IL6, CXCL1, CXCL3, CXCL8/IL8, CCL2, and PTGS2. These results provide the first insights into genome-wide regulation of mRNA stability in RA FLS and highlight the potential contribution of dynamic regulation of the mRNA stabilome by TNF to chronic synovitis.During rheumatoid arthritis (RA), Tumor Necrosis Factor (TNF) activates fibroblast-like synoviocytes (FLS) inducing in a temporal order a constellation of genes, which perpetuate synovial inflammation. Although the molecular mechanisms regulating TNF-induced transcription are well characterized, little is known about the impact of mRNA stability on gene expression and the impact of TNF on decay rates of mRNA transcripts in FLS. To address these issues we performed RNA sequencing and genome-wide analysis of the mRNA stabilome in RA FLS. We found that TNF induces a biphasic gene expression program: initially, the inducible transcriptome consists primarily of unstable transcripts but progressively switches and becomes dominated by very stable transcripts. This temporal switch is due to: a) TNF-induced prolonged stabilization of previously unstable transcripts that enables progressive transcript accumulation over days and b) sustained expression and late induction of very stable transcripts. TNF-induced mRNA stabilization in RA FLS occurs during the late phase of TNF response, is MAPK-dependent, and involves several genes with pathogenic potential such as  Pannus artilage . It is hartilage . During artilage ,5. Theseartilage .IL6 and CXCL10, non-terminating transcription of IL6, and continuous expression of cytokines, chemokines and tissue destructive enzymes [In target cells, TNF triggers a series of molecular events that unfold in a stereotypic temporal order including an immediate-early phase followed by a later phase ,7–9. The enzymes . Within In this study, we further investigate the molecular events induced in RA FLS during their transition from early to the late phases of TNF response, and elucidate the dynamics of regulation of gene expression. As our previous studies explored upstream signaling, chromatin modifications, and gene transcription ,11, hereSynovial tissues were obtained from RA patients who underwent, as part of standard medical care, total knee replacement or elbow synovectomy, using a protocol approved by the Hospital for Special Surgery Institutional Review Board that adheres to NIH guidelines and regulations. De-identified specimens that would otherwise have been discarded were used in this study and were obtained under a waiver of consent. The diagnosis of RA was based on the 1987 American College of Rheumatology criteria . The resSynovial tissue fragments were incubated with liberase. Cells were allowed to adhere to tissue culture dishes and passaged every 3–5 days. 4–5 passages yielded a relatively homogeneous population of FLS.FLS were cultured in alpha-MEM . The following reagents were used as indicated: TNF , Actinomycin D , Triptolide , Flavopiridol , SB202190 , JNK Inhibitor II SP600125 , U0126 . Dimethyl sulphoxide (DMSO) (Sigma-Aldrich) was used as a vehicle control.6 FLS using RNeasy mini kit (Qiagen) and 1μg was reverse transcribed using a First Strand cDNA synthesis kit (Fermentas). qPCR was performed using SYBR Green supermix following the manufacturer's protocols, and triplicate reactions were run for each sample. The human oligonucleotide primers used were:RNA was extracted from 0.5x105-ATCAAGAAGGTGGTGAAGCA-3 and 5-GTCGCTGTTGAAGTCAGAGGA-3;GAPDH: 5-TAATGGGCATTCCTTCTTCT-3 and 5-TGTCCTAACGCTCATACTTTT-3IL-6: 5-GGACAACTCAGGGATGCAAT-3 and 5-GCAGAAGAGAGCCAACCAAC-3IL-6 Primary Transcript (PT): 5-TTTTGCCAAGGAGTGCTAAAGA-3 and 5-AACCCTCTGCACCCAGTTTTC-3IL8: 5-AGCAGCAAGTGTCCCAAAGA-3 and 5- GTGTCTGGGGAAAGCTAGGGCCL2: 5-GAGGCTTCCCCTCACTATCC-3 and 5-CTCAAGTGATCCACCCACCT-3CCL5: 5-TCCTGAGACCCTGAGACAGC-3 and 5- TGTGCCAAAATCAGCACAAT-3CCL5 PT: 5-AGTCATAGCCACACTCAAGAATGG-3 and 5-GATGCAGGATTGAGGCAAGC-3CXCL1: 5-TCCCCCATGGTTCAGAAAATC-3 and 5-GGTGCTCCCCTTGTTCAGTATCT-3CXCL3: 5-GTGGCTGAACAAATTAACGAA-3 and 5-AGCCTGAATGTGCCATAAGA-3PTGS2: 5-TCTCCTGCCTGTGCTGTG-3 and 5-CAGATTCACGCTCAAGTTCCMMP3:5-ACAGGTTGATTCCTGGGTCA-3 and 5- CCCATATATGCCTGCTGTCC-3MMP3 PT: Total RNA was extracted from synovial fibroblasts derived from two different RA patients using the RNeasy mini kit (Qiagen). RNA-sequencing libraries were generated from two patient donor FLS using Tru-Seq kits (Illumina) for poly-A selected transcripts. Single end sequencing was performed . Reads were aligned to the human reference hg19 using TopHat2 . QualityCount tables generated from HTSeq were processed and filtered based on expression in TNF treated conditions (>100 counts per gene). Differential testing was performed between TNF treated libraries in 1, 3, 24, 72 h, and the control libraries using DESeq2 . DESeq2 St,i represents the stability at time point t for gene i. Formulating stability ratios was informative for determining mRNA stability at individual time-points. However, comparing these ratios between two time-points of TNF stimulation could be challenging to interpret due to the confounding effects of gene expression changes resulting from the time-dependent effects of TNF and Act D. Therefore, we utilized the RiboDiff statistical framework as a tool to determine differentially stable genes between time points of TNF stimulation (i from the TNF samples [i from the TNF+Act D treated samples [C represents either the early (1 hour) TNF stimulation (C = 0) or the late (72 hours) TNF stimulation (C = 1) time-point. The shared parameter C = 0) and the late time-point . Statistical significance for each gene is determined by testing the log-likelihood of the null model  were performed according to the manufacturer’s instruction. Duplicate wells of cell culture supernatant samples (25 μl) were analyzed by Luminex MagPix . Cytokine concentrations were determined by Luminex Xponent 4.2 and EMD-Millipore Milliplex Analyst v5.1 using 5-p log analysis.Results are expressed as mean ± SEM and GraphPad Prism Analytical Software Version 5 for Windows was used. For statistical analysis of qPCR and multiplex cytokine assay results, the Analysis Of Variance (ANOVA) with Tukey post analysis test was used as appropriate.IL6, resulting in accumulation of IL-6 mRNA and protein [IL6 expression  of TNF stimulation . We est protein , IL-6 mRpression . Similarpression . Notablymulation . We havemulation . These rmulation . These dAiming to characterize genome-wide the mRNA stability in RA FLS, we performed RNA sequencing and analyzed the effects of Act D in two biological replicates (derived from two different RA patients) of TNF-stimulated FLS. More specifically, RA FLS were stimulated with TNF (10 ng/ml) for 1, 3, 24 or 72 hours and then Act D (10 μg/ml) was added for 3 hours to disrupt transcription. The mRNA stability status was calculated as the ratio of RPKM levels at the TNF+Act D condition divided by the RPKM levels at the TNF condition. This ratio ranges from 0 to 1 and classifies genes along a spectrum ranging from very unstable to very stable transcripts. In Inflammation-induced stabilization of arthritogenic transcripts, such as IL-6, might contribute to perpetuation of synovitis. Aiming to identify additional gene-transcripts amenable to stabilization by TNF, we analyzed genome-wide the differential mRNA stability status of genes between the early (1 hour) and the late (72 hours) phases of TNF response. DESeq2 was used to analyze the differential gene expression between TNF and TNF + Act D conditions and calculate statistical significance of differences  using two biological replicates (derived from two different RA patients) of RNA sequencing. During the early phase of TNF stimulation transcripts encoded by 2,365 genes were found to be significantly  down-regulated by Act D . Out of We then performed an alternative and more rigorous statistical analysis to identify in a genome-wide manner the genes that were significantly stabilized by TNF. For this purpose we used RiboDiff as a tool to evaluate the stabilization effect of TNF and calculate statistical significance of this effect as described in Methods and To investigate the potential biological consequences of the observed TNF-induced mRNA stabilization, GSEA/MSigDB gene ontology analysis was performed for the top 10% of the stabilized transcripts (593 genes). Interestingly, the stabilized gene-set was highly enriched for a number of potentially pathogenic biologic processes . It is oNext, we investigated the molecular mechanisms that drive the observed TNF-induced mRNA stabilization. Initially, we tested the hypothesis that TNF induces a transcriptional program that orchestrates late phase stabilization of expressed mRNA transcripts. Using DESeq2, we identified the genes in RA FLS that were regulated by TNF-stimulation in a statistically significant manner . This obCCL5 is intrinsically very stable and its stability is not regulated by MAPKs [IL6  out of >300 expressed genes. These data suggest that only genes with very stable transcripts achieve the very top expression levels in RA FLS. At 72 hours, among the very top highly expressed genes are included the highly stabilized IL-6 (ranked 39th by expression level), IL-8 (ranked 32nd by expression level), CCL2 (ranked 20th by expression level), and CXCL1 (ranked 61st by expression level). Monitoring the TNF-induced expression kinetics relative to the TNF-induced stabilization dynamics for IL-6, IL-8 and CCL2 mRNA, it was observed that all three genes achieve their highest expression levels when their transcripts have been stabilized by TNF  ,7–9. We CCL5) and IP-10 (encoded by CXCL10) proteins were measured in culture supernatants  and by kinetics of expression (transient or sustained). We analyzed the time course of TNF-induced gene expression in our experimental system focusing on 386 genes that were identified as highly induced by DESeq2 . Hierarchical clustering of the gene expression values led to the classification of highly induced genes into 6 clusters with distinct timing of peak gene expression and diverse expression duration kinetics . ClusterTo further explore this concept, we monitored in parallel the TNF-induced kinetics of total mRNA expression and