL21(DE3) cells in a 2 L LB media at 37 °C and 220 rpm, induced with 0.4 mm IPTG after reaching an OD600 (0.8) and expressed at room temperature overnight. The resulting cells were pelleted by centrifugation, and re‐suspended in a 100 mL ice‐cold buffer , and lysed by ultrasonication. The recombinant hTNF‐α was overexpressed as soluble proteins, whereas hTNFR1‐ECD formed inclusion bodies.The coding sequences of the extracellular domain of the human TNF‐hTNF‐α were biotinylated using the EZ‐LINK NHS‐PEG4‐BIOTIN kit  following the manufacturer's instruction. Briefly, the above recombinant hTNF‐α was incubated with newly reconstituted sulfo‐N‐hydroxysuccinimide (NHS) esters of biotin (molar ratio of 1:20) at 4 °C for 2 h. Excess biotin was then removed using a desalting column .Next, primary amines (‐NH2) on hTNFR1‐ECD inclusion bodies were pelleted twice by centrifugation at 4000 g for 15 min at 4 °C, dissolved in a 40 mL denaturing solution  for 40 min at room temperature, and kept in the denaturing solution supplemented with 20 mm DTT. After 30 min incubation, 30 mm oxidized glutathione was added. After an additional 30 min incubation, the resulting solution was slowly diluted ten‐fold with a buffer , and incubated overnight with gentle stirring at 4 °C. The final solution was centrifuged at 20 000 g for 20 min at 4 °C to remove insoluble debris, dialyzed twice against a 4 L 50 mm Tris buffer (pH 7.5) at 4 °C, and dialyzed twice again with a 4 L buffer  at 4 °C.The hTNF‐α and the renatured hTNFR1‐ECD were subject to affinity column purification. For typical affinity purification, insoluble debris was first removed by centrifugation. The supernatant was loaded to a Ni‐NTA agarose (GE) column, rinsed with a washing buffer , and eluted with an elution buffer . The elution fractions containing the desired protein were combined, dialyzed to remove imidazole, concentrated by ultrafiltration, aliquoted, and stored at −80 °C.The supernatant containing recombinant hTNFR1‐ECD was further conjugated to horse‐radish peroxidase using HRP Conjugation Kit  aliquoted, and stored at −20 °C.The purified hTNF‐α was dissolved at 30 µg mL−1 in an acetate buffer (pH 4.5), and coupled to a CM5 chip  following the manufacturer's instruction. The running buffer  contained 1% instead of 5% DMSO in order to keep the activity of hTNF‐α  and injected at a flow rate of 30 µL min−1 for 90 s for the association step followed by dissociation for an additional 90 s using the LWM multi‐cycle kinetics/affinity method provided by GE Healthcare. Solvent correction was carried out before and after each analysis with eight different concentrations of DMSO solution per cycle. The KD value was calculated using Evaluation Software .Binding affinity of small molecule to protein was measured on a BIAcore 8K instrument  by SPR assay. For a typical assay, purified −1 avidin  in a 50 mm sodium carbonate buffer (pH 9.0) at 4 °C. After removal of the coating solution, the plates were incubated at 275 µL/well with a blocking solution containing 5% milk v/v in a PBST buffer (pH 7.4) at 37 °C for 1 h. The resulting plates were rinsed once with PBST and incubated at 100 µL/well with a 7.5 nm sparsely biotinylated hTNF‐α solution  for 1 h with shaking. After incubation, plates were rinsed four times with a washing buffer .Microtiter plates (Nunc 96F Maxisorp) were coated overnight with 100 µL/well of 10 µg mLm to 1 nm using a solution containing 25 mm MES (pH = 6.2), 25 mm NaCl, 1% DMSO, and 4.6 nm TNFR‐HRP. The final dilution mixture was added to each well of the above plates (100 µL/well) and incubated with shaking for 90 min. Then the plates were washed three times with PBST followed by the addition of 50 µL TMB peroxidase substrate of HRP  into each well. After incubation at room temperature for 30 min, the reaction was quenched with 2 m sulfuric acid, and absorbance at 450 nm was measured on a microplate reader .Testing compounds were 1:10 serial diluted from 100 µα induced cell apoptosis measurement, L929 cells that overexpress TNFR1 receptor were seeded at a density of 104 cells/well in a 96‐well microtiter plate and incubated for 24 h in a DMEM media with 10% FBS at 37 °C. The overnight L929 cells were switched into a fresh media containing 0.3 µg mL−1 actinomycin D, 1 ng mL−1hTNF‐α, and/or 10 nm adalimumab (positive control), and/or indicated concentrations of various compounds (in 1% DMSO), and incubated at 37 °C for an additional 20 h. Cell survival was measured by the CCK8 assay . Cells without hTNF‐α or any testing compound were used as blank, while cells with only hTNF‐α were used as the negative control.The Cell Counting Kit‐8  was applied to measure cell proliferation and survival. For TNF‐4 cells/well L929 cells were seeded in a 96‐well microtiter plate and incubated in a DMEM media with 10% FBS