HCT116 cells and LS174 cells were employed to investigate the inhibiting effect of (+)-UA (<10 μM) on SCF-mediated migration of c-KIT(+) CRC cells. HCT116 cells were employed to investigate the molecular mechanisms. The results indicated that firstly, 8 μM (+)-UA decreased ATP content via uncoupling; secondly, 8 μM (+)-UA induced mTOR inhibition, thereby mediated activation suppression of PKC-A, and induced the autophagy of the completed autophagic flux that resulted in the autophagic degradation and transcriptional inhibition of c-KIT and the increase in LDH release; ultimately, 8 μM (+)-UA inhibited SCF-mediated migration of CRC c-KIT(+) cells. Taken together, 8 μM could be determined as the effective concentration for (+)-UA to inhibit SCF-mediated migration of CRC c-KIT(+) cells.Inhibition of tumor cell migration is a treatment strategy for patients with colorectal cancer (CRC). SCF-dependent activation of c-KIT is responsible for migration of c-KIT positive [c-KIT(+)] cells of CRC. Drug resistance to Imatinib Mesylate (c-KIT inhibitor) has emerged. Inhibition of mTOR can induce autophagic degradation of c-KIT. (+)-usnic acid [(+)-UA], isolated from lichens, has two major functions including induction of proton shuttle and targeting inhibition of mTOR. To reduce hepatotoxicity, the treatment concentration of (+)-UA should be lower than 10  Colorectal cancer (CRC) is characterized by the fourth most familiar cause of cancer-related death in the world . MetastaMast/stem cell growth factor receptor Kit (c-KIT), a tyrosine-protein kinase-type receptor for cytokine Kit ligand (SCF), plays a significant role in the regulation of cells migration . A previμM of (+)-UA can effectively mediate autophagic death or apoptosis in a time and dose dependent manner [μM seems to be the concentration threshold for hepatocytes to overcome (+)-UA mediated hepatocyte toxicity by increasing oxidative phosphorylation [μM. Nevertheless, there is still no research that involves whether or not the treatment of c-KIT(+) CRC cells with (+)-UA (<10 μM) can inhibit SCF-mediated cells migration.(+)-UA (GAS: 7562-61-0) is isolated from lichens that is widely used as an antimicrobial agent in Traditional Chinese Medicine . In factt manner , 21, 22.t manner . Furtherrylation . TherefoμM and 15.66 μM, respectively [μM) on SCF-mediated migration of c-KIT(+) CRC cells. And because HCT116 cell line is a suitable transfection host, HCT116 cells would be employed to investigate the molecular mechanisms for (+)-UA (<10 μM) to inhibit SCF-mediated migration of c-KIT(+) CRC cells.Notably, cell viability assays had indicated that the IC50 values for (+)-UA against human CRC HCT116 cells and LS174 cells were 17.7 ectively , 26. Furectively . In thisμsnic acid, Dulbecco's Modified Eagle's Medium (DMEM), William's E medium, thiazolyl blue tetrazolium bromide (MTT), Kit ligand (SCF), Go-6976, dimethylsulfoxide (DMSO), and fetal bovine serum (FBS) were purchased from Sigma-Aldrich . Antibiotic-antimycotic and puromycin were purchased from Life Technologies . Antibodies against c-KIT, PKC-A, phospho-PKC-A (Thr638), phospho-c-KIT (Tyr703), Tuberin (TSC2), phospho-ribosomal protein S6 kinase beta-1  at Thr389 site, phospho-Eukaryotic translation initiation factor 4E-binding protein 1  at Thr37/46 sites, Sequestosome-1 , microtubule-associated proteins 1A/1B light chain 3B , UBC9, TFAP2A, and autophagy protein 5 (ATG5) were purchased from Sigma-Aldrich .(+)-2 at 37°C.The human CRC HCT116 cells and LS174 cells were gifts from Laboratory of Molecular Biology, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, China. HCT116 cells and LS174 cells were cultured in RPMI1640 medium supplemented with 10% BSA in PBS, 100 U/mL penicillin, 100 mg/ml streptomycin, and 2 mM glutamine and kept in a humidified atmosphere of 5% COμM). After the treatments for 24 or 48 hours, Wound Healing Assay and Proliferation Assay were implemented, respectively. Before Migration Assay, cells were pretreated with 2, 4, or 8 μM of (+)-UA for 24 or 48 hours, respectively. Migration Assay was carried out using the transwell plates. Cells were seeded on upper chamber, and chemotaxis medium containing 10% FBS with or without SCF was added to lower chamber. Cells were divided into eight groups: (1) 10% FBS medium + cells without pretreatment, (2) 10% FBS medium + SCF (100 ng/ml) + cells without pretreatment, and (3-8) 10% FBS medium + SCF (100 ng/ml) + the cells after pretreatments -UA for 24 or 48 hours, respectively). After 12 hours of cells migration, migration indexes would be obtained. After that, the effective concentration and time for (+)-UA (<10 μM) to inhibit the proliferation and migration of HCT116 cells and LS174 cells would be determined. The results indicated that the effective concentration was 8 μM, and the effective time was 24 and 48 hours, respectively.HCT116 cells and LS174 cells were divided into five groups: (1) 1% FBS medium, (2) 1% FBS medium + SCF (100 ng/ml), (3-5) 1% FBS medium + SCF (100 ng/ml) + (+)-UA -UA or DSMO for 6, 12, 24, or 48 hours, respectively. Cellular ATP level and LDH level were detected. Then, cells were pretreated with 2, 4, or 8 μM of (+)-UA or DSMO for 24 or 48 hours, respectively. Caspase-3/7 (CASP3/7) activity measurements were implemented.Cells were pretreated with 2, 4, or 8 μM (+)-UA or DSMO for 24 or 48 hours, respectively. Since inhibition of TSC2 can induce the continuous activation of mTOR [μM, 48 hours), (3) siRNA targeting TSC2 , and (4) siRNA targeting TSC2  + (+)-UA . The levels of above-mentioned proteins were detected in these two experiments, respectively.Phosphorylated TSC2 can inhibit mTOR activity . Phospho of mTOR . SilenciμM, 24 or 48 hours), (4) SCF , (5-6) (+)-UA  + SCF , and (7) SCF  + Go-6976 . The levels of p-c-KIT, c-KIT, p-PKC-A, and p-PKC-A were detected, respectively. The results indicated that the treatment with 8 μM (+)-UA mediated the downregulation of c-KIT and p-c-KIT in 48 hours and attenuated SCF-mediated activation of PKC-A in 24 or 48 hours, respectively.Exogenous SCF was employed to induce phosphorylation of c-KIT (T703 site) and PKC-A (Thr638) . Go-6976μM, 48 hours), (5) (+)-UA  + SCF , and (6) siRNA targeting ATG5  + (+)-UA  + SCF . The levels of c-KIT and p-c-KIT were detected, respectively.ATG5 is a key protein that is responsible for autophagosome formation . SilenciμM, 48 hours), (5) (+)-UA  + SCF , and (6) siRNA targeting UBC9  + (+)-UA  + SCF . The protein levels of TFAP2A and SUMO-TFAP2A and the levels of protein and mRNA of UBC9 and c-KIT were detected, respectively.In autophagy process, upregulated UBC9-mediated increase in sumoylation of TFAP2A (SUMO-TFAP2A) will