active transcription (measured by the expression of primary transcripts) of very stable transcripts, such as MMP3 and CCL5. Interestingly, both genes displayed late peaks and protracted expression kinetics, but the kinetics of active transcription were discordant with the total mRNA expression kinetics . For MMPIs the mRNA degradation rate per gene constant (‘Constant degradation hypothesis’) or is it changing over time (‘Varying degradation hypothesis’)? Next generation sequencing and metabolic labeling of RNA, combined with computational modeling have resolved the debate by providing evidence that favors the varying degradation hypothesis in T-lymphocytes, dendritic cells, and foreskin fibroblasts –33. To oA plausible explanation for TNF-triggered stabilization of transcripts is the induction of molecular pathways that finally inhibit the mRNA degradation machinery . NotablyA very interesting finding of our study was the discovery of a temporal switch in the stability states of TNF-induced transcripts in RA FLS. Unstable transcripts dominate the early response to TNF, while during the late TNF response the gene expression program is outbalanced by very stable transcripts . WhereasS1 Table(PDF)Click here for additional data file.S1 FigC which in the current study is either 1h or 72h); left grey circle is for a given gene i the read count from the TNF condition at the time-point C ; right grey circle is for a given gene i the read count from the TNF+Act D condition at the time-point C . The r term denotes replicates for the TNF and TNF+Act D libraries. Empty circles represent unobserved variables that include the dispersion parameters (denoted by K), which are estimated by performing a gamma regression on the raw dispersions, and normalized counts (denoted by μ) that are estimated independently for the TNF and TNF+Act D libraries. Black squares are equations that estimate the expected log read count and model the relationship between TNF and TNF+Act D read count abundances. The i, under time condition C for the TNF library. The i, under time condition C for the TNF+Act D library. The term i the differential effect of the time condition C on the TNF+Act D library. RiboDiff tests the significance of the The graphical model explains the RiboDiff method applied to evaluate time-dependent, TNF induced changes in mRNA stability. Gray circles depict observed variables: top grey circle indicates the time-point of TNF stimulation ((PDF)Click here for additional data file.S2 Fig2 fold change of the TNF + Act D normalized counts over the TNF 1h normalized counts. (b) Of the significantly downregulated genes depicted in (a), 2248 were also expressed at the TNF 72h time point. Differential testing of the TNF72h against the TNF72h with Act D was performed. The corresponding log2 fold change at TNF72h (y-axis) is plotted against the log2 fold change at TNF1h (x-axis). Genes with an increased log2 fold change (orange) represent genes that were stabilized at 72 hours (compared to 1 hour). Destabilized genes demonstrate reduced log2 fold change at 72 hours (compared to 1 hour) (blue).(a), Graph illustrating genes expressed at 1 hour of TNF-stimulation and downregulated by actinomycin D (Act D). Gene expression was measured by RNA sequencing. Genes were filtered for expression (raw reads > 100). Differential testing was performed using DESeq2 for the TNF 1h condition against the TNF + Act D condition using two biological replicates. Significance from DESeq2 is presented as the adjusted p-values < 0.1 (red dots) and ≥ 0.1 (grey dots). The x-axis represents the expression level at TNF 1 hour as the mean of normalized counts from two biological replicates. The y axis represents the log(PDF)Click here for additional data file.S3 FigP values were calculated by one-way ANOVA and Tukey post-test analysis .RA FLS were exposed to a single dose of TNF (10 ng/ml) for 1, 3, 24 and 72 hours. Subsequently, actinomycin D  was added for 1 or 3 hours to block active transcription. Real-time quantitative reverse transcription polymerase chain reaction was used to measure the mRNA levels of IL-8, CXCL1, CXCL3, PTGS2 (a), and CCL2 (b) mRNA. Cumulative results from seven independent experiments are shown. Values were normalized relative to mRNA for GAPDH and are presented as mean ±SEM. (PDF)Click here for additional data file.S4 Fig2 fold change (up- or down-regulation) compared to unstimulated cells (Control). The x axis represents expression level as normalized counts . DESeq2 was used to analyze the TNF-induced differential gene expression and to calculate statistical significance. Red color visualizes genes up- or down-regulated by TNF to a statistically significant degree .(a-d), Bland-Altman plots of TNF regulated genes at 1 (a), 3 (b), 24 (c) and 72 (d) hours of TNF-stimulation. The y axis represents the log(PDF)Click here for additional data file.S5 Fig2 RPKM) to their mRNA stability states .RA FLS were exposed to a single dose of TNF (10 ng/ml) for 3 or 24 hours. Subsequently, actinomycin D  was added for 3 hours to block active transcription. Gene expression was measured by RNA sequencing in two biological replicates and RPKM values were generated using CuffDiff2. The mRNA stability status was calculated genome-wide as the ratio of RPKM levels at the TNF+Act D condition divided to the RPKM levels at the TNF condition. This ratio ranges from 0 to 1 and classifies genes to a spectrum from very unstable to very stable transcripts. Genes induced ≥2-fold by TNF at 3 hours (a) and 24 hours (b), were plotted comparing their expression levels Click here for additional data file.S6 FigTwo biological replicates of RA FLS (derived from two different RA patients) were exposed to TNF (10ng/ml) for 1-72h and gene expression was measured by RNA sequencing. 386 genes were identified as highly induced (≥5-fold) by TNF at any time point and were clustered into 6 clusters with distinct kinetics of peak expression. For (a-d), line graphs of mean expression (read counts) at 0-72h of TNF stimulation for each cluster.(PDF)Click here for additional data file.


TNF-α-induced inflammatory cytokines were strongly reduced in presence of Atsttrin in primary human disc. Mechanism study indicated Atsttrin protected against intervertebral disc degeneration by inhibiting TNF-α-induced inflammation. These findings show that Atsttrin is a potential molecular target for disc degenerative diseases.Atsttrin, an engineered molecule composed of three fragments of progranulin(PGRN), exerts comparable anti-inflammation ability. Intervertebral disc degeneration (IDD) is involved in inflammation in which TNF-α plays a key role. This study aims to examine the effect and the mechanism of Atsttrin in the pathogenesis of intervertebral disc degeneration. For this purpose, we took advantage of murine and human intervertebral disc (IVD) and examined the expression of TNF-α in IVD tissues using immunohistochemistry and TNF-α level in peripheral sera by ELISA assay. Moreover, murine IVD was taken to undergo the Safranin O and HE staining. Furthermore, primary human nucleus pulposus cells were used for immunohistochemistry staining, fluorescent staining, Western Blot, ELISA assay and RT-PCR assay. Herein we found TNF-α expression was elevated in intervertebral disc and peripheral sera in patients with IDD. Interestingly, Atsttrin effectively inhibited TNF-α-mediated catabolism in murine disc by  Intervertebral disc degeneration(IDD) is an irreversible degenerative disease which is very common in the elderly, characterized by disc inflammation, endplate degeneration, vertebral osteophyte formation, spinal canal stenosis , 2. The Tumor necrosis factor-α (TNF-α) plays an important role in the process of intervertebral disc degeneration, and it also be the key reason for the low back pain , 5. SeruProgranulin, which is also known as PGRN, is a growth factor which has a unique structure with “beeds-on-a-string”. PGRN is expressed in various cells and plays a critical role in various physiological and pathological processes, including wound healing, tumorigenesis and inflammation –11. AtstGiven the indication that previous study demonstrated that loss-of-PGRN accelerated degeneration in the aging mice model and the importance of Atsttrin’s anti-inflammation ability, in this study, we determined to examine whether Atsttrin as PGRN-derived engineered molecule, has a protective effect in intervertebral disc degeneration as well as the molecular mechanism involved.To investigate expression of TNF-α in the pathogenesis of IDD, we performed immunohistochemistry staining in IVD tissues from normal people and patients with IDD respectively. As shown in Figure ex vivo study. For this purpose, we cultured the mice disc with or without 50 ng/ml TNF-α in the presence or absence of 1ug/ml Atsttrin for 7 days. Followed by Safranin O staining and HE staining. As is shown in Figure Given the finding that TNF-α is increased in the pathogenesis of IDD and the importance of TNF-α in IDD process, to further determine whether Atsttrin could suppress TNF-α-mediated catabolism in disc, we isolated murine disc and did ex vivo study. It’s widely accepted that TNF-α plays a critical role in the intervertebral disc degeneration process. In addition, TNF-α-induced inflammatory cytokine such as IL-17, IL-1, COX-2 and iNOS could cause further destruction. To investigate whether Atsttrin could inhibit TNF-α-induced inflammation in human disc, we cultured the nucleus tissues with or without TNF-α in presence or absence of Atsttrin for 7 days. After harvested the tissues, immunohistochemistry staining was performed for the expression of MMP-13, COX-2, iNOS and IL-17, As is illustrated in Figure in vitro  is one of the most popular degenerative diseases which highly affect people’s daily life . So far,It’s widely accepted that inflammation plays a critical role in the pathogenesis of IDD , 17. In Among these molecules, TNF-α attract researchers’ attention owing to its peak site of the inflammatory cascade , 24. PreProgranulin, also known as PGRN, is a growth factor with potent anti-inflammation ability. PGRN consists of seven and half units (P-G-F-B-A-C-E-D) . While PPrevious study indicated