at 37 °C for 24 h. Compounds at different concentrations were next added and incubated for an additional 20 h at 37 °C. Cell survival was measured by CCK8.For cytotoxicity assay of compounds, 104 cells/well in a 96‐well plate and incubated in a DMEM media with 10% FBS at 37 °C for 24 h. The overnight L929 cells were incubated into a fresh DMEM media containing 0.3 µg mL−1 actinomycin D, 1 ng mL−1hTNF‐α, and 50 µmKB or Kae (in 1% DMSO), or 100 µmMHCl (in 1% DMSO), or 20 µmGCN (in 1% DMSO), or 10 nm adalimumab , and incubated at 37 °C for an additional 14 h. Cells in each well were lysed, and the cellular proteins were extracted and subject to analysis using BCA Assay Kit . The amount of β‐actin, total Caspase‐3, Cleaved Caspase‐3, and IκBα was determined by western‐blot analysis using an anti‐β‐actin antibody , anti‐Caspase‐3 antibody , anti‐Cleaved Caspase‐3 antibody , and anti‐IκBα antibody , respectively.L929 cells were seeded at a density of 10All animal experiments were approved by the Institutional Animal Care and Use Committee of ShanghaiTech University. Female C57BL/6 mice (6‐ to 8‐week‐old) were purchased from Shanghai Model Organisms Center. All animals were maintained in a pathogen‐free, temperature‐controlled environment with a 12 h light/dark cycle and provided chow and water ad libitum. All animals were allowed to acclimate for 1 week in the animal facility before handling.Kae, and indomethacin were dissolved in neat DMSO (vehicle). Atopic treatment (10 µL in volume) was carried out on the inner and outer surfaces of one ear for each mouse. Acute irritant contact dermatitis was induced by 2.4 µg TPA. After 30 min, vehicle, Indo (0.5 mg), and Kae (0.5 mg) were applied to the TPA treated mouse ear of each cohort group, respectively. After an additional 3 h, a second atopic treatment was carried out to the corresponding mouse ear of each cohort group. After an additional 3 h, mice were sacrificed. Peripheral blood was collected from the canthus vein of mice. Φ 6 mm ear punch biopsies were taken for ear thickness measured with a thickness gauge and for weight. Ear biopsies were stored at −80 °C for the following gene expression experiments.Mice were randomly divided into different cohort study groups. TPA, Biopsies from negative control and treated mice ears in each group were collected and fixed in 4% paraformaldehyde solution overnight at 4 °C. The tissues were embedded in paraffin and then sectioned at a thickness of 6 µm. Tissue sections were stained with hematoxylin and eosin prior to observation with a light microscope.Peripheral blood was collected from each mouse and treated with red blood cell (RBC) lysis buffer . Whole cells except erythrocytes were collected by centrifuged at 500 g for 10 min and incubated with anti‐Mo Ly‐6G/Ly‐6C  antibody for 20 min. Cells were washed several times and analyzed on a flow cytometer .α, IL‐1β, and CXCL2 were measured and normalized to the internal control genes (β‐actin). The primer sequences are listed in Table The above ear biopsies were lysed by TRIzol Reagent (Invitrogen) regent and homogenized by a homogenizer . Total RNA was obtained from lysed cells and then reverse‐transcribed into cDNA using the Vazyme kit (R212‐02) following the manufacturer's protocol. RT‐qPCR was performed in triplicates with 2 × SYBR Green qPCR Master Mix  on CFX384 Real‐Time System (BIO‐RAD). Messenger RNA expression levels of TNF‐IC50 calculation. Significance was assumed at a p‐value <0.05 by using a two‐tailed Student's t‐test or one‐way ANOVA for comparisons of two groups or more than two groups, respectively, in the software for normally distributed data sets with equal variances, .Experiments were repeated at least three times, and the results were expressed as means ± standard deviation (S.D.) unless otherwise indicated. Sample numbers are indicated in figure legends. Data analysis was performed with GraphPad Prism software. Four parameter non‐linear regression was used for fitting and The authors declare no conflict of interest.S.W., X.S., and J.L. contributed equally to this work. Prof. Richard A. Lerner directed and provided insight and method to this study, a work he viewed highly for DEL application before his passing away. S.W. performed the selection, the biochemical experiments, in vivo experiments, and analyzed the data. J.L. synthesized the DEL library and contributed to the SPR experiments. Q.H., X.S., Q.J., L.L., Y.Y., and Y.D. helped in the experiments. X.S. and T.W. helped in the in vivo experiments. M.Y. provided the GCN chemicals. S.W., P.M., H.X., and J.L. analyzed the data. P.M., H.X., and G.Y. designed the study and wrote the manuscript. All authors read and approved the final manuscript.Supporting InformationClick here for additional data file.Suplemental Table 4Click here for additional data file.