result in transcriptional inhibition of c-KIT –13. Sile5cells/well and were fed with 5% FBS. Until the cells grown to 95% confluence, monolayer cells were scratched to create a wound. The floating cells and 5% FBS medium were washed and removed. Then, the monolayer cells were fed with 1% FBS and treated with 2, 4, or 8 μM of (+)-UA or DSMO as control for 24 or 48 h, respectively. Photographs were taken to detect the relative wound width under high power microscope. Experiments were repeated three times.HCT116 cells or LS174 cells were plated on 6-well culture plates at the density of 3 × 106 cells/ml HCT116 cells or LS174 cells were seeded into the upper chamber of 24-well transwell plates and were starved in FBS-free medium for 2 h. After that, chemotaxis medium containing 10% FBS with or without SCF was added to lower chamber. Cells were incubated in FBS-free medium for 12 h at 37°C. Then, the cells on the upper chamber were removed. The cells in the lower chamber were stained. Photographs of cells migrated to the lower chamber were taken under an inverted microscope. The numbers of the cells were counted, and the data were expressed as Migration Index: compared to the number of cells in 10% FBS medium without SCF.After the pretreatments, 2 × 10μL) and cultured at 37°C for 4 h. Then BrdU solution was washed and removed. FixDenat (200 μl) was added. After incubation for 30 min, FixDenat was removed, and then anti-BrdU-POD (100 μl) was added to experimental wells. After washing for three times, substrate solution (100 μl) was added to experimental wells. And the optical density would be measured at 450 nm.HCT116 cells or LS174 cells were seeded into 96-well plates. After the pretreatments, proliferation assay was evaluated by BrdU Cell Proliferation Assay Kit  according to the manufacturer's protocol. Briefly, cells were treated with bromodeoxyuridine , according to the manufacturer's protocol.The activity of CASP3/7 was assayed by Caspase-Glo® 3/7 Assay Systems , according to the manufacturer's protocol.The cytotoxicity of (+)-UA was detected by Cytotoxicity LDH Assay Kit-WST® , according to the manufacturer's instructions.4 cells per well) and allowed to grow overnight to reach 60%~70% confluence. siRNA was mixed with Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) and Opti-MEM medium (Thermo Fisher Scientific) and incubated at room temperature for 30 min. siRNA-reagent complex was added to cells and cultured for 48 hr. Individual experiments were repeated 3 times. Transient transfection of siRNAs was performed according to the manufacturer's protocol.The small interfering RNA  for Human TSC2 (HSS111011), Human ATG5 (HSS190366), and Human UBC9 (HSS111130), and scrambled siRNA were from Thermo Fisher Scientific Inc. . HCT116 cells were seeded in 24-well plates . Prior to use on real-time PCR assays, the synthesized cDNA samples were diluted with nuclease-free water to a final concentration of 100 ng/μL (roughly a 1:20 dilution). After that, cDNA was amplified using the SYBR Premix and following SYBR gene expression assays . Primers were designed and shown as follows:  c-KIT: 5′‐TATACAACCCTGGCATTATGTCC‐3′ (forward), 5′‐TGCGAAGGAGGCTAAACCTA‐3′ (reverse);  UBC9: 5′‐TTATGAACTGGGAATGTGC‐3′ (forward),  5′‐CTTTGGAGGTGATGAGGG‐3′ (reverse).Total RNA was extracted from HCT116 cells using RNeasy Mini Kit (Qiagen), as recommended by the manufacturer's protocol. An on-column DNase digestion with the RNase-free DNase Set (Qiagen) was included to remove any genomic DNA contamination in RNA samples. RNA integrity was analyzed by agarose gel electrophoresis and its purity and concentration were calculated by measuring the optical density of the samples at 260 and 280 nm using a spectrophotometer. For single strand cDNA synthesis, 1 β-Actin RNA was described using the equation 2−ΔCT where ΔCT=CT of mRNA-CT of β-Actin [ The relative amount of mRNA to  β-Actin .Total protein was extracted from the HCT116 cells, and the cells were washed twice with PBS and resuspended with an ice-cold Tris-mannitol buffer  containing protease inhibitors and 0.02% sodium azide. Samples were centrifuged for 10 minutes at 950 g. After the concentrations of the proteins were determined, equal amounts of protein were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene difluoride membranes . The membranes were blocked with 5% non-fat dry milk in Tis-buffer saline-0.05% Tween (pH 7.4) and incubated with target antibodies (Abcam) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at 37°C for 2 h. The signals of targeted protein were tested by Amersham ECL . And glyceraldehydes-3-phosphate dehydrogenase  was used as an internal control to correct variation among the different samples.P < 0.05.Data were indicated as mean ± standard deviation (SD). The data were analyzed by one-way ANOVA followed by the least significant difference (LSD) test using SPSS 19.0 software. Statistical difference was accepted at μM of (+)-UA for 24 or 48 hours evidently inhibited SCF-mediated increases in the proliferation and migration of cells -UA for 24 or 48 hours demonstrated the significant inhibiting effects on SCF-mediated wound closure -UA on SCF-mediated wound closure of HCT116 cells and LS174 cells were visualized in Figures  Figures . Consist Figures .μM of (+)-UA for 24 or 48 hours significantly decreased cellular ATP content -UA for 48 hours evidently increased LDH release -UA did not significantly induce increase in CASP3/7 activity -UA for 24 or 48 hours markedly upregulated phosphorylation level of TSC2 and downregulated the phosphorylation levels of S6K1 and 4E-BP1 and induced a significant transformation of LC3B from cytosolic form (LC3B-I) to membrane-bound form (LC3B-II) -UA for 24 hours, but that occurred under the treatment with 8 μM (+)-UA for 48 hours -UA for 24 hours mediated the significant downregulation of p-PKC-A and the distinct upregulation of p-c-KIT; (2) the treatment of HCT116 cells with 8 μM (+)-UA for 48 hours induced the notable downregulation of p-PKC-A, p-c-KIT, and c-KIT; (3) the treatment of HCT116 cells with SCF for 30 minutes mediated the significant upregulation of p-c-KIT and p-PKC-A; (4) the treatment of HCT116 cells with 8 μM (+)-UA for 24 hours evidently attenuated SCF-induced upregulation of p-PKC-A but further enhanced SCF-induced upregulation of p-c-KIT; (5) the treatment of HCT116 cells with 8 μM (+)-UA for 48 hours not only reversed SCF-induced upregulation of p-c-KIT but also mediated a notable downregulation of c-KIT; (6) the effects of Go-6976 on p-PKC-A and p-c-KIT showed the same trend as that of the treatment with (+)-UA for 24 hours.Western blot Figures  revealedμM of (+)-UA for 48 hours not only reversed SCF-induced upregulation of p-c-KIT but also mediated a notable downregulation of c-KIT; (3) the downregulation of p-c-KIT and c-KIT, induced by (+)-UA, was rescued by silencing of ATG5.Western blot Figures  revealedμM, 48 hours) or (+)-UA  + SCF  evidently induced upregulation of UBC9, thereby mediating the increase in the upper band of TFAP2A (SUMO-TFAP2A), which ultimately resulted in downregulation of c-KIT; (3) the expression changes of UBC9 and SUMO-TFAP2A as well as c-KIT, induced by the treatment with (+)-UA + SCF, were reversed by silencing UBC9. Consistent with the results in Figures μM, 48 hours) or (+)-UA  + SCF  mediated a significant upregulation of UBC9 mRNA, which were remarkably attenuated by silencing UBC9  or (+)-UA  + SCF  induced an outstanding downregulation of c-KIT mRNA, which were significantly rescued by silencing UBC9  theing UBC9 .