that Atsttrin had a therapeutic effect in the inflammatory arthritis mice model in which TNF-α plays a predominant role –14. In aex vivo study by using murine intervertebral disc. Herein, we found additional use of TNF-α accelerated cartilaginous tissue loss in the murine intervertebral disc. Additionally, TNF-α indeed affected the construction of intervertebral disc, however, in present study we did not see the osteophyte formation which may due to the less duration of treatment or the different microenvironments between ex vivo and in vivo. Intervertebral disc is full of matrix (aggrecan and collagen II)), in the pathogenesis of IDD, inflammatory cytokines penetrated and cause destruction. Metalloproteinase 13 (MMP-13) is the main enzyme which digesting type II collagen (Col II) [ex vivo study, we confirmed our findings by in vitro assay. Fluorescent staining indicated in the human nucleus cells, TNF-α remarkably increased the expression of iNOS and COX-2, however, iNOS and COX-2’s expressions were significantly decreased in presence of Atsttrin. Given the findings that recombinant Atsttrin has a potential therapeutic effect in the intervertebral disc degeneration, and the clue that Atsttrin exerted a protective effect via targeting TNF-α pathway in the inflammatory arthritis mice model, we next determined that Atsttrin inhibited this pathogenesis through interfering with TNF-α signaling. In present study, we took advantage of primary human nucleus cells and mimic in vivo stimulation. We found Atsttrin significantly down-regulated TNF-α-induced inflammatory cytokines’ expression at transcriptional level. Sequentially, the protein expressions of inflammatory molecules were dramatically reduced. Collectively, Atsttrin has a therapeutic effect in IDD, and this effect is through inhibiting TNF-α signaling.In the present study, we found TNF-α was up-regulated in local nucleus cells and peripheral blood , 39. Thi(Col II) , causing(Col II) . We confAtsttrin is a new engineered molecule derived from the growth factor PGRN. Nowadays, more and more studies indicated Atsttrin exhibited a strong anti-inflammation ability and exerted a protective effect in some pathological processes. It’s reported Atsttrin was therapeutic in inflammatory arthritis mice model; moreover, overexpression of Atsttrin in local articular cartilage protected against osteoarthritis , 36; andCollectively, Atsttrin is therapeutic in the intervertebral disc degenerative disease via targeting TNF-α and Atsttrin could be a noval drug candidate for the inflammatory degenerative diseases.40 patients with single level lumbar disc herniation were enrolled between 2017.1.1 and 2017.5.31 at Qilu Hospital of Shandong University. Collection of lumbar disc tissue and peripheral blood samples was performed according to the medical ethics regulations of Medical Ethical Committee of Qilu Hospital of Shandong University. This research was approved by the Qilu Hospital of Shandong University Review Board, and written informed consent document was requested and received from all patients and healthy individuals in the study. The animal experimental procedures were performed with the formal approval of The International Guiding Principles for Animal Research, as approved by the Laboratory Animal Centre of Shandong University.All the patients enrolled in this study signed the informed consent and agreed to participate in this study, 40 patients with lumber disc herniation (LDH)  were enrolled in our study as the experimental group. The selection criteria were as follows: 1) persistent lumbosacral pain accompanied with lower limb radiating pain at least 3 months ,accompanied by or without limb numbness; 2) lumber disc MRI and CT imaging studies showed significant disc herniation performance and compression of the nerve; 3) Lasegue’s sign (+) or positive for femoral nerve traction test (+). The exclusion criteria were as follows: 1) LDH combined with osteoarthritis, rheumatoid osteoarthritis; (2) LDH combined with malignant tumor; (3) recent history of trauma and surgery; and (4) LDH combined with infections of other organs and systems. And we selected 10 normal people  as the control group.Four milliliters of blood were extracted with a heparin sodium anticoagulant tube from all the people early in the morning after fasting. Blood samples were sent to the laboratory immediately and centrifuged at 4°C at 2,000 g for 10 minutes. Supernatant was extracted and used for the detection of TNF-α.2 cell culture flask at a density of approximately 25,000cells/cm2 in DMEM/F12 culture medium  supplemented with 10% fetal bovine serum , 100 U/mL penicillin and 100 mg/mL streptomycin  and then incubated in the incubator with 5% CO2, 37°C. Replaced the culture medium every 3 days, after the NP cells reached 80-90% confluence then passaged with 0.25% trypsin. In this study, we used second or third generation cells for all experiments.All procedures are in compliance with the requirement of Qilu Hospital’s Research Ethic Committee, Shandong University, China. All the intervertebral disc tissues were harvest from the lumber spine surgery of the patients with IDD. After washed with sterile phosphate buffer (PBS) 3 times and then removed the endplate cartilage and annulus fibrosus (AF) carefully using a dissecting microscope. The nucleus pulposus (NP) was digested with 0.25% trypsin  for 30 minutes and 0.2% type II collagenenzyme  for 4 hours. Then seeded the NP cell suspension in the 25 cmThe procedure was performed as previously described –44. The The specimen of mouse spine were taken from the 3-month-old mice, after complete dissected the spine and removed the surrounding soft tissues, cut the lumbar spine into the length of 5 mm and keep the integrity of the intervertebral disc. Soaked the lumbar spine of the mouse with 100 U/ml penicillin/streptomycin 30 minutes, then culture the lumbar spine in the medium as described above. For Safranin O/Fast Green staining and HE staining, the explants of lumbar spine of the mouse were fixed in 4% paraformaldehyde for 48 h and processed for paraffin embedding. Sections were stained with Modified Safranine O-Fast Green FCF Cartilage Stain Kit  according to the manufacture instruction.Degenerated IVD tissues obtained from the patients with lumber disc herniation, the disc samples were fixed in 4% formaldehyde and then embedded with paraffin. The paraffin was cut into 4 um sections. After deparaffinized and rehydrated, 0.125% trypsin  was used to antigen repair, then incubated the section with 3% hydrogen peroxide to eliminate endogenous peroxidase activity. Then use 20% goat serum  to close the nonspecific protein binding sites. The sections were incubated with rabbit anti-human iNOS , rabbit anti-human MMP-13 and rabbit anti-human COX-2 ) primary antibodies overnight at 4°C. Then the incubated the sections with a goat anti-rabbit immunoglobulin(IgG)-horseradish peroxidase (HRP) secondary antibody  at 37°C for 1 hour. IX71-SIF type microscope  was used to take the images, image-Pro Plus software  was used to quantify the mean densities (MDs) of the expression intensities of all the cytokines.For immunofluorescence staining The NP cells were treated with 10 ng/ml of TNF-α  in the presence or absence of 200 ng/ml of Atsttrin  for 48 h. Then fixed the cells with 4% formaldehyde for 10 minutes, and permeabilized in 0.2% Triton-X 100 for 15 minutes and then blocked in 1% BSA for 30 minutes at room temperature. Then incubate the cells with rabbit anti-human COX-2  and rabbit anti-human iNOS  primary antibody overnight at 4°C. The next day cells were washed with PBS and then incubated in a secondary fluorescently labeled goat anti-rabbit immunoglobulin (IgG) antibody  for 1 hour at room temperature. The image was taken by a fluorescent microscope .To investigate the effects of Atsttrin on TNF-α, human NP cells were seeded into a six-well plate and stimulated with TNF-α (10 ng/ml) with or without Atsttrin  for 48 hours. Then harvest the NP cells for western blot analysis and real-time PCR (RT-PCR) to observe the expression level of COX-2, MMP-13 and iNOS. The supernatant were harvested for ELISA test of IL-6.After the NP cells were confluence in the six-well plate then lysed the cells with RIPA buffer  on ice for 40 minutes. Then centrifuged at 4°C, 12,000 rpm. Carefully absorb the supernatant and measure the protein concentration with BCA protein Assay Kit  according to the manufacture instructions. Separated each group of protein in 10% acrylamide–SDS–PAGE and subsequently transferred to PVDF membranes . Blocked the membranes with TBST which contain 5% milk powder and followed incubate with primary antibodies  overnight at 4°C. Then incubate the PVDF membranes in goat anti-rabbit immunoglobulin (IgG)-horseradish peroxidase (HRP) secondary antibody  at room temperature for 1 hour. Equal protein loading was confirmed by reprobing the membranes with the rabbit anti-GAPDH-HRP antibody . Protein bands were detected using a FluorChem E Chemiluminescent Western Blot Imaging System  and image-Pro Plus software  was used to analyze the optical density of each strip .After treated with TNF-α and Atsttrin, the human NP cells were disintegrated with TRIzol reagent . After total RNA was extracted then reverse transcribed to cDNA according to the manufacturer’s instructions of cDNA Synthesis Kit . Real-time PCR reactions were carried out on Roche LightCycler  utilizing a SYBR Green PCR Matrix Mix . The nucleotide sequences of MMP-13, COX-2, iNOS and GAPDH primers are listed in Table We obtained the serum sample from the patients with LDH and stored at −20°C. The supernatants harvested from NP cell culture and stored at −20°C until assay. The serum concentration of TNF-α was measured by ELISA according to the manufacturer’s instructions , IL-6 in the supernatants from conditioned NP cells cultures were detected utilizing Human IL-6 Elisa kit  follow the instructions protocol. The color reaction was assayed at 450 nm using a Varioskan flash multifunction plate reader .Statistics were analyzed using SPSS 22.0 , all data were conducted as mean values ± standard error of mean (SEM). P<0.05 was considered as statistically significant.