Heart Failure (HF) is the most common cause of hospitalization in the Western societies. HF is a heterogeneous and complex syndrome that may result from any dysfunction of systolic or diastolic capacity. Abnormal diastolic left ventricular function with impaired relaxation and increased diastolic stiffness is characteristic of heart failure with preserved ejection fraction (HFpEF). HFpEF accounts for more than 50% of all cases of HF. The prevalence increases with age: from around 1% for those aged <55 years to >10% in those aged 70 years or over. Nearly 50% of HF patients have HFrEF and the other 50% have HFpEF/HFmrEF, mainly based on studies in hospitalized patients. The ESC Long-Term Registry, in the outpatient setting, reports that 60% have HFrEF, 24% have HFmrEF, and 16% have HFpEF. To some extent, more than 50% of HF patients are female. HFpEF is closely associated with co-morbidities, age, and gender. Epidemiological evidence suggests that HFpEF is highly represented in older obese women and proposed as ‘obese female HFpEF phenotype’. While HFrEF phenotype is more a male phenotype. In addition, metabolic abnormalities and hemodynamic perturbations in obese HFpEF patients appear to have a greater impact in women then in men , 22. To  Worldwide there are increasing number of patients with signs of cardiac decompensation such as dyspnea, lung congestion and ankle oedema, fatigue and exercise intolerance, and objective proof of heart failure (HF) such as increased B-type natriuretic peptide (BNP) level, while the left ventricular (LV) ejection fraction (EF) is normal . According to the actual HF guidelines both in Europe  and UnitDespite the enormous studies that attempted to cover the cellular and molecular basis of HFpEF development, a full understanding of its pathophysiology remains elusive. Consequently, the pharmacological management of the disease includes agents that counteract the deleterious effects of the maladaptive compensatory mechanisms, in the absence of evidence-based treatment .The lack of a comprehensive and detailed understanding of the underlying mechanisms stems from the complex interplay between multiple factors that are involved in the pathophysiology of HFpEF. Such multi-factorial contribution gives rise to the heterogenic nature of disease presentation and progression among patients . AlthougSeveral studies have reported different profiles in patients with HFrEF and HFpEF . HFpEF iDiabetes mellites is often prevalent in HFpEF patients. In addition to the systemic inflammatory state and increased oxidative stress, diabetes can contribute to the development of the disease through other pathways such as accelerating atherosclerosis, thereby leading to myocardial ischemia . The proVisceral adipose tissue is associated with high levels of cytokines secretion . MoreoveHypertension coexists with HFpEF and found to be a risk factor for increased LV afterload and pressure-induced hypertrophy. It correlated also with chronic proinflammatory state, arterial stiffness, and titin-dependent stiffness . FurtherThe prevalence of HFpEF increases with age. Like hypertension and diabetes, aging is linked to elevated ventricular-vascular stiffening , which lThe incidence of HFpEF is higher in women , indicat. Similarly, diastolic dysfunction is present in the vast majority of HF patients  is highly beneficial in HFrEF, but appears to be less important in HFpEF  and AT1-blockers (ARB) in HFpEF patients may have an important impact. In a meta-analysis  higher dA hallmark in myocardial cells obtained from animal models of HFpEF  with diastolic dysfunction or human heart biopsies from patients with HFpEF appears to be a reduced activity of protein kinase G (PKG), possibly because of increased inflammatory and oxidative stress . It is tThe RELAX trial  randomizAn alternative approach for boosting the cGMP-PKG pathway may be a therapy with the direct stimulator of the sGC vericiguat. In a phase II dose-finding study, the effect of vericiguat (1.25 up to 10 mg/d for 12 weeks) on N-terminal pro-B type natriuretic peptide (NT-proBNP) blood levels was studied in 351 patients with HFrEF  . The treAs a matter of fact, HFpEF clinical trials conducted to date have shown some important inconsistencies regarding inclusion criteria that per se implicate the complexity and clinical heterogeneity of HFpEF patients. On the other hand, non-uniform trial designs also limit the comparison of the outcomes of certain drugs/classes. For this reason, different scoring systems have been proposed to help the diagnosis . NeverthWithin the clinical heterogeneity of HFpEF , prevaleFurthermore, and as previously discussed in this review, comorbidities and risk factors are major determinants of HFpEF prognosis. Hence, HFpEF treatment must include lifestyle interventions for comorbidities and risk factors