μM [μM) could effectively inhibit migration of c-KIT(+) CRC cells.Inhibition of tumor cells migration is a therapeutic strategy for CRC patients . SCF-depμM . This st(+)-UA Inhibited SCF-Mediated Migration of c-KIT(+) CRC Cells. c-KIT is expressed in HCT116 cell line and LS174 cell line, respectively [μM of (+)-UA for 24 or 48 hours could effectively inhibit SCF-mediated cell proliferation -UA for 24 or 48 hours significantly inhibited SCF-mediated cell migration -UA to inhibit SCF-mediated migration of CRC c-KIT(+) cells.ectively , 27. The Figures , which c Figures . More im Figures . The evi (+)-UA Induced ATP Decrease via Uncoupling. Lipophilic- and weakly acidic- (+)-UA would mediate mitochondrial proton shuttle (uncoupling) to induce ATP decrease [μM of (+)-UA for 24 or 48 hours observably decreased ATP levels -UA could mediate inhibition of cells migration probably via uncoupling-induced ATP decrease.decrease . ATP decdecrease . This st Figures , thereby Figures . These r(+)-UA Induced Inhibition of mTORC1 through the Functional Synergy between Uncoupling and the Targeting Inhibition of mTOR. Firstly, (+)-UA could mediate suppression of mTOR via the target-binding of mTOR [μM of (+)-UA for 24 or 48 h evidently upregulated TSC2 and downregulated the phosphorylation levels of S6K1 and 4E-BP1 -UA-Induced LDH Release Was Dependent on the Functional Synergy between Targeting Inhibition of mTOR and the Inductions of Proton Shuttle in Lysosomes and Autophagosome. (+)-UA could mediate inhibition of mTOR [μM (+)-UA for 24 hours, but that occurred under the treatment with 8 μM (+)-UA for 48 hours -UA for 48 hours evidently induced LDH release -UA for 24 or 48 hours did not remarkably induce the increase in CASP3/7 activity -UA for 24 or 48 h may not induce apoptosis, but probably mediate necrosis death. Therefore, the treatment of HCT116 cells with 8 μM of (+)-UA-mediated inhibition of cells migration should also be partially attributed to necrosis death pathway that was initiated by the functional synergy between targeting inhibition of mTOR and the inductions of proton shuttle in lysosomes and autophagosome. of mTOR . Meanwhi of mTOR . This st Figures , which i Figures , which s Figures , which s(+)-UA Induced Activity Suppression of PKC-A. Treatment of HCT116 cells with 8 μM (+)-UA within the early 24 or 48 hours evidently reversed SCF-induced upregulation of p-PKC-A -UA-mediated PKC-A activity inhibition in 24 hours further enhanced SCF-induced tyrosine autophosphorylation of c-KIT -UA for 24 or 48 hours remarkably reversed SCF-mediated cells migration -UA in 24 or 48 hours could inhibit SCF-mediated cells migration probably via activity suppression of PKC-A. Figures , which s Figures . Crucial Figures . Hence, Autophagic Degradation of c-KIT. In 24 h, 8 μM of (+)-UA-mediated proton shuttle in lysosomes and autophagosome resulted in the autophagy only with the incomplete autophagic flux -UA for 48 h not only attenuated SCF-induced upregulation of p-c-KIT but also downregulated c-KIT, which were rescued by silencing of ATG5 -UA for 48 h effectively induced autophagic degradation of c-KIT. Figures , which w Figures . With pr Figures . Sequest Figures . These eTranscriptional Inhibition of c-KIT in Autophagy Process. The upregulation of UBC9 is required for increasing autophagic flux in autophagy process [μM of (+)-UA for 48 h induced the significant upregulation of mRNA and protein of UBC9 -UA for 48 hours mediated a balance between transcription and degradation of UBC9 that maintains upregulated level of UBC9. UBC9 interacts with the C terminal region of TFAP2A, which lead to sumoylation of TFAP2A on lysine-10 [μM (+)-UA-mediated UBC9 upregulation effectively induced the increase in SUMO-TFAP2A -UA for 48 hours induced the notable downregulation of mRNA and protein of c-KIT, which were significantly rescued by silencing of UBC9. These data suggested that treatment of HCT116 cells with 8 μM of (+)-UA for 48 h induced transcriptional inhibition of c-KIT via upregulated UBC9-mediated suppression of AP-2 transcriptional activity. process . However process . This st Figures . This evysine-10 . Therefo Figures . After t Figures  and West Figures  verifiedμM, a relative low hepatocyte toxicity concentration, could be determined as the effective concentration to inhibit SCF-mediated migration of c-KIT(+) CRC cells. (+)-UA-mediated functional synergy between induction of proton shuttle and targeting inhibition of mTOR effectively regulated many processes related to cell migration, thereby inhibiting SCF-mediated migration of c-KIT(+) CRC cells.Repression of c-KIT is closely associated with reduced migration of c-KIT(+) cells . Therefo


Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are a family of structurally related proteins that transduces signals from members of TNFR superfamily and various other immune receptors. Major downstream signaling events mediated by the TRAF molecules include activation of the transcription factor nuclear factor κB (NF-κB) and the mitogen-activated protein kinases (MAPKs). In addition, some TRAF family members, particularly TRAF2 and TRAF3, serve as negative regulators of specific signaling pathways, such as the noncanonical NF-κB and proinflammatory toll-like receptor pathways. Thus, TRAFs possess important and complex signaling functions in the immune system and play an important role in regulating immune and inflammatory responses. This review will focus on the role of TRAF proteins in the regulation of NF-κB and MAPK signaling pathways. Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) form a family of intracellular signaling molecules, which in mammalian cells includes six typical members TRAF1–6) and an atypical member (TRAF7) –6 and an. The typOriginally identified as signaling adaptors of TNFR2 , the TRANuclear factor κB is a family of inducible transcription factors that regulate multiple biological processes, including immune and inflammatory responses, cell growth and survival, and oncogenesis , 10, 11.Two major signaling pathways, the canonical and noncanonical pathways, mediate activation of the NF-κB members . The canvia the ubiquitin/proteasome pathway, which prevents induction of p100 processing , where MAPKs mediate induction of various proinflammatory cytokines and chemokines required for host defense and inflammation  via the adaptor MyD88 to trigger the formation of a receptor-associated signaling complex that also contains TRAF6   . It is uin vivo along with TLR stimulation.TRAF6 regulation also involves various other factors. A member of the NOD-like receptors (NLR) family, NLRC3, has been shown to inhibit signaling from MyD88-dependent TLRs . NLRC3 ivia the adaptor protein TNF receptor-associated death domain (TRADD)  , 75. Theof TRAF2 , 76. TNFof TRAF2   deficient in either TRAF2 or TRAF5 are fully functional in TNFα-stimulated NF-κB activation, whereas MEFs lacking both TRAF members are severely attenuated, although not completely defective, in the NF-κB activation . These fvia binding to K63 ubiquitin chains conjugated to RIP1 by the cIAPs (via its ubiquitin-binding domain, UBAN (via binding to linear ubiquitin chains and contributes to the inhibition of NF-κB activation possibly by preventing NEMO recruitment (Recent studies suggest that the signaling function of TNFR1 complex I also involves another E3 ubiquitin ligase, LUBAC, which specifically conjugates linear ubiquitin chains , 90. LUBhe cIAPs  (Figure he cIAPs . Within he cIAPs , 92. Thein, UBAN , 93–95. in, UBAN . It has in, UBAN . The sigin, UBAN –100. In ruitment , 102.TRAF2 is also a mediator of TNFR2 signaling. In fact, TRAF2, along with TRAF1, was originally identified as signaling adaptors physically associated with TNFR2 . TRAF2 bWhile TRAF proteins are generally known as signaling adaptors that mediate activation of NF-κB and MAPK pathways, it is now clear that TRAF2 and TRAF3 are pivotal negative regulators of the noncanonical NF-κB pathway . An initvia a C-terminal region (via TRAF2–TRAF3 dimerization (Unlike TRAF3, TRAF2 does not bind the N-terminal region of NIK and only weakly interacts with NIK l region , 116. Inl region , 117. Mol region , 119. Bil region , 121. Itrization , 121. Thrization –125. Therization .Precisely how the TRAF–cIAP complex is assembled and regulates NIK stability is incompletely understood. More recent studies suggest that another TRAF member, TRAF1, may also be involved in cIAP E3 complex function and NIK regulation , 127. TRin vitro induction of proinflammatory cytokines by the TLR4 and TLR3 ligands LPS and polyIC (Compared to TRAF6, much less is known about the function of TRAF2 and TRAF3 in regulating TLR signaling. Nevertheless, recent studies suggest a role for both TRAF2 and TRAF3 in negatively regulating TLR-stimulated expression of proinflammatory cytokines . TRAF3 dd polyIC , 131. AtAn unexpected finding is the anti-inflammatory function of TRAF2 in the TLR signaling pathway . Like TRThe finding that both TRAF2 and TRAF3 negatively regulate proinflammatory cytokine induction by TLRs raises the question of whether these TRAF members have a common mechanism of action. TRAF3 has been shown to play a role in regulating receptor-proximal signaling in the MyD88 pathway  Figure . InteresIl23a induction (The role of TRAF2 and TRAF3 in MAPK regulation may be cell type specific, since bone marrow-derived macrophages and peritoneal macrophages derived from TRAF2-MKO and TRAF3-MKO mice do not display hyper-activation of MAPKs upon LPS stimulation , 131. Annduction . It appenduction . IRF5 isnduction , 18. Botnduction –145. TRAnduction . The conEmerging evidence suggests that the anti-inflammatory function of TRAF–cIAP complex in macrophages plays an important role in regulating antitumor immunity. Myeloid cell-specific deletion of TRAF2 promotes the generation of M1 type of macrophages in tumor microenvironment, which promotes recruitment of CD4 and CD8 effector T cells and enhances antitumor immunity . ConsistTumor necrosis factor receptor-associated factor proteins, particularly TRAF6 and TRAF2, have been established as pivotal mediators of NF-κB and MAPK pathways. In addition, recent studies have identified TRAF2 and TRAF3 as negative regulators of the noncanonical NF-κB pathway and proinflammatory TLR signaling pathways. Accumulating studies have also revealed potential molecular mechanisms that regulate the fate and signaling functions of TRAFs. These research progresses provide novel insights into the mechanism underlying the signaling function of TRAFs and shed light on the molecular basis of human diseases associated with TRAF-dependent signaling pathways. These scientific advances have also suggested new opportunities for designing therapeutic approaches in the treatment of inflammatory diseases and cancer. For example, the IAP antagonists, which induce degradation of cIAPs and, thereby, disruption of the TRAF–cIAP E3 complex, have been tested in animal models of cancer immunotherapy and obtained promising results.The recent progresses also raise a number of questions to be addressed in future studies. For example, the molecular mechanism by which TRAF6 mediates activation of NF-κB and MAPK pathways is incompletely understood. Although the E3 ligase activity of TRAF6 is important, controversies exist regarding how TRAF6 exerts the ubiquitin-dependent mechanism for activating the NF-κB and MAPK pathways. The signaling mechanism of TRAF2 is also elusive. In particular, whether the E3 ligase activity TRAF2 is required for TNFα-induced NF-κB activation is in debate. Furthermore, our current knowledge is largely based on cell line studies, and it will be important to have a mouse model for studying the role of ubiquitination in TRAF2 function under physiological conditions. Another important question is about the negative roles of TRAFs in regulating signaling. It is important to further define the mechanism by which TRAF2 and TRAF3 function together with cIAPs to mediate ubiquitin-dependent protein degradation and characterize the target proteins of this ubiquitin ligase complex. Finally, how TRAF proteins are regulated in different receptor pathways represents another important area for future research.Both authors have made significant contributions to this review.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. The reviewers HH and WZ and the handling Editor declared their shared affiliation.