The effects of environmental and dispersal processes on macrofungi community assembly remain unclear. Further, it is not well understood if community assembly differs for different functional guilds of macrofungi, e.g., soil and rotten-wood macrofungi. In this study, using 2433 macrofungi sporocarps belonging to 217 species located within a forest dynamics plot in temperate mountain forest (China), we examined the explanatory power of topography, spatial eigenvectors , plant community, and light availability for local spatial variation in the macrofungi community through variance partitioning and partial least squares path modeling. We found spatial eigenvectors and light as the most important factors for explaining species richness and composition of macrofungi. Light was negatively correlated with species richness of macrofungi. Furthermore, species richness and composition of soil macrofungi were best explained by light, and species richness and composition of rotten-wood macrofungi were best explained by spatial eigenvectors. Woody plant community structure was not an important factor for species richness and composition of macrofungi. Our findings suggest that spatial processes, perhaps dispersal limitation, and light availability were the most important factors affecting macrofungi community in temperate deciduous broad-leaved forest. Major differences in influencing factors between soil and rotten-wood macrofungi were observed, with light as the major driver for soil macrofungi and unknown spatial processes as the major driver for rotten-wood macrofungi. These findings shed new light to the processes shaping community assembly in macrofungi in temperate deciduous broad-leaved forest and point to the potential importance of both intrinsic dynamics, such as dispersal, and external forcing, such as forest dynamics, via its effect on light availability. Macrofungi exert profound biological and economic impacts . The maiConservation awareness and measures of macrofungi resources are largely lacking . Some maEcological drift and dispersal probably are important drivers for community dynamics of woody plants, in supplement to environmental filtering . DispersThe growth of most fungi is related to light, and strong light may inhibit or even kill mycelia . Forest Rotten wood is an important habitat that can sustain the growth of macrofungi . TherefoTemperate mountain forests, widely distributed in northern and eastern China, are dominated by deciduous trees and show wide variation in community structure in time and space scales. Macrofungi are species rich in these ecosystems . In this2, is located in Songxian County in the extreme north of Henan Province, East China . The reserve was set up in the temperate zone in 1982 to preserve a portion of the deciduous broad-leaved forest in the region. The reserve includes 2404 known species of vascular plants, belonging to 815 genera and 170 families. A total of 17 species are included in the Chinese list of rare and endangered species. The annual mean temperature in the region is 13.5°C, the annual mean precipitation is 1200 mm, and forest cover is 81.2%. The dominant tree species in the deciduous broad-leaved forest and in the plot are Quercus aliena var. acuteserrata, Toxicodendron vernicifluum, and Sorbus alnifolia.The Baiyunshan National Nature Reserve (BNNR), with an area of approximately 168 kmIn 2015, a permanent plot covering 5 ha  was established within the deciduous broad-leaved forest in the BNNR. Data were collected following the plot standards of the Center for Tropical Forest Science network . All treAll macrofungi sporocarps in the 5 ha plot were collected based on the 20 m × 20 m subplots monthly from the beginning of May to the end of October 2016. Macrofungi began to grow when the weather became warm in early summer (May) and disappeared when the weather became cold in late autumn (October) in this temperate area. Macrofungi were investigated six times at the end of each month, and substrate types, including rotten wood, living tree, soil, and litterfall, were recorded. Each month, all macrofungi were counted and harvested from each of the 20 m × 20 m subplots. Before specimen collection, the collectors underwent professional training with regard to the basic characteristics of macrofungi and different methods of collection in the field.In the laboratory, the macrofungi sporocarps collected were identified mainly based on macroscopic morphology  and microscopic characteristics . Initial identification was applied to genus or subgenus level based on macroscopic morphology. The microscopic characteristics were observed under the microscope by using freehand slicing. Then, identification was applied to species level through consultation with appropriate literature . A few sTable 1). These parameters were measured following the methods described by Four topographic attributes were measured for each 20 m × 20 m subplot in the field, namely, elevation , slope , aspect , and convex and concave   monthly from May to October 2016. Three replicate photos were taken, and photos showing the highest contrast between sky and foliage for each 10 m × 10 m subplot were selected. The camera was equipped with an ultra-wide-angle fisheye lens  arranged horizontally. Data were collected early in the morning, during late dusk, or on overcast days whenever possible to avoid inaccurate readings caused by direct sunlight . FinallyQ. aliena var. acuteserrata is the dominant species . PC analysis was applied to reduce the basal areas of the 93 plant species into a convenient number of predictors, which was transformed by “Hellinger.” The first six PCs (largely reflecting variables in parentheses) explained 80.14% of the total variation of plant basal area: PC1 , 42.1%; PC2 , 13.6%; PC3 , 9.6%; PC4 , 6.3%; PC5 , 5.0%; and PC6 , 3.3%  were used to determine spatial eigenvectors by using the method reported by Variance partitioning  can be u1).Forest ecological systems are complex and are characterized by multiple interacting processes. Target variables, such as species richness, may be influenced directly and indirectly by a set of variables. In contrast to ordination analysis and variance partitioning, partial least squares path modeling (PLS-PM) allows the user to determine the indirect effects of two explanatory variables . PLS-PM Figure 1A). Moreover, the species diversity of macrofungi differs among different substrates, and 89.4% species tended to be distributed in one kind of substrate . A total of 149, 64, 15, and 15 macrofungi species were recorded in the soil, rotten wood, living tree, and litterfall, respectively. The most abundant macrofungi species were soil macrofungi, followed by rotten-wood macrofungi. The spatial distribution characteristics of the overall soil and rotten-wood macrofungi are shown in Supplementary Figure We recorded 2433 macrofungi sporocarps from 217 species in 33 families between May and October 2016. The species richness of macrofungi hump-shaped distributed from May to October. The species richness and composition of macrofungi remarkably differed in the different months . For the macrofungi species richness, 18.0% of the variation was attributed to pure spatial eigenvectors, and 14.0% was attributed to pure light. For macrofungi species composition, 11.0% of the variation could be attributed to pure spatial eigenvectors, and 9.0% was attributed to pure light.Variation partitioning showed that topography, spatial eigenvectors, plant community, and light explained 62.0 and 47.0% of the variation in species richness and composition for the overall macrofungi, respectively . For macrofungi species richness, the path coefficients of PCNM eigenvectors and light were 33.42 and -23.07%, respectively. For macrofungi species composition, the path coefficients of PCNM eigenvectors and light were 29.80 and -19.70%, respectively.The PLS-PM results indicated that the two highest path coefficients were for PCNM eigenvectors and the light was for macrofungi species richness and composition . For species richness of soil macrofungi, the effects of pure light (16.0%) were higher than those of pure spatial eigenvectors (11.0%). For species richness of rotten-wood macrofungi, the effects of pure spatial eigenvectors (19.0%) were higher than those of pure light (8.0%). For species composition, the analysis of variation partitioning showed that all environmental factors explained 33.0 and 25.0% of the variations in the soil and rotten-wood macrofungi, respectively . For the species composition of soil macrofungi, the effects of pure light (12.0%) are higher than those of pure spatial eigenvectors (7.0%). For the species composition of rotten-wood macrofungi, the effects of pure spatial eigenvectors are the highest among the factors (9.0%).For species richness, the analysis of variation partitioning showed that all environmental factors explained 53.0 and 44.0% of the variations in the soil and rotten-wood macrofungi, respectively . For species richness of rotten-wood macrofungi, the path coefficients of PCNM variables (35.17%) are higher than those of light (6.60%). For species composition of soil macrofungi, the path coefficients of light are the highest (30.75%). For species composition of rotten-wood macrofungi, the path coefficients of PCNM eigenvectors are the highest (20.91%). The outer models (relationships among indicators) of PLS-PM are illustrated in Supplementary Figure The PLS-PM results indicated that the path coefficients of light (30.67%) are higher than those of PCNM eigenvectors (13.17%) for species richness of soil macrofungi (The present study took an important step forward toward the understanding of the assembly mechanisms of macrofungi community, with respect to species richness and composition of macrofungi in temperate deciduous broad-leaved forest. Furthermore, dispersal limitation and light availability could be the major drivers of the species richness and composition of macrofungi in temperate deciduous broad-leaved forest. The major influencing factors differ between soil and rotten-wood macrofungi community.Variation in the species richness and compositions of macrofungi is mainly explained by light and spatial eigenvectors. In particular, light availability was negatively correlated with the species richness and composition of macrofungi. This conclusion is supported by In this study, spatial eigenvectors were considered an important factor affecting the species richness and species composition of the overall macrofungi. Spatial eigenvectors exhibited high explanatory power after controlling for the effects of topography, plant community, and light by variance partitioning. The PLS-PM also indicated the significant effect of spatial eigenvectors on the species richness and composition of overall macrofungi. The dispersability of macrofungi and the suitability of habitat are two important conditions for the successful colonization of macrofungi . StudiesIn forest ecosystems, topography is the most common factor that determines distinct species communities . HoweverPrevious studies found that woody plants are an important factor for the species composition of macrofungi . With inMacrofungi species in the plot were categorized into two main groups, namely, soil and rotten-wood fungi, to determine their major influencing factors. Light is the major driver for soil macrofungi, and spatial eigenvectors are the major driver for rotten-wood macrofungi. This finding could be caused by a random and fragment distribution of rotten wood . Rotten Spatial processes, perhaps dispersal limitation, and light availability were the most important factors affecting macrofungi community in temperate deciduous broad-leaved forest. Woody plant community structure was not an important factor for species richness and composition of macrofungi in this study. Major differences in influencing factors between soil and rotten-wood macrofungi were observed, with light as the major driver for soil macrofungi and unknown spatial processes as the major driver for rotten-wood macrofungi. These findings shed new light to the processes shaping community assembly in macrofungi in temperate deciduous broad-leaved forest and point to the potential importance of both intrinsic dynamics, such as dispersal, and external forcing, such as forest dynamics, via its effect on light availability.This study elucidated the factors influencing macrofungi community structure and provided an opportunity to improve biodiversity conservation in temperate deciduous broad-leaved forest for sustainable forest management . Our finYY conceived the ideas. YC developed the methodology. YC and J-CS led the writing of the manuscript. YC, XW, RC, and ZY conducted the fieldwork.