management. In HFpEF patients, exercise intolerance is a primary symptom associated with low quality of life. In the Inspiratory Muscle Training and Functional Electrical Stimulation for Treatment of HFpEF (TRAINING-HF) Trial, HFpEF patients who received inspiratory muscle training (IMT) and functional electrical stimulation (FES), exhibited an increase in peak exercise oxygen uptake and improved quality of life , suggestFunctional and structural changes underlying HFpEF are partially due to the clustering of risk factors that lead to endothelial dysfunction, myocyte dysfunction, tissue fibrosis, altered cellular signaling, abnormal energetics, myocardial ischemia, systemic inflammation. Various molecular and cellular mechanisms leading to the development and progression of HFpEF and epigenetic regulation may play an importnat role in the pathogenesis of HFpEF. Different mechanisms of epigenetic modifications may relate to various HF-depended settings, i.e. cardiac hypertrophy, myocardial infarction, increase in susceptibility to ischemic injury, fetal phenotype of cardiac tissue, reduce pump function, atrial fibrillation, and sudden death, and play a controversial role. For example epigenetic modifications affecting DNA methylation , ATP-depOxidative stress and inflammation are evident in the pathophysiology of HFpEF . The oxi2O2), nitroxyl and NO , and cytochrome p450 . Severall and NO .ROS can directly impair contractile function by modifying proteins involved in excitation-contraction coupling and myocyte contraction. These modifications include disulphide bridges, S-glutathionylation, S-nitrosylation and tyrosine nitration and chlorination leading to changes in protein function and activity . In addiTaken together, oxidative stress drives cardiomyocytes, endothelial cells, and ECM dysfunction. All of which are major determinants of diastolic dysfunction in HFpEF patients and animal models of HFpEF .The impaired LV filling that occurs in HFpEF patients is influenced by several factors, with ventricular stiffness playing a central role in the development of diastolic dysfunction. Conceptually, a differentiation can be made between ECM-based and cardiomyocyte-based stiffness. Studies showed, that there is an interplay between ECM and cardiomyocytes . Therefovia a crosstalk that leads to cardiac remodeling and HF , viral myocarditis or ischemia, and is therefore associated with biomechanical cardiac stress markers such as BNP . In contDuring inflammation, proinflammatory cytokines are released in the renal or pulmonary vasculature due to increased arterial pressure, which has been studied in hypertensive HFpEF patients and also in animal models. Similarly, the release of proinflammatory cytokines and their increase in serum are observed in patients with obesity. Here, cytokine release is mediated by infiltration of monocytes and macrophages into the increased visceral white adipose tissue . In typeThe release of proinflammatory cytokines also leads to ROS and NOS production by macrophages and monocytes, which causes an increase in oxidative stress. This oxidative stress induces mitochondrial damage, resulting in increased ROS production. In addition, mitochondrial damage also has significant effects on energy provision processes and programmed cell death (apoptosis) .During the inflammation process, proinflammatory signaling is triggered. A common cause associated with cormobidates is metabolic stress (metabolic load). Systemic inflammation is manifested by an increase in plasma biomarkers such as CRP (C-reactive protein), TNFα, TNFαR1 (tumor necrosis factor α receptor 1), GDF15 (growth differentiation factor 15), IL6, soluble IL1R1 (interleukin 1 receptor 1), IL1RL1 (interleukin 1 receptor like 1), and integrin subunit beta 2. These biomarkers promote systemic inflammation in HFpEF, and are therefore associated with the disease .Systemic inflammation also triggers the expression of endothelial adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule 1(VCAM-1), the latter being an important regulator involved in inflammation . VCAM-1 In addition to metabolic stress, hemodynamic stress is also an important contributor to the initiation of proinflammatory processes. Hemodynamic stress is caused by arterial hypertension, while profibrotic signaling pathways are activated in addition to proinflammatory cascades, as reflected by differences in biomarkers , such as proteins, peptides, and microRNAs (miRNAs) .The signaling pathways are regulated by microRNAs, which in turn are controlled by alterations in the balance of histone acetylation (HDAC). Moreover, cardiac-specific miRNAs play an important role in cardiac remodeling processes leading to HF phenotypes. Specific HF phenotypes, e.g., HFpEF or HFrEF, might be distinguished by differences in the expression of miRNA-1, miRNA-29, miRNA133, and