NRG1 controls the formation of excitatory and inhibitory synapses in cortical circuits. While the expression of different NRG1 isoforms occurs during development, adult neurons primarily express the CRD-NRG1 isoform characterized by a highly conserved intracellular domain (NRG1-ICD). We and others have demonstrated that Nrg1 intracellular signaling promotes dendrite elongation and excitatory connections during neuronal development. However, the role of Nrg1 intracellular signaling in adult neurons and pathological conditions remains largely unaddressed. Here, we investigated the role of Nrg1 intracellular signaling in neuroprotection and stroke. Our bioinformatic analysis revealed the evolutionary conservation of the NRG1-ICD and a decrease in NRG1 expression with age in the human frontal cortex. Hence, we first evaluated whether Nrg1 signaling may affect pathological hallmarks in an in vitro model of neuronal senescence; however, our data failed to reveal a role for Nrg1 in the activation of the stress-related pathway p38 MAPK and DNA damage. Previous studies demonstrated that the soluble EGF domain of Nrg1 alleviated brain ischemia, a pathological process involving the generation of free radicals, reactive oxygen species (ROS), and excitotoxicity. Hence, we tested the hypothesis that Nrg1 intracellular signaling could be neuroprotective in stroke. We discovered that Nrg1 expression significantly increased neuronal survival upon oxygen-glucose deprivation (OGD), an established in vitro model for stroke. Notably, the specific activation of Nrg1 intracellular signaling by expression of the Nrg1-ICD protected neurons from OGD. Additionally, time-lapse experiments confirmed that Nrg1 intracellular signaling increased the survival of neurons exposed to OGD. Finally, we investigated the relevance of Nrg1 intracellular signaling in stroke in vivo. Using viral vectors, we expressed the Nrg1-ICD in cortical neurons and subsequently challenged them by a focal hemorrhagic stroke; our data indicated that Nrg1 intracellular signaling improved neuronal survival in the infarcted area. Altogether, these data highlight Nrg1 intracellular signaling as neuroprotective upon ischemic lesion both in vitro and in vivo. Given the complexity of the neurotoxic effects of stroke and the involvement of various mechanisms, such as the generation of ROS, excitotoxicity, and inflammation, further studies are required to determine the molecular bases of the neuroprotective effect of Nrg1 intracellular signaling. In conclusion, our research highlights the stimulation of Nrg1 intracellular signaling as a promising target for cortical stroke treatment.The schizophrenia risk gene  Nrg1 gene encodes more than 20 isoforms grouped into six types of proteins. While the Nrg1 extracellular domain displays high variability, all isoforms contain the epidermal growth factor- (EGF-) like domain located in the extracellular domain that is necessary and sufficient for activation of the ErbB4 receptor. In addition, many Nrg1 isoforms possess common transmembrane and intracellular domains that elicit Nrg1 intracellular  signaling [The major synaptogenic protein Neuregulin 1 (Nrg1) controls the formation of excitatory and inhibitory synapses in the cortex . The Nrgignaling . Neuronaignaling –8, releaDifferent studies have demonstrated that Nrg1/ErbB4 canonical signaling controls both the formation of cortical inhibitory circuits and the synchronization of neuronal activity , 9–12. Min vitro experiments, Nrg1 intracellular signaling failed to affect the tested hallmarks of neuronal senescence, namely, DNA damage and p38 mitogen-activated protein kinase (MAPK) activation, a pathway activated by age-related stressors such as inflammation and oxidative stress.Herein, we investigated the role of Nrg1-ICD signaling in mature neurons and tested a working hypothesis that this pathway may exert a neuroprotective role in mature cortical neurons. As our bioinformatic query revealed a decrease in NRG1 expression with age, we first hypothesized a link between Nrg1 intracellular signaling and aging. In our As previous studies indicated an increase in Nrg1 expression in response to hypoxia and that ErbB4 activation reduced ischemic infarct, we hypothesized that Nrg1 intracellular signaling might be neuroprotective in hypoxia and stroke.in vitro, while the activation of Nrg1 intracellular signaling reduced neuronal death following OGD. Notably, we discovered that Nrg1 also improved neuronal survival in an experimental model of cortical hemorrhagic stroke in vivo.Our data revealed that OGD conditions triggered the activation of Nrg1 intracellular signaling in cortical neurons NRG1 were downloaded from the Ensembl website (https://www.ensembl.org) and aligned using ClustalW (McGettigan PA 2007). We aligned the following sequences: panda , dog , cat , ferret , chimpanzee , human , gorilla , Macacus , marmoset , cavia , mouse , and sheep . The meta-analysis of NRG1 expression in human aging was described previously [The sequences of eviously . The meteviously .NRG1, for the human gene; NRG1, for the human protein; Nrg1, for the mouse gene; and Nrg1, for the mouse protein.We followed the naming of Neuregulin 1 used in NCBI and Ensembl websites for the use of capital letters and the NCBI Style Guide for the use of italic and nonitalic letters. Therefore, we used the following: Mus musculus, were previously generated and fully described [https://www.addgene.org/26976). Briefly, the original open reading frame was excised and the Nrg1 sequence was inserted after the human Synapsin promoter. Correct cloning was verified via sequencing. The vector for GFP expression under the human Synapsin promoter was kindly deposited into the Addgene repository by Bryan Roth (https://www.addgene.org/50465). The adeno-associated viral particles (AAVs), serotype 1, were produced and purchased by the Viral Vector Production Unit (UPV) of the Universitat Autonoma de Barcelona, Spain, according to standard protocols. The physical titer of the viral particle was also evaluated by immunofluorescence in neurons to assess the biological activity and obtain the same level of infection for the three viruses. The correct expression of the constructs was also verified by Western blot (not shown).The original constructs for the expression of GFP-tagged Nrg1-ICD and CRD-Nrg1 (Nrg1-FL), aka type III Nrg1, from escribed . The conμg mL−1; Sigma-Aldrich) for 1 min at 37°C. Cortices were washed Hank's solution, dissociated