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We hypothesised that TNF-α has pro- and anti-inflammatory effects on monocytes, occurring differentially via TNFR1 and TNFR2. Monocytes were isolated from healthy human subjects and exposed to LPS, plus/minus the addition of blocking antibodies to TNF-α or its receptors. Pro- and anti-inflammatory cytokine production was quantified using real-time PCR and ELISAs. Cell surface expression of TNFR1/2 was measured by flow cytometry. We demonstrated that monocytes vary in the expression patterns of TNFR1 and TNFR2. Autocrine binding of TNF-α led to sustained upregulation of proinflammatory cytokines via TNFR1. In contrast, autocrine binding via TNFR2 upregulated the anti-inflammatory cytokine, IL-10, without proinflammatory effect. TNFR2 was responsible for binding soluble TNF-α secreted by monocytes, clearing the cytokine from the pericellular environment. TNFR1 blockade did not change the cell surface expression of TNFR2, leaving this receptor free to upregulate IL-10. These novel results support the concept of selective TNFR1 blockade in vivo in order that positive anti-inflammatory effects of TNF-α can be retained via TNFR2 ligation.Selective TNFR1 blockade in inflammatory diseases is emerging as a clinical strategy. We studied the roles of the two TNF- Anti-TNopathies , 3 and iopathies , 5. Desp unclear . Intrigumstances , 7.α in humans and are believed to be central in many diseases, including inflammatory bowel disease, arthropathies, and septic shock [α receptors in humans; TNFR1 is expressed on most human cells whereas TNFR2 is found only on immune cells and vascular endothelial cells [Monocytes are the principal source of TNF-ic shock , 8. Monoal cells . TNFR1 ial cells . The rolal cells . Howeveral cells .α signalling via TNFR2, specifically upregulation of the adhesion molecule P-selectin leading to platelet-neutrophil complex formation [The importance of characterising the respective roles of TNFR1 and TNFR2 in human cells is being increasingly recognised. If TNFR1, ubiquitously expressed on almost all cell types, is the “proinflammatory” receptor, with TNFR2 responsible for a more immunomodulatory role, selective TNFR1 blockade would seem a more appropriate strategy in chronic inflammatory diseases. Indeed, a phase one study of Atrosab, a humanized monoclonal antibody that specifically blocks TNFR1, has demonstrated an acceptable safety profile and phase two proof-of-concept trials in rheumatoid arthritis and psoriasis are planned for 2016 . In addiormation .α signalling in monocytes has both pro- and anti-inflammatory effects, occurring differentially through TNFR1 and TNFR2. Our experimental aims were as follows: to study the respective roles of the two receptors on autocrine regulation of pro- and anti-inflammatory cytokine production, to assess the expression patterns of both receptors on monocytes, and to study the effects of TNF-α on their cell surface expression pattern.We hypothesised that TNF-Peripheral blood monocytes were obtained from 19 healthy subjects  who were nonsmokers and not receiving regular medication. The median age was 30 years (range: 21 to 45) and all subjects provided informed consent. The studies had received approval from a research ethics committee (Research and Ethics Committee number 3359a).™ , and centrifuged to obtain a buffy coat layer of PBMCs. The cells were resuspended in sterile PBS containing 2 mM EDTA and 0.1% BSA and CD14+ CD16− monocytes were extracted using the Dynabeads® Untouched™ Human Monocytes kit , following the product protocol. The monocytes (>95% viable by exclusion of trypan blue) were resuspended in culture medium  supplemented with 10% foetal calf serum, 10% L-glutamine, and 10% penicillin V and streptomycin. Throughout all stages of the isolation, the cells were kept on ice to minimise cellular activation.Whole blood was collected into heparinised tubes, layered over Lymphoprep2. Monocytes were plated at a concentration of between 0.25 and 0.45 million per mL of culture medium. The number of replicates used is specified in individual experiments. Salmonella Enteritidis derived LPS  was used as the TNF-α stimulant. The blocking mAbs used in relevant experiments are shown in μg/mL to 1 mL of culture medium per well , following initial concentration response experiments. An irrelevant mouse IgG1 antibody was used as a control in validation experiments to exclude any nonspecific effect of the mAbs (data not shown). Culture plates containing cells incubated with mAbs were gently agitated for 20 minutes prior to the addition of LPS.Cell culture was conducted in 12 or 24-well tissue culture plates  at 37°C and in 5% COConcentrations of the mediators were measured in monocyte supernatants using specific ELISAs according to manufacturer instructions and read using a Synergy HT microplate reader . All samples and standards were run in duplicate and the average was taken as the sample result. Manufacturers' details and lower and upper limits of quantification are given in later®  at −80°C for future study. Messenger RNA was extracted from each cell pellet using the Isolate RNA Minikit  and adequate quantity and purity of extracted RNA were determined using a Nanodrop 2000c spectrophotometer . RNA samples were reverse transcribed to cDNA using a High Capacity RNA-to-cDNA Kit  and real-time quantitative PCR carried out for each gene of interest using TaqMan™ gene expression assays , as listed in −ΔCT formula was used to calculate the relative expression of mRNA of the genes of interest [Adherent cells were pelleted and stored in RNAinterest .6 per sample) underwent flow cytometry either immediately after isolation or after a relevant period in culture and were labelled with fluorophore-labelled mAbs to TNFR1 and TNFR2, as described in [Monocytes .Data are presented as median and interquartile range/range or mean and standard deviation where percentages are presented. Friedman's test was employed to detect overall differences between experimental conditions or time points, followed byα had a positive feedback effect on monocytes, cells (from n = 4) were stimulated with LPS in the presence or absence of TNF-α mAb. α, CXCL8, IL-1β, IL-6, IL-10, and TGF-β after stimulation with LPS and LPS in the presence of TNF-α mAb. α mAb were observed for CXCL8, IL-1β, IL-6, and IL-10  at each time point by ELISA. A significant reduction in CXCL8 in the LPS-stimulated cell supernatants was observed from 32 hours onwards in the presence of TNF-α mAb, from 172.7 ng/mL (range: 150.6 to 237.0 ng/mL) to 138.3 ng/mL (range: 122.3 to 159.4 ng/mL) at 32 hours (p = 0.03).To determine whether autocrine binding of TNF-α receptors following autocrine binding of TNF-α, monocytes (from n = 7) were cultured following stimulation with 100 ng/mL LPS in the presence or absence of 10 μg/mL TNFR1 mAb or TNFR2 mAb or both. No effect was observed for TNF-α mRNA expression  or a mAb specific to either of the two TNF-α receptors .We wished to determine whether TNF-α led to a significant decrease in TNFR1 surface expression and conversely a small but significant increase in TNFR2. In addition, α itself. The results suggest that there is a positive feedback loop in monocytes, where TNFR1 expression is increased following signalling through TNFR2 by TNF-α. An example of flow cytometry overlay histograms from one subject are shown in Figures α autocrine feedback via TNFR2 could not occur. p = 0.09) and shows that by 6 and 22 hours after stimulation TNFR1 MFI ratio was significantly greater in the monocytes expressing both TNFR1 and TNFR2 than in those expressing TNFR1 only. In contrast, although there was a significant increase in TNFR2 expression over time in monocytes expressing TNFR2 alone or both receptors, there was no difference between those two monocyte types . Figures α mAb caused a small but significant increase in soluble TNFR1 consistent with an autocrine effect but had no effect on soluble TNFR2 concentration.To determine if the reduction in cell surface TNFR1 following TNF-α in the culture medium of monocytes initially increased, in the absence of . The relevance of this increase in TNFR1 secondary to TNF-α signalling via TNFR2 on monocytes, which can express both receptors, is unknown but would not negate the clinical efficacy of selectively blocking TNFR1 as any increase in TNFR1 expression on cells by TNFR2 ligation would still be blocked by the TNFR1 mAb. It is also important to note that TNFR1 blockade did not reduce TNFR2 expression on the monocyte surface thereby leaving it available to bind TNF-α and induce IL-10 production. The increase in TNFR2 receptors over time is likely to have occurred as a direct result of LPS stimulation .  Figurα disease is to become a viable strategy, it will be necessary to replicate and extend our experiments within the relevant disease cohorts.There are some limitations to our study. Firstly, as a practical issue, the monocyte yield is low compared to other leukocytes, and hence the number of cellular functions and experimental conditions we could study at one time was limited, thus requiring separate experiments to generate a global view. Secondly, it was not possible to include a negative control arm to experiments, whereby monocytes were not exposed to LPS. However, validation experiments revealed that monocytes cultured in the absence of LPS became apoptotic, especially at later time points. This meant that obtaining a sufficient number of events for each flow cytometry sample for viable unstimulated cells would not have been possible. Thirdly, if targeted TNFR1 blockade in TNF-Further delineation of the TNFR1 and TNFR2 intracellular signalling pathways in monocytes is also needed. The ability of TNFR2 ligation to induce signalling pathways that initiate IL-10 upregulation in the absence of any upregulation of proinflammatory cytokines, whilst TNFR1 ligation can do both, is of importance in abrogation of the inflammatory/anti-inflammatory balance and hence damage versus resolution. Determining the steps in the pathways should make it possible to separate these effects.