miRNA-208, which are due to an imbalance in regulatory mechanisms. In this context, miRNAs play a crucial role as gene regulators in the control of inflammation, as they are thought to act as signaling regulators to fine-tune inflammatory responses and prevent their uncontrolled progression .Proinflammatory signals triggered by hemodynamic stress release cytokines or chemokines that induce infiltration of the myocardium by monocytes, T/B cells, and activated macrophages. Macrophage activation leads to collagen formation and fibroblast activation, resulting in remodeling of the interstitial ECM and stiffness of the myocardium. Structural remodeling of the basal lamina includes changes in laminin isoform and increases in perlecan, nidogen, and collagen IV .While the ECM-based changes are likely to be the main factor in the advanced stages of the disease, the cardiomyocyte-based changes occur in the early stages of HFpEF . A commoFurthermore, the phosphorylation of titin plays a significant role in the modulation of passive stiffness. Not only the kinase, but also the region of phosphorylation has a major influence. While phosphorylation of the N2Bus region reduces stiffness, passive tension is increased by phosphorylation of the PEVK region. These opposite effects occur, because the negatively charged phosphate group increases the negative charge by the acidic amino acids of the N2Bus region and thus increases elasticity. The negative charge of the phosphate group, on the other hand, reduces the positive charge of the PEVK region caused by basic amino acids and decreases elasticity . So far,It has been evident that oxidative stress and inflammation drive HFpEF development through detrimental effects on protein signalling that regulate cardiomyocyte function  Zhazykb.passive in hypertrophic cardiomyopathy and HFpEF cardiomyocytes from patients and animal models, suggesting the oxidative stress-induced modifications in PKG pathway to play a crucial role in HFpEF and hypertrophic cardiomyopathy pathology , sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA), ryanodine receptors (RyR), cTnI, cMyBPC, and titin. PKA dependent phosphorylation of cTnI and cMyBPC is responsible for myofilament Ca2+ desensitization and the subsequent Ca2+ reuptake into the sarcoplasmic reticulum  as well . TherebyThis EA/EEs ratio is reduced in HFpEF and hypertensive patients compared to a young control group . HoweverThe interaction of LV systolic stiffening and arterial stiffening increases therefore systolic workload, prolongs relaxation, impairs filling and increases end-diastolic pressure (EDP), which in turn impairs diastole . Besidesß (TGF-β). This promotes furthermore the development of fibrosis (In general, the ECM is not only important for structural support and maintenance, but also influences cardiac homeostasis and is responsible for force transmission and transducing key signals to cardiomyocytes . Under nfibrosis . In addifibrosis . In summfibrosis .The present review provides an overview about the current knowledge of HFpEF pathophysiology. As previously described, the guideline-directed medical therapy is the only existing option for disease management . Better


Then, we analysed whether papers included specific recommendations for natural system management, nature conservation, policies or governance structures, or science in general. Besides, we studied whether the papers addressed socio-ecological studies related to flora and fauna and from what specific group of organisms or systems. Data were compared using the chi-square (χ2) test (Pearson p < 0.005). A total of 467 papers were analysed, where 34% were from the Southern Hemisphere  and 66% from the Northern Hemisphere . The Northern Hemisphere (mainly North America and Europe) played a major role in the socio-ecological knowledge exchange than the Southern Hemisphere (South America and Africa). The results showed socio-ecological studies focused mainly on generating management recommendations in social and environmental science fields. The number of studies coming from the Northern Hemisphere was significantly higher than those from the Southern Hemisphere. Most of them were conducted at a local level  in three different systems (i) terrestrial , (ii) freshwater  and (iii) marine . Most of the studies (70%) were conducted in production systems, where the majority included livestock (mainly bovine) and aquatic fisheries . Most vegetation papers (65%) were on native forests. Papers on wildlife made up 30% of all animal-related studies, with mammals, birds, and marine invertebrates (such as collars) being the most extensively researched species. This work highlighted the socio-ecological approach that was used in the analysed countries with greater HDI to develop management options for natural systems.Socio-ecology studies