by mechanical disaggregation in 5 mL of plating medium  supplemented with 10% horse serum and 20% glucose), and counted in a Neubauer chamber. Cells were plated into precoated dishes with poly D-lysine (Sigma-Aldrich)  and placed into a humidified incubator containing 95% air and 5% CO2. The plating medium was replaced with equilibrated neurobasal media supplemented with B27 and GlutaMAX . Infection with viral vectors was performed on day 3 (D3). On D10, the culture medium was replaced with medium without GlutaMAX. For the paradigm of in vitro aging, the culture medium was substituted at D14 with a medium with B27 minus antioxidants  and cells were analyzed at D21.Primary cultures of cortical neurons were prepared from embryonic day 17-18 (E17-18) C57Bl6J mice, as previously described , 5. Brie2, 5 mM KCl, 137 mM NaCl, 0.4 mM KH2 PO4, 0.3 mM Na2HPO4, 0.5 mM MgCl2, 0.4 mM MgSO4, 25 mM HEPES, 4 mM NaHCO3) was equilibrated at 37°C and saturated with bubbling nitrogen. The culture medium was substituted with the saturated OGD medium in a custom-made hypoxia chamber at conditions with 0% oxygen. After the hypoxic stress, the OGD medium was changed again for the original conditioned culture medium.Oxygen-glucose deprivation (OGD) was performed as previously described . BrieflyImmunocytochemistry was performed according to a standard protocol as previously described . BrieflyThe images of the nuclear localization of Nrg1-ICD upon OGD were acquired with a Leica TCS SP2 AOBS  inverted laser scanning confocal microscope using the oil objective 63X Plan-Apochromat-Lambda Blue 1.4 N.A. The excitation wavelengths for fluorochromes were 488 nm (argon laser) for GFP and 405 nm (blue diode) for DAPI. Two-dimensional pseudocolor images were gathered with a size of 1024 × 1024 pixels and Airy 1 pinhole diameter. All confocal images were acquired using the same settings. ImageJ was used to analyze the confocal images. First, a Z-stack was generated and an image showing a nuclear plane was selected for each stack. GFP fluorescence intensity was measured in the soma and in the nucleus of each neuron using DAPI staining as a mask for delimiting the nucleus. A total of 27 neurons (9 neurons per well) were analyzed in three independent wells per each experimental condition.Antibodies used were phopsho-p38 MAPK , phospho-histone-h2a-x-ser139-antibody, phospho-H2AX ), GFP , and MAP2 . All secondary antibodies are Alexa conjugated from Life Technologies.https://pga.mgh.harvard.edu/primerbank/) [The neurons were cultured in normoxic conditions and analyzed at D14. Neuronal cultures were homogenized with TRIzol Reagent (Ambion/RNA Life Technologies Co.), and RNA was extracted with Direct-zolTM RNA minipreps (ZIMO research ref. R2052) following the manufacturer's instructions. RNA was quantified by absorbance at 260 nm using a NanoDrop ND-100 . Retrotranscription to first-strand cDNA was performed using a RevertAid H Minus First Strand cDNA Synthesis Kit . Briefly, 5 ng of synthesized cDNA was used to perform the qPCR using GoTaq® qPCR Master Mix  in ABI PRISM 7900HT SDS . All values were normalized with the housekeeping gene Gapdh. The primer pairs used in this study were designed and validated by PrimerBank (erbank/) .Primer sequences:Casp2 Casp3 Casp7 DAPK1 Apaf1 MAPK1 BAD BAX Bcl-2 BCL2I1 Akt1 MAPK14 Rbfox3 Gapdh The experiments were performed in 3-month-old C57Bl6J female mice provided by Charles River at the Centro de Biología Molecular “Severo Ochoa” (CSIC), Madrid, Spain. Experiments were supervised by the bioethics committee of the institute and performed in compliance with bioethical regulations of the European Commission. Animals were group housed with food and water ad libitum.μL of the virus was injected at a flow rate of 0.2 μL per minute with a Hamilton syringe. At the end of the injection, the needle was left in place for 5 minutes to allow the diffusion of the virus and then gently withdrawn. The injection of collagenase was performed with a similar procedure: 18 mU of collagenase  in a volume of 1 μL was injected at stereotaxic coordinates relative to bregma as follows: anteroposterior, 1.7; mediolateral, 1.0; and dorsoventral axes, 0.8. 1 μL of collagenase was injected at a flow rate of 0.2 μL per minute with a Hamilton syringe. At the end of the injection, the needle was left in place for 5 minutes to allow the diffusion of the collagenase and then slowly withdrawn. Mice were allowed to recover and sacrificed 24 hours after the injection of the collagenase.The stereotactic injection of viral vectors for the expression of GFP and Nrg1-ICD was performed 30 days before experimental stroke. The operation was performed according to standard procedure as previously described . Brieflyμm. Primary and secondary antibodies were diluted in PBS with 0.25% Triton and 4% BSA and incubated overnight in floating sections. The antibody for GFP staining  was diluted 1/500. The Alexa488-conjugated anti-chicken secondary antibody was purchased from Life Technologies. Pictures were taken with a Leica DM6000B microscope equipped with a Leica DFC35FX camera and an HCX PL FLUOTAR 10X 0.3 dry objective.Mouse brains were fixed and processed as previously described . BrieflyNRG1 gene generates six types of protein and more than 20 isoforms that are differentially expressed during development and regulated by neuronal activity [The activity . These iWe compared NRG1 sequences in different mammals, including mouse, human, and four nonhuman primates. Notably, we discovered the full conservation of the amino acids of the TM domain and the phosphorylation sites that control the processing of NRG1 in the species tested , 31, 32,n = 716individuals > 25 years of age. In all four cohorts, there existed a negative correlation between age and NRG1/ERBB4 expression, indicating a decrease in both NRG1 and ERBB4 expression with increasing age .Recent studies suggested the involvement of NRG1 in age-related neurodegenerative disorders –20 and tsing age . The decWe reasoned that the conservation of NRG1-ICD signaling, the decreased expression of NRG1/ERBB4 during aging, and the involvement of NRG1 in neurodegeneration might indicate a role of NRG1 intracellular signaling in neuroprotection in the aging brain.To address this working hypothesis, we first generated viral vectors to express CRD-Nrg1 full-length (Nrg1-FL) and Nrg1-ICD.We and others have previously established that the expression of the Nrg1-ICD mimics the end product of CRD-Nrg1 full-length processing by