α, present as a direct result of TNFR2 blockade, could enhance release of neutrophil products such as reactive oxygen species or the expression of adhesion molecules on endothelial cells. This would support the hypothesis that membrane bound TNFR2 has an important homeostatic role in clearing TNF-α from the pericellular environment and further support selective TNFR1 blockade in vivo.In addition, further work is needed to clarify the role of TNFR2 in monocytes. Whether monocytes that express both TNFR1 and TNFR2 differ substantially in function from those only expressing TNFR1 or TNFR2 and whether expression patterns change in response to a stimulus or as part of an illness are of particular interest. Intriguingly, approximately 15% to 25% of monocytes in both the freshly isolated and stimulated state expressed neither TNFR1 nor TNFR2 and the percentage of monocytes with this subtype increased slightly in response to LPS stimulation. The significance of this finding in the context of monocyte function is unclear and could be investigated further by separating monocytes by TNFR1/TNFR2 status by flow cytometry and tracking changes to receptor expression within each subgroup. Furthermore, it would be informative to examine the functional effects of selective TNFR2 blockade (both soluble and membrane bound). The cell-free supernatants from monocytes cultured in the presence or absence of TNFR2 mAb could be harvested and used as a stimulus for neutrophils or endothelial cells. This would identify if the excess pericellular soluble TNF- in vivo in TNF-α associated inflammatory diseases. Both macrophage phenotypes can be produced in vitro and hence the relative roles of TNFR1 and TNFR2 could be studied by macrophage subtype. However, this may not be relevant in vivo as recent studies suggest that macrophages are rarely derived from monocytes [Lastly, it would be informative to determine whether the imbalance in favour of IL-10 secretion in the presence of TNFR1 blockade has a beneficial effect on macrophage differentiation. IL-10 drives monocytes towards the more immunomodulatory M2 macrophage phenotype (rather than the type 1 proinflammatory M1 phenotype ), which onocytes . Thus, iα signalling leads to chronic inflammatory disease. TNFR1 is ubiquitous and its ligation by TNF-α has widespread proinflammatory effects, whereas TNFR2 expression is limited mainly to leukocytes and its function in humans has been less clear. The data presented here highlights that monocytes vary in their expression patterns of TNFR1 and TNFR2 but support the concept that proinflammatory effects of TNF-α occur via TNFR1 whilst TNFR2 predominantly plays an anti-inflammatory role by increasing IL-10 output and removing sTNF-α from the pericellular environment. These findings may support the current strategy of selective TNFR1 blockade for TNF-α driven chronic inflammatory disease.In susceptible individuals, disordered and excessive TNF-


Drosophila AD model expressing human BACE and APP695. Transgenic GULP1 significantly lowered the levels of both Aβ1-40 and Aβ1-42 without decreasing the BACE and APP695 levels. Overexpression of GULP1 also reduced APP/BACE-mediated retinal degeneration, rescued motor dysfunction and extended longevity of the flies. Our results indicate that GULP1 regulate APP processing and reduce neurotoxicity in a Drosophila AD model.Amyloidogenic processing of APP by β- and γ-secretases leads to the generation of amyloid-β peptide (Aβ), and the accumulation of Aβ in senile plaques is a hallmark of Alzheimer’s disease (AD). Understanding the mechanisms of APP processing is therefore paramount. Increasing evidence suggests that APP intracellular domain (AICD) interacting proteins influence APP processing. In this study, we characterized the overexpression of AICD interactor GULP1 in a  Caenorhabditis elegans CED-6, is an adaptor protein with multiple protein interaction domains/regions including an N-terminal phosphotyrosine-binding (PTB) domain, a centrally located leucine zipper and a carboxyl terminal proline/serine rich region [Human GULP1 (engulfment adaptor PTB-domain-containing 1), the homologue of h region . GULP1/Ch region -6. Recenh region , 8.in vivo remains to be determined.APP is a large type I transmembrane protein contains a large ectodomain and a small intracellular domain, namely AICD. Amyloidogenic processing of APP by β- and γ-secretases leads to the generation of amyloid-β peptide (Aβ), and accumulation of Aβ to form senile plaques is a hallmark of Alzheimer’s disease (AD). Although the mechanisms by which APP processing is regulated are still not fully understood, increasing evidence suggests that AICD interacting proteins can influence APP metabolism and Aβ generation . Inin vivo, we utilized the fruit fly, Drosophila melanogaster. Drosophila is a popular model for studying neurodegenerative diseases, as approximately 75% of all known human disease-associated genes are conserved in flies, including those implicated in AD [Drosophila [Drosophila AD model which expresses human APP695 wildtype and BACE genes. This Drosophila model is a powerful tool for AD research as the files exhibit a number of clinical AD neuropathology and symptomology for both familial/sporadic AD including Aβ aggregation and memory defects [To understand the effect of GULP1 ed in AD . In addiosophila , 13. In  defects . In thisin vivo, we created UAS-GULP1 transgenic flies, and crossed them to gmr-GAL4 to overexpress GULP1 in the fly eyes. Expression of GULP1 in gmr-GAL4/+; UAS-GULP1/+ animals (gmr > GULP1) was confirmed by Western blot analysis (data not shown), and there was no significant change in the number of rhabdomeres per ommatidium between gmr > GULP1 heterozygous and control gmr driver flies  [UAS-GULP1 flies to the gmr > APP, BACE flies to produce gmr-GAL4/+; UAS-APP695, UAS-BACE/UAS-GULP1 heterozygous flies . Western blot analysis revealed the expression of GULP1, APP and BACE transgenes in several independent lines  . We cross Figure . There ws Figure .elav-GAL4 driver was used to overexpress two independent UAS-GULP1 fly lines (3M and 5M) together with UAS-APP695 and UAS-BACE. Similar to the previous report, we observed that elav > APP, BACE heterozygous flies have reduced climbing activities, indicating that APP and BACE compromised CNS functions [Progressive decline in locomotor ability is one of the surrogate markers of neurotoxicity in a number of AD models -16. To iunctions    Figure  and 3D. ) Figure .Drosophila compound eye has been widely used for monitoring neurotoxicity and neurodegeneration [Drosophila AD model, we examined the ommatidial organization of gmr > GULP1, APP, BACE fly by pseudopupil assay. As shown in Figure Drosophila retinal structure. Yet, GULP1 overexpression reduced the effect of APP/BACE-mediated degeneration as gmr > GULP1, APP, BACE flies showed significant higher average number of rhabdomeres per ommatidium than gmr > APP, BACE flies  by which GULP1 alters APP processing is largely unknown. GULP1 has been shown to interact with ARF6 , a membe oocytes -28. As A oocytes , it is pDrosophila, dCED-6 is a key molecule in the Draper pathway mediating the glial engulfment of dying/injured neurons as well as presynaptic debris at the larval neuromuscular junction [elav-GAL4’s expression includes both neuronal and glial cells [In junction , 30, 31.al cells , it is pal cells -38. Addial cells , a neuroal cells .On the other hand, conflicting effect of GULP1 on APP processing are reported , 8. SimiDrosophila model does not only improve our understanding of the function of GULP1 in APP processing in vivo, but also opens a novel avenue for investigating the possibility of targeting GULP1-APP interaction to alter Aβ production.Although lowering Aβ by the enhancement of Aβ clearance using recombinant Aβ antibodies have shown promises in mouse model , Eli LilDrosophila embryos . The expressions of GULP1 in 5 independent lines  were determined by Western blotting.The full length human GULP1 cDNA was amplified by PCR and subcloned into GAL4-responsive pUAST expression vector, and microinjected into elav-GAL4 (458), gmr-GAL4 (1104), UAS-APP695, UAS-BACE (33797) were obtained from Bloomington Drosophila Stock Center, USA. Virgin females for crosses were collected within 8 hours at 25°C or 16 hours at 18°C.Fly stocks and crosses were raised on standard cornmeal medium with 1.25% agar; 10.5% dextrose; 10.5% cornmeal and 2.1% yeast. Flies were kept in incubators  maintained at 18 °C, 19°C, 25°C or 28°C as specified. Fifteen fly heads were collected and homogenized in 75 µl 2X SDS sample buffer containing 100mM Tris, 4% SDS, 0.2% Bromophenol blue, 20% Glycerol and 15 μl/ml β-mercaptoethanol. Lysates were then boiled for 10 minutes and separated by SDS/PAGE gels. Protein on the gels was transferred to nitrocellulose blotting membrane (PALL) using a wet blotting system (Bio-Rad). Blots were probed with the following antibodies: Anti-GULP1 ; Anti-BAHuman Aβ1-40 and Aβ1-42 in the fly heads were determined by using the human β40 and β42 ELISA kits (Millipore). In brief, 15 fly heads were homogenized in 50 µl ice-cold 1X RIPA buffer containing 50 mM Tris, 150 mM NaCl, 1% SDS, 1% NP-40, 0.5% sodium deoxycholate, pH 8.0 and Complete™ protease inhibitor (Roche). The homogenates were diluted 10-fold with 450 µl sample diluent and then cleared by centrifugation at 15,000 rpm for 5 minutes at 4°C. 200 µl of supernatant of each sample was added to ELISA plate with primary antibody. After overnight incubation at 4°C, the ELISA plate was washed with wash buffer. Streptavidin-peroxidase-conjugate was then added for colorimetric signal development at room temperature. The colorimetric reaction was stopped by adding stop solution, and signals were measured at 450 nm by using a microplate reader (Bio-Rad).Pseudopupil assay was performed essentially as described previously , 59. In Locomotor activity of the flies were determined at 20 dpe. In brief, group of 10 flies were placed at the bottom of a 15mL falcon tube. The number of flies that successfully climbed up a vertical distance of 8 cm or more was recorded. At least 80 flies of each genotype were analyzed in an experiment. Three independent experiment were performed.Groups of 15 flies were placed in a food vial. Dead flies were counted every 3 days. At least 70 flies were assayed for each genotype from three independent crosses.ARF6 activation assay was performed as described previously  by an acp < 0.05, **p < 0.01.The Mann-Whitney rank sum test was performed to compare mean differences between the average numbers of rhabdomeres per ommatidium in pseudopupil assay. One-way ANOVA test with Tukey post-hoc analysis was performed for ELISA analysis and climbing assay. Log-rank (Mantel-Cox) Test was performed for survival assay. A P-value of less than 0.05 was considered statistically significant. Significance is indicated as *


The data presented here are manually encoded characteristics of research papers in the area of Energy-Economy Nexus  that describe the methods, samples, and other details related to the individual estimations done in the examined empirical papers.Data cover papers indexed by Scopus, published in economic journals, written in English, after year 2000. In addition, papers were manually filtered to only those that deal with Energy-Economy Nexus investigation and have at least 10 citations at (at the time of query – November 2015). This data are to be used to conduct meta-analysis – associated dataset was used in Hajko  Specifications TableValue of the data•Data cover 104 top-cited Energy-Economy Nexus articles.•Suitable for meta-analysis.•Recorded for studies as a whole  and for individual estimation samples reported in the studies .12The data represent manually encoded characteristics of research papers in the area of Energy-Economy Nexus .All covered papers are indexed by Scopus, published in economic journals, written in English, after year 2000. In addition, papers were manually filtered to only those that deal with Energy-Economy Nexus investigation and have at least 10 citations at (at the time of query – November 2015).Variables describe both real-world characteristics and estimation characteristics used in the estimation samples in individual studies. ANDPUBYEAR > 1999 ) AND AND ) AND ) AND )Original Scopus query:


Neutrophils are the most important effector cells during the development of chronic obstructive pulmonary disease (COPD). Although neutrophil elastase is critical in cigarette smoke (CS)-induced lung parenchyma, the mechanism by which CS triggers elastase release from neutrophils remains unclear. Here we report that CS induction of autophagy in neutrophils by activating platelet- activating factor receptor (PAFR) promotes COPD progression in mouse. We found that the dead neutrophils were increased in bronchoalveolar lavage fluid from CS-exposed mice. Blocking PAFR suppressed the CS-induced autophagy in neutrophils, protected neutrophils from death, and reduced elastase release. Mechanistically, CS enhanced ROS production and High mobility group box 1 (HMGB1) expression through activation of PAFR. The elevated HMGB1 interacted with beclin1, which promoted the dissociation of Bcl-2 from beclin1 and the assembly of autophagy core complex. Moreover, the antagonism of PAFR by rupatadine, a prescription PAFR inhibitor, protected against the development of emphysema, and reduced the autophagic death of neutrophils after CS exposure. These results suggest that CS contributes to the pathogenesis of COPD partly by inducing a PAFR-dependent autophagic death of neutrophils. Therefore, PAFR may be a therapeutic target for COPD and inhibition of PAFR may provide potential therapeutic benefits in the treatment of patients with COPD. Chronic obstructive pulmonary disease (COPD) is one of the most common respiratory diseases that threaten the lives of the patients . The mosAutophagy is a conserved lysosomal degradation process in eukaryotic cells, which can be activated by many stimuli, including pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs) and endoplasmic reticulum (ER) stress . Basal aIn this study, we proposed that PAFR participates in CS-induced autophagy in neutrophils during the development of COPD. We found that CSE-induced neutrophils death and NE releasing depend partly on the activation of PAFR. Activation of PAFR in response to CSE exposure enhances the expression of HMGB1 by inducing reactive oxygen species (ROS) generation in neutrophils, which decreases the association of Bcl-2 and Beclin-1, promotes autophagic flux and autophagic cell death. Pharmacologic inhibition of PAFR by rupatadine, a prescription PAFR antagonist, protects against the development of the CS induced COPD in mouse. Collectively, these findings not only reveal a new mechanism of COPD development, but also suggest that PAFR on neutrophils is a promising target for COPD therapy.A 16-week chronic model of CS exposure was used to mimic COPD in C57BL/6 mice –26. HistIt has been confirmed that CS can induce autophagy in lung epithelial cells, fibroblasts and macrophages , 29. We Neutrophil death is an important source for neutrophil-derived proteinases such as elastase and cathepsin G . Indeed,To investigate the mechanism of PAFR-induced autophagy in CS-caused neutrophil death, we analyzed autophagic flux in CSE- and C-PAF- stimulated neutrophils using a LC3B-II/I ratio assay. CSE or C-PAF stimulation increased the level of LC3B-II, co-stimulation of the cells with CSE plus Bafilomycin A1 or C-PAF plus Bafilomycin A1 further increased LC3B-II, soluble and insoluble p62 levels in neutrophils, suggesting that both CSE and C-PAF caused LC3B-II accumulation is due to an enhanced formation of autophagosome Figure  and 5B. To verify whether PAFR could be a therapeutic target for the treatment of COPD, we observed the effects of rupatadine, a prescription dual antagonist for PAFR and histamine1 receptor (H1R), in mouse model of CS-induced COPD. H1R inhibitor loratadine was used as a control. Rupatadine, but not loratadine treatment significantly attenuated the enlargement of alveolar space Figure , reducedCigarette smoke is the most important independent risk factor for the development of COPD as well as lung cancer and idiopathic pulmonary fibrosis. However, CS contains thousands of chemicals, which results in extremely complicated network of the mechanism involved in the pathogenesis of CS induced COPD. The present study illustrates that CS induced neutrophil death and elastase release are partly dependent on PAFR-medicated autophagy. The association of Bcl-2 and beclin1 was suppressed by HMGB1 whose expression was increased in neutrophil after CS stimulation. Inhibition of PAFR not only decreased HMGB1 level, suppressed autophagy activity, decreased NE release, but also produced therapeutic efficacy in mouse model of CS induced COPD.Streptococcus pneumoniae is the most important bacteria during pathogenesis of COPD, which adherence to airway is mediated by PAFR [Previous findings indicated that the content of PAF is increased in BALF and blood from COPD patients, and CS exposure promotes the synthesis of PAFR agonist in CS-exposed mice . In addi by PAFR , but whe by PAFR , 41. It  by PAFR . Moreove by PAFR . In this by PAFR . Thus, f-/- mice shows resistance to CS induced emphysema and apoptosis [A number of studies indicate autophagy involved in the development of COPD. CS or CSE induces autophagy in human bronchial epithelial and in mouse airway cells by increasing the LC3B-II/I ratio , 44. In poptosis . Howeverpoptosis , 47, sugpoptosis . It alsopoptosis . Our resNeutrophil is often considered as the effector cells in the pathogenesis of COPD through releasing proteinases such as elastase . In addiDespite recent mechanistic insights into the pathogenesis of COPD, therapeutics for this chronic diseases remain a significant clinical unmet need. COPD is already the third most common cause of death in world with the pathologic changes of progressive airway obstruction, emphysema, and fibrosis of small airways , 2. Bron3 in a chamber  for 4 hr a day, 5 days per week, for 16 weeks. Cigarette smoke exposure was performed using The University of Kentucky reference research cigarettes 3R4F . Rupatadine (3 mg/kg) or Loratadine (3 mg/kg) was administered to the mice by oral gavage once a day during the last 4 weeks of CS exposure. 24 hr after the last administration, all the mice were anesthetized by avertin, and the lung function was measured. Lungs were excised and fixed for morphological evaluation or for measurement of protein expression.All animal procedures were conducted in accordance with the guidelines of ARRIVE and approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College (Permit No. 002802). All protocols were in accordance with the approved guidelines. Male C57 BL/6 mice   were maintained in animal facility at the Institute of Materia Medica under Specific Pathogen Free (SPF) conditions. For animal studies, mice were earmarked before grouping and then were randomly separated into groups by an independent person. COPD mouse model was done according to previously published methods with modification –26. BrieSaponin, 3-MA and C-PAF  was purchased from Sigma-Aldrich . CV-3988 was purchased from ENZO life sciences . Bafilomycin A1 was purchased from Calbiochem . Rupatadine was a generous gift from Zhejiang CiFu Pharmaceutics Inc. . Loratadine was purchased from National institutes for Food and Drug Control . Mouse neutrophil elastase, PAF and histamine ELISA kits were obtained from Cusabio Biotech Co., Ltd , whereas the other ELISA kits were purchased from eBioscience . Anti-mouse LC3B, neutrophil elastase antibodies were obtained from Abcam ; anti-mouse p62 antibody was purchased from Sigma- Aldrich. Anti-GAPDH antibody was purchased from Kangcheng Bio-tch . All the other antibodies were obtained from Cell Signaling technology.Mice were anesthetized with 400 mg/kg avertin by i.p injection and placed on a flexivent system . Then the mice were mechanically ventilated with a tidal volume of 10 ml/kg and respiratory rate of 150 breaths/min; 3 cmH2O positive end-expiratory pressure (PEEP) for evaluating lung function as described previously .After the lung tissues were removed, the specimens were fixed with 4% paraformaldehyde then embedded in paraffin. Hematoxylin and eosin (H&E) staining was performed to determine the mean linear intercept. For detecting the airway wall thickness in the lung, all the sections were scanned at × 200 magnification, and the airway thickness was calculated using software Image-Pro plus 5.1.BALF was obtained from CS or air exposed mice, and the PAF-AH activity in BALF was detected using PAF acetylhydrolase inhibitor screening kit (BioVision).Neutrophils in BALF and bone marrow (BM) from mice were isolated using Neutrophil Isolation Kit (Miltenyi Biotec) according to manufacturer’s guideline. To obtain BALF, mice were sacrificed by over dosage avertin. The trachea was exposed using scissors. Using a 23 G needle to slowly inject 1 ml cold PBS with 0.1 mM EDTA into the lung, then collect the BALF from lungs. To obtain BM, femurs and shins were isolated with scissors from sacrificed mice, and then the bits of bone were removed. Flush the femurs and shins with 3 ml cold RPMI-1640 medium using a 25 G needle. The BM neutrophils were cultured in RPMI-1640 medium supplemented with 10% FBS and 1000 U/ml penicillin and 1000 μg/ml streptomycin. For plasmid transfection, the expression vector was transfected into BM neutrophils using the monocyte nucleofector kit (Lonza) according to the manufacturer’s instructions. Delivery of HMGB1-siRNAs (RIBOBIO) or PAFR-siRNAs (Santa Cruz) into cells was carried out with Lipofectamine RNA interference MAX Transfection Reagent (Life Technologies) following the manufacturer’s instructions.Co-immunoprecipitation experiments were per-formed as described previously . BrieflyCigarette smoke extract (CSE) was prepared as previous described  by bubblCells were stained with both FITC-Annexin V and PI (Beyotime). After 15 min incubation at 37 °C, stained samples were analyzed with flow cytometer (BD). For analysis apoptotic neutrophil in BALF, the cells were also stained with APC-Ly-6G antibody (Biolegend), and then the Ly-6G positive cells were detected with FITC-Annexin V and PI by flow cytometer.For detect autophagic flux, bone marrow neutrophils were infected with mRFP-GFP-LC3 adenoviral , and incProteins were extracted from cells or lung tissues using RIPA buffer , and the protein concentrations were determined with BCA Protein Assay Kit. The RIPA buffer-insoluble fraction was washed with PBS and resuspended in Laemmli SDS sample buffer. Protein sample were separated using SDS-PAGE, transferred to a PVDF membrane, and subjected to immunoblot analysis using specific antibody. The signaling was captured by a LAS4000 Image Station . To evaluate the quantity of NE releasing from neutrophils, the culture supernatants were mixed with 5 × SDS loading, and the NE level was analyzed with immunoblot. The analysis of insoluble p62 was performed as previously report . For immBone marrow neutrophils were treated with CSE (5%), C-PAF (5 μg/ml), or CV3988 (30 μM) for 24 hr, and intercellular ROS level was evaluated with dichlorofluorescin diacetate (DCFDA) (Abcam). Cells were stained with 20 μM DCFDA at 37 °C for 30 min. Then the cells were washed once, and the dichlorofluorescein (DCF) level was detected with confocal or flow cytometer.Data are represented as the mean ± standard error of the mean (SEM). Student’s t test (2-tailed) was used to compare difference between two groups. One-way ANOVA with Tukey-Kramer’s comparison test was used to analyze difference among multiple groups. Generally, all assays were carried out with n ≥ 3 biological replicates. P < 0.05 was considered statistically significant.


Zerumbone is a known anti-cancer herbal compound. However, the actual protein target is not fully understood or known. This investigation focus on the association of zerumbone in HCT116 colon cancer cell proliferation and its link with TNF-alpha. The study shows that with the increasing concentration of zerumbone, there was a reduction of HCT116 cells proliferation based on the cell line study and hence higher TNF-alpha inhibition based on the TNF-alpha assay. The study also emphasizes on the computational aspect by investigating the molecular docking analysis of zerumbone against TNF-alpha. The docked complex was further validated using molecular dynamics simulation studies. The docking analysis observed that alpha-beta unsaturated carbonyl scaffold is an important moiety for the anti-cancer activity of zerumbone. Furthermore, the DFT analysis also confirms the reactivity nature of zerumbone based on the frontier molecular orbital analysis. It is also the third-leading cause of cancer-related deaths in US3. The risk factors include smoking, obesity, unhealthy diet etc4. The risk factors also include Crohn’s disease and ulcerative colitis which are part of inflammatory bowel disease5.Colorectal or colon cancers occur because of unhealthy lifestyle and old age and in some cases because of genetic factorsZingiber zerumbet Smith6. Its rhizomes have been used traditionally for folk medicine and pain relief 7.On the other hand, zerumbone has been tested for various anti-cancer activities including colon cancer. Zerumbone is a cyclic sesquiterpene extracted from the rhizomes of 8. It has also shown to inhibit human colonic adeno-carcinoma cell proliferation9. The present investigation deals with the anti-cancer activity of zerumbone against HCT116 cells (colon cancer cell line) and its subsequent role in TNF-alpha inhibition in cancer cell proliferation. The linkage of zerumbone inhibiting TNF-alpha and its role in the proliferation of colon cancer cell has never been reported or study.Moreover, recent study on zerumbone observed that zerumbone possess unique and potent anticancer activity against colon cancers cells11. Because of this chromosomal arrangement and their sequence homology TNF has also become known as TNF-alpha12. Although the cytotoxic activity of TNF has been well documented, the mechanism of TNF-induced lysis is not well understood13. In fact, the TNF is cytotoxic for many tumoral cell lines, whereas normal cells generally are considered resistant to this action14. In F17 cells, TNF treatment induced a classical form of apoptosis, while TNF induced a necrotic form of cell death in L-M cells13. The TNF-alpha is also a pleiotropic cytokine that activates T-cells via both TNF-Receptor (R-I and R-II) and mediates both apoptotic and survival signals17. The TNF-alpha mediates its biological functions predominantly via TNFR-I. Following binding of TNF-alpha to TNFR-I, the TNFR-associated death domain (TRADD) is recruited to TNFR-I forming a platform for downstream signaling19. Then, the TNRF-associated factor 2 (TRAF2) and receptor-interacting protein kinase 1 (RIPK1) are recruited to TRADD forming a signaling complex. The TRADD also recruits fas-associated death domain (FADD), which initiates activation of apical caspases resulting in activation of effector caspases, and apoptosis21. However, signaling through TNFR1 might also induce apoptosis in a caspase-independent manner22.The TNF gene, in both the mouse and human, is positioned in the major histocompatibility complex, close to the LT3 geneThe present study involves morphological changes of HCT116 Cells treated with zerumbone in various concentrations and the supernatant was utilize for the inhibition of TNF-alpha using Mouse TNF-alpha ELISA kit. The study was cross examined using molecular docking techniques with zerumbone against TNF-alpha enzyme (PDB ID 5MU8) and the stability of the protein-ligand docked complex confirmed by molecular dynamics simulation. Furthermore, the frontier molecular orbital study was carried out to understand the HOMO and LUMO energies of zerumbone that might contribute in the reactivity of the molecule at the active site of the enzyme.Z. zerumbet sample was collected from Moreh, Manipur, India (94.34217° E and 24.35172° N) and given the institutional accession number as IBSD/Z-42-23. Zerumbone was extracted from the oil extract of the fresh rhizomes and identified by FT-IR, Proton and 13 C NMR and mass spectrophotometer.4 cells/well. To adhere the cells it was incubated for 16 h and treated with cisplatin (25 µg/µL)  and zerumbone in various concentration  for 24 h. The cells from the media was collected and centrifuged for 15 min at 5000 rpm and the supernatant was utilize for the TNF-alpha assay using Mouse TNF-alpha ELISA kit  as per the standard protocol of the manufacturer.HCT116 cells were purchased from ATCC  and cultured in McCoy’s 5 A medium supplemented with 10% FBS having 100 units/ml penicillin and 100 μg/ml streptomycin solution in a humidified atmosphere of 5% CO2 at 37 °C. Cells were seeded in 24-well plate with a density of 5 × 10The release of TNF-alpha level in the HCT116 cells was examined for the zerumbone concentrations . Cisplatin, a known anti-cancer drug was used as positive control and the untreated cells were used as the negative control.https://www.graphpad.com/). All the graphs and figures were prepared using the mean values in GraphPad Prism 7.The results were expressed as the mean ± SEM for three replicates. The analysis was done in GraphPad Prism 7 (http://www.rcsb.org/pdb/explore.do?structureId = 5mu8) and loaded in Molegro Virtual Docker 6.02 (MVD). The binding site was predicted and set at the coordinates   was retrieved from Protein Data Bank ′′− 0.594128 * Csp2 − 0.061912 * ′′E-Intra (vdw)′′ + 0.464056 * HBond + 0.483368 * HeavyAtoms + 0.953672 * ′′E-Solvation′′ + 3.0229 * Nplus − 3.9493 * halogen − 1.00763 * N − 3.10696 * OS + 1.61426 * OH).24. The system was solvated using the equilibrated 3-point solvent model (spc216) and ions were added. The assembled system was relaxed by energy minimization and equilibrated using the NVT and NPT scale for 100 ps. The well-equilibrated was finally run for 30 ns MD production. The generated trajectory file was analyzed for RMSD, RMSF, SASA, Hbond and Radius of gyration.MD simulations were carried out for 5MU8, 5MU8-zerumbone and 5MU8-α-humulene docked complex. MD simulation was carried out using GROMACS version 5.1.2 operated on Ubuntu 16.04. The topology file for PDB ID 5MU8 was processed with OPLS-AA/L force field while 5MU8-zerumbone complex was processed with GROMOS96 43a1bind) calculations were carried out for the 30 ns simulated complexes of the 5MU8-Zerumbone complex and 5MU8-α-humulene complex using g_mmpbsa (http://rashmikumari.github.io/g_mmpbsa/). The Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) estimates the free energy interactions as defined by Kollman et al.25. The equation of the ΔGbind is represented by the following equationTo adhere the molecular docking simulation results and MD simulation trajectory analysis, the binding free-energy  charges for full natural bond order (NBO) analysis. The molecular orbital energies were considered for calculating the band energy gap ΔELUMO-HOMO.The conformation of the best docking pose of zerumbone and α-humulene was exported and DFT calculations were carried out using Gaussian 0927 to estimate the pharmacokinetics and their medicinal chemistry property. These parameters were calculated because drug development and discovery process involve assessment of absorption, distribution, metabolism and excretion and their toxic effects27. The newly introduced methods such as BOILED-Egg diagram, iLOGP and Bioavailability Radar map was also calculated for zerumbone and α-humulene. Lastly, LD50 and health effect analysis was carried out using ACD Ilab 2.0 (https://ilab.acdlabs.com/iLab2/) and comparative a chart was plotted to compare the two molecules.The Physicochemical property and ADME-Toxicity parameters of zerumbone and α-humulene were computed using SwissADMEThis article does not contain any studies with human participants or animals performed by any of the authors.The cell line assay of HCT116 observed the death of the cells after 24 hours of treatment with 20–30 μM zerumbone concentration Fig. . The celThe docking score of the best top five conformational pose of zerumbone is presented in Table Figures in vitro techniques is the first such report which is supported by computational studies. Moreover, the investigated enzyme is a TNF-α promotion signaling pathway that interferes with the proliferation of colon cancer cells. The strong interaction of zerumbone is because of the alpha-beta unsaturated carbonyl group which is a notable moiety that theater in certain unidentified targets and enzyme28. Similarly, Murakami et al. observed that the absence of alpha-beta unsaturated carbonyl group in compounds such as alpha-humulene, were unable to disrupt various tumour promoter enzymes such as 12-O-Tetradecanoylphorbol-13-acetate (TPA)7. In fact the difference in their structure lies in the carbonyl group present in zerumbone but not in alpha-humulene29. Thus, indicating that the alpha-beta unsaturated carbonyl group is the key component of zerumbone for its biological activity against cancer cell lines and protein targets30. The overall score revealed favourable binding energy and strong bonding which were confirmed from the docking scores with the TNF-α enzyme  values of the 5MU8-zerumbone complex and 5MU8-α-humulene complex were −52.10 kJ/mol and −40.23 kJ/mol respectively. The ΔGbind values suggest that 5MU8-zerumbone complex have better binding free-energy than the 5MU8-α-humulene complex.The binding free energy calculation of the protein-ligand interaction from the 100 snapshots extracted every 100 ps from the last 30 ns MD simulation stable trajectory is shown in Table In Table Moreover, the electrostatic energy and SASA energy were also favourable for the binding. These results also suggested that hydrophobic interaction played an important role in protein-ligand binding.LUMO - HOMO) was −0.12 eV and −0.08 eV for zerumbone and α-humulene which confirms that zerumbone had a lower band gap and will definitely have a strong binding affinity at the active site of TNF-alpha (5MU8). Additionally, the predicted NMR and IR spectra of zerumbone and α-humulene which optimized at DFT/B3LYP/LanL2DLZ level of theory are presented in Figs SF7–SF10 in supplementary materialFigures 50 of zerumbone is much higher than α-humulene.The Boiled egg diagram and bioavailability radar map of zerumbone and α-humulene are presented in Fig. 50.The present investigation confirms the inhibition of TNF-alpha by zerumbone and the decrease in HCT116 Cell proliferation in controlling colon cancer. The molecular docking study also confirms the binding mode of zerumbone at the active site of the enzyme. Moreover, the computational investigation such as the binding free-energy analysis on zerumbone and α-humulene point out that the presence of the alpha-beta unsaturated carbonyl group is a major driving force. Because, the compound α-humulene which lacks the alpha-beta unsaturated carbonyl group proved to be a bad inhibitor from the computational investigation as well as from the physiochemical property analysis and ADME-Toxicity analysis. In fact, the strong binding affinity and favourable docking score observed in zerumbone is because of the alpha-beta unsaturated carbonyl group which is also responsible for apoptosis. The 30 ns MD simulation confirmed the conformational stability based on the trajectory analysis and the DFT analysis confirms the reactivity nature of zerumbone based on the frontier molecular orbital analysis. Additionally, the physiochemical property and ADME-Toxicity analysis also confirms that it has good oral bioavailability and higher LDSupplementary Information