the relationships between human activities and natural systems and their importance in management and public policy. Our objective was to analyse how published papers in countries with a high Human Development Index (HDI) perform socio-ecological studies and compare them between the Northern and Southern Hemispheres. To do this, we used the Scopus platform as a source for searching and obtaining scientific papers about socio-ecological studies conducted in countries from the Northern and Southern Hemispheres. We calculated the number ( The socio-ecological approach is inherently complex  perform socio-ecological studies and to evaluate differences between the Northern and Southern Hemispheres. Our specific questions were: (i) Is there a predominant main subject area that occurs in developed countries in both hemispheres? Or is there a difference between hemispheres? (ii) What is the exchange of socio-ecological studies between both hemispheres? (iii) What are the main recommendations  derived from socio-ecological studies? Are there different recommendations associated with different hemispheres or countries? (iv) What are the predominant natural systems  and spatial scales  analysed in each hemisphere? (v) What kind of nature  are mostly included in the socio-ecological studies of each hemisphere? With this in mind, we expected that this analysis of scientific papers on socio-ecology generated in high HDI countries could reveal if there were differences between the Northern and Southern Hemispheres regarding some characteristics , highlighting the focus until now and the main gaps that need to be filled.www.scopus.com) as a source for searching and collecting scientific studies. We searched for the papers using four keywords. The first three words focused on the concept of socio-ecology and its potential combinations, and the fourth keyword was the name of the country we were looking for. We wrote the keywords in the singular  to obtain more results, and we omitted the “social-ecology” term in the search to avoid articles that misuse the concept, although this term appears in many selected papers  that provides dimensions of social and economic development worldwide and we used it to identify countries with more expected scientific production. Many authors prefer this index to represent people’s quality of life and the economic standard of living in countries worldwide . We selected countries with the highest HDI and categorized them by hemisphere and continent (Annex n = 18), considering the order of the HDI ranking from highest to lowest, with Norway being the highest HDI and France at the limit. Countries near the Equator line were not selected because we assumed they share characteristics with the Northern and Southern Hemispheres . For the African continent, only one country (South Africa) is a developed country according to HDI; therefore, we included Botswana, the African country with the highest medium HDI, to achieve a better representation of this continent.Once the search results were displayed, we reviewed each paper by country and selected those linked to socio-ecological studies in the Northern and Southern Hemispheres. Selection criteria for countries were based on the Human Development Index (HDI) and its components. The HDI is a metric developed by the United Nations UNDP,   thewww.scimagojr.com): (i) Agricultural and Biological Sciences, (ii) Arts and Humanities, (iii) Decision Sciences, (iv) Earth and Planetary Sciences, (v) Economics, Econometrics and Finance, (vi) Environmental Sciences, (vii) Medicine, and (viii) Social Sciences. Likewise, we analysed whether the papers had specific recommendations or not for (i) Management of natural systems, (ii) Conservation of nature, (iii) Policies or governance structures, and (iv) Scientific-general knowledge . We also evaluated as exclusive categories the three main spatial scales  and the three different predominant natural systems .For all the papers reviewed (see Annex n) of papers published each year to evaluate the timeline of socio-ecology papers. We also estimated the number of observations in YES or NO classes for each category of subject areas and recommendations, expressed in absolute frequencies and frequencies relative to the total (in percentage). To assess statistical differences among Countries and between hemispheres in the number of papers produced for some subject area or recommendation, we used chi-square (χ2) test (Pearson p < 0.005). The significance value followed the suggestion of Benjamin et al.  that addressed socio-ecological studies related to flora and fauna.We calculated the quantity .In the analysed dataset, the earliest socio-ecological articles were published in the 1990s Fig. . The firn = 467 papers) considering the three criteria of relevance that we established in the methodology