gamma-secretase and consistently activates Nrg1 intracellular signaling , 6. To iin vitro. In long-term culture, neurons accumulate various alterations related to senescence, including oxidative stress, DNA damage, and changes in lipid composition [Next, we began to evaluate the working hypothesis that Nrg1 intracellular signaling may be neuroprotective during neuronal aging position –37.Here, we focused on two hallmarks of neuronal senescence: the activation of the stress sensor p38 MAPK and DNA damage  to weaken the extracellular matrix of the cerebral capillaries, thereby causing focal blood extravasation and hemorrhage. We allowed the animals to recover for 24 hours before being sacrificed.Briefly, we injected brain cortices with a small amount of collagenase . Even given the limited number of surviving neurons, these data provide proof-of-principle for a neuroprotective role for Nrg1 intracellular signaling following hemorrhagic cortical stroke in vivo.We analyzed the infarcted areas and scored the survival of neurons expressing Nrg1-ICD in comparison to controls in the core and the peri-infarct area  share the ICD that is released upon proteolytic processing to elicit Nrg1 intracellular signaling. Our data indicates the phylogenetic conservation of the regions that regulate the activation of NRG1 intracellular signaling and the nuclear localization of Nrg1-ICD, supporting the relevance of this signaling pathway. Also, our bioinformatic analyses suggest that NRG1 mRNA expression decreases during aging. Given previous studies that implicate Nrg1 in age-related diseases such as neuroinflammation, neurodegeneration, and stroke , 18–24, in vitro model of neuronal senescence. Various pathways associated with aging in neurons, such as inflammation and oxidative stress, activate p38 MAPK signaling [in vitro experimental paradigm, Nrg1 intracellular signaling did not regulate these neuronal senescence hallmarks. However, these observations do not exclude Nrg1 intracellular signaling from being involved in other aspects of aging in cortical neurons or under different experimental conditions and further studies will be required to investigate this hypothesis.As Nrg1 expression decreased with aging, our first working hypothesis stated that Nrg1 intracellular signaling might be involved in senescence. To gain a preliminary insight into the role of Nrg1 in senescence, we tested whether Nrg1 intracellular signaling modulated the activation of p38 MAPK or DNA damage in an ignaling . Similarignaling . In our We next tested the hypothesis that Nrg1 intracellular signaling may be involved in stroke. Stroke is the second cause of death and disability worldwide, and as the risk of brain stroke increases with age, the societal burden of cortical infarct will increase significantly in the coming decades . Interesin vitro and in vivo. The expression of Nrg1-ICD delayed the onset of neural degeneration and improved neuronal survival in vitro. To address the role of Nrg1-ICD signaling in vivo, we employed collagenase injection as an experimental model of focal hemorrhagic stroke. Compared to other experimental setups, this model has the advantage of the controlled size and location of stroke by stereotactic injection. Thus, we provoked stroke in an area adjacent to the region previously infected to express Nrg1-ICD or GFP as control. Our data indicated that the activation of Nrg1 intracellular signaling improved neuronal survival in vivo. These findings support previous studies that indicate a crucial role for Nrg1 in stroke and unveil a novel function of Nrg1 intracellular signaling in neuroprotection in this pathology.Moreover, our data indicate the neuroprotective function of Nrg1 intracellular signaling in vitro [β leads to the enrichment and activation of LIMK1 in the synaptosomes, with this activation involved in synaptic transmission [Mechanistically, the molecular machinery underlying Nrg1 intracellular signaling also remains largely unknown. In particular, the direct molecular interactors of Nrg1-ICD remain poorly understood. The Nrg1-ICD can interact with LIM domain kinase 1 (LIMK1) in vitro , while tsmission . Consistsmission . Therefo2+, which in turn promotes the generation of ROS by inducing mitochondrial depolarization and the activation of ROS-generating enzymes [The fact that the Nrg1-ICD sequence does not contain overt DNA binding or transcriptional motifs has complicated the understanding of Nrg1 nuclear signaling . In spir enzymes . OxidatiAs the Nrg1-ICD promotes neurotransmission and the formation of excitatory connections , we specin vitro and in vivo. The finding that the highly conserved Nrg1-ICD has a neuroprotective role may foster the identification of new therapeutic targets to treat neurodegeneration following brain infarct.In conclusion, our work establishes a novel role for intracellular Nrg1 signaling in neuroprotection upon stroke


Background and Objective: Exosome component 5 (EXOSC5) is a novel cancer-related gene that is aberrantly expressed in various malignances. However, the molecular mechanism and biological role of EXOSC5 have not been explored in colorectal cancer (CRC). In this study, we investigated the functions and mechanisms by which EXOSC5 promotes the progression of CRC.Methods: EXOSC5 expressions in CRC cell lines and paired CRC and adjacent normal tissues were measured via quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry (IHC). In vitro experiments including colony formation, Cell Counting Kit-8 (CCK-8), and flow cytometry and in vivo tumorigenesis assay were performed to explore the effects of EXOSC5 on growth of CRC. The impacts of EXOSC5 on ERK and Akt signaling pathways were measured by Western blot.Results: The mRNA and protein expression levels of EXOSC5 were up-regulated in CRC as compared to adjacent normal tissues. IHC analysis indicated that high EXOSC5 level was positively associated with poor prognosis. EXOSC5 overexpression facilitated the growth of CRC cells, while EXOSC5 knockdown led to decreased proliferation, G1/S phase transition arrest. The oncogenic functions of EXOSC5 were associated with activation of the ERK and Akt pathways in CRC.Conclusion: EXOSC5 is overexpressed in CRC and promotes CRC growth partly through activation of ERK and Akt signaling pathways. Accordingly, EXOSC5 may be a novel oncogene, and acts as a therapeutic target, or prognostic factor for CRC. Colorectal cancer is one of the major threats to public health; it is the third most common malignancy and the third-leading cause of cancer-related deaths globally . CRC is The exosome complex is a multi-protein intracellular complex capable of degrading various types of RNA molecules. Apart from their function in RNAs degradation, components of the exosome complex have been associated in carcinogenesis. Goodarzi et al. indicated that transfer RNAs (tRNA) were promoters of breast cancer metastasis, in which tRNAGluUUC promoted metastasis by directly enhancing EXOSC2 expression . Stefansin vivo and in vitro experiments. Moreover, we have shown that EXOSC5 promoted CRC progression via ERK and Akt signaling pathways. As far as we know, this is the first study reporting the oncogenic and prognostic roles of EXOSC5 in the development of CRC.Our study gave evidence of a significant correlation between the up-regulation of EXOSC5 and survival outcomes of patients with CRC, and the oncogenic function was investigated via A total of 53 primary CRC tissues and paired normal tissues were collected from the Department of Gastrointestinal Surgery, Beijing Hospital (BJH). All tissues were frozen immediately in liquid nitrogen after surgical excision and stored at −80°C. From June 2011 to April 2013, archival formalin fixed and paraffin-embedded specimens of 159 patients with CRC who underwent colorectal surgery at BJH were obtained from the Department of Pathology. Written informed consent was obtained from all patients and the study was approved by the ethics committees of the BJH.The immortal normal colon cell lines (NCM460) and CRC cell lines  were purchased from American Type Culture Collection . Cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 10% fetal bovine serum (FBS). Dissolved by DMSO, AKT inhibitor MK-2206, or ERK inhibitor GDC-0994  were administered to suppress the phosphorylation of AKT or ERK signaling pathways. All cells were kept with 5% CO2 humidified atmosphere at 37°C.EXOSC5 short hairpin RNAs (shEXOSC5), EXOSC5 overexpression plasmid, and its control were purchased from GeneChem Company . The target sequences of the shRNAs were as follows: EXOSC5 shRNA#1: 5′- GAAGGTCAGCAAAGAGATT−3′; EXOSC5 shRNA#2: 5′- CGAAGTGATCCTGAGGCCGAAGATT−3′. HT29 and SW480 cells were transfected with the shEXOSC5 plasmid, and CACO2 and LOVO cells were transfected with the EXOSC5 overexpression plasmid. Cells transfected with vector were used as controls. The shRNAs and plasmids were transfected into cells using Lipofectamine 2,000 reagent (Invitrogen) following the manufacturer's protocol. Knockdown or overexpression of EXOSC5 was measured by qRT-PCR and western blot.TRIzol reagent  was used to extract the total RNA from tumor and normal tissue samples and cultured cells. PrimeScript RT Reagent Kit  was used to synthesize complementary DNA (cDNA). SYBR Green Premix Ex Taq  on an ABI 7,900 PCR system (Applied Biosystems) was used to conduct qRT-PCR. The primer sequences used were: EXOSC5 forward: 5'- ACTTTGCCTGCGAACAGAACC−3′, EXOSC5 reverse: 5′- CTCTTTGCTGACCTTCACCTC−3′; GAPDH forward: 5′- TGACTTCAACAGCGACACCCA−3′, and GAPDH reverse: 5′- CACCCTGTTGCTGTAGCCAAA−3′. GAPDH was used as endogenous control. The relative expression level of the target gene was calculated by 2-ΔCT and normalized to control cells. For the correlation study, the fold change of EXOSC5 expression level was calculated by 2-ΔΔCT.4 g, 4°C for 20 min. The concentrations of the supernatants were detected using the BCA Protein assay kit . Identical quantities of proteins were electrophoresed by SDS-PAGE , transferred onto PVDF membranes . After that, total proteins were incubated with primary antibodies at 4°C overnight. EXOSC5 was detected with a polyclonal antiEXOSC5 antibody . Then, the membrane was washed with PBS three times and incubated with secondary antibody. GAPDH  was used as standard loading control.The total proteins were extracted on ice for 60 min in RIPA buffer  with phosphatase inhibitors and protease . Cell lysates were centrifuged at 1.2 × 10The IHC was conducted using a standard immunoperoxidase staining procedure. The primary antibodies against EXOSC5  were used at concentrations of 1:200. The percentage of stained cells was scored as 0 (no staining), 1 (1–25%), 2 (26–50%), or 3 (51–100%). The staining intensity was scored as 0 (negative), 1 (weak), 2 (intermediate), or 3 (strong). The IHC score was calculated as the sum of both parameters, and the samples were grouped as negative (0), weak (1–2), moderate (3), and strong (4–6) staining. The tissues scored 0–3 were considered low expression, while those scored 4–6 were deemed high expression.The cell proliferation assays were performed with a CCK-8 Assay. At 48 h after transfection, cells were trypsinized and reseeded into 96-well plates . Then, 10 μL of CCK-8  solution was added to each well and absorbance at 450 nm was measured after 2 h of incubation. Anchorage-independent growth was determined by colony formation assays. 500 cells were seeded in 6-well plates, and were cultured for 2 weeks. The cells were then fixed using 4% paraformaldehyde, and stained with 0.1% crystal violet . The number of colonies with more than 50 cells was counted. The experiments were repeated in triplicate.6 CRC cells were injected subcutaneously into the dorsal region of nude mice. Tumor volume (mm3) was measured every 5th day, and tumor weight (mg) was measured at the end of the experiment.Four to six weeks old nude mice were obtained from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences . The animal studies were approved by the Fudan University Animal Ethics Committee. All the animals were housed and maintained under specific pathogen-free conditions. Hundred microliter PBS with 1 × 10The cells were fixed with 75% ethanol at 4°C overnight. Cells were stained with 50 μg/mL propidium iodide  containing RNaseI . The stained cells were analyzed by flow cytometry (Guava® easyCyte™). The experiments were performed in triplicate independently.P < 0.05 was considered statistically significant. All statistical analyses were performed by SPSS version 21.0 .ANOVA test was performed to compare the differences between groups. Kaplan-Meier with the log-rank test were used to analyze Survival data. Cox proportional hazards regression model was used to identify independent prognostic factors associated with overall survival. The correlations between IHC scores and clinicopathologic characteristics were tested using chi-square test and Logistic multivariate analysis. A EXOSC5 mRNA expression levels were determined