section. Concerning geographical origin, we found socio-ecological studies mainly coming from North American and European countries. Geographically, institutions based in Southern Hemisphere countries produced 34% of the selected papers, with an average of 9.4 (±SD 7.1) papers per country (160 papers from 2002 to 2021), corresponding 58 to South America, 39 to Africa, and 63 to Oceania. The countries with the most papers in the Southern Hemisphere were Australia (n = 47), Chile (n = 35), South Africa (n = 32), and Argentina (n = 21). The Northern Hemisphere produced 66% of socio-ecological papers, with an average of 14.6 (±SD 15.2) papers per country (307 papers from 1992 to 2021), corresponding 93 to North America, 127 to Europe and 87 to Asia. The countries with the most papers in the Northern Hemisphere were the United States (n = 69), China (n = 53), Spain (n = 44), Canada (n = 24), and France (n = 20). Figure We found 737 papers that somehow dealt with socio-ecology, of which we selected 63% of the studies n = 67 papersχ2 test, and the subject areas  and Earth and Planetary Sciences . Between hemispheres, the differences were significant for Arts and Humanities , Agricultural and Biological Sciences , Environmental Sciences , Economics, Econometrics, and Finance , where the number of articles studied in these areas was notably higher in the Northern Hemisphere than in the Southern Hemisphere , Agricultural and Biological Sciences, Economics, Econometrics and Finance they had a medium amount (between 36 and 45 papers), and Medicine had the least amount (only 7) Table . The anaas Table , showed χ2 test, Table The recommendations derived from the largest number of papers were Management, followed in decreasing order by Policies, General Knowledge, and Conservation in both hemispheres. The Northern Hemisphere presented higher trends and was closer to the total than the Southern Hemisphere, where 136 papers resulted in Management recommendations and fewer than 100 papers resulted in Policies, General Knowledge, and Conservation recommendations Table . In the Most of the studies were at the local level (47%), followed by regional (33%) and national (20%) studies Fig. . In turnn = 100 papers) or fauna (n = 79 papers). The highest proportion of the vegetation papers was conducted on natural systems (mainly native forests) (65%), and to a lesser extent, on forest plantations or crops (35%). Regarding the fauna, 70% of the studies were conducted in terrestrial (n = 22) and aquatic (n = 33) production systems, where livestock (mainly bovine) and aquatic fisheries  were the most studied groups. Wildlife represented 30% of the studies on animals, where marine invertebrates , mammals and birds were the most studied groups.Among the 467 selected papers, 38% were focused on the relationship between humans and aspects of nature . Despite the growing importance of ocean protection and conservation globally, the scientific community makes efforts to understand the management of the terrestrial ecosystem, where socio-ecology plays a preponderant role in environmental and social issues in countries such as South Africa . The socio-ecological approach describes the relationship among various social and environmental aspects of a specific location . In both hemispheres, socio-ecology has evolved in high HDI countries, where it is employed as a normative term and to foster the essential laws and regulations that direct the formation of international agreements concerning social and environmental issues on a global scale  and their social perceptions are emphasised  through scores based on the economic freedom of each country . Overall, the studies in terrestrial systems are the ones that have been carried out the most compared to freshwater and marine systems. Our analysis shows that studies focused on socio-ecology have increased in recent years (since 2006), where most socio-ecological studies generated management recommendations, followed by policy recommendations in both hemispheres. However, these subject areas and recommendations come mainly from studies in the northern hemisphere with the largest number of papers. The relative importance of these subject areas was broadly used across multiple spatial scales. Such scales in socio-ecological studies provide opportunities to engage people with flora and fauna, where native forests generated better social spaces to improve the sustainable management of the natural systems than forest plantations or crops. The reviewed studies highlight socio-ecological critical research on productive systems such as livestock and marine fisheries. Based on the results, the socio-ecology approach was used in the analysed countries with greater social and economic development indicators to develop management options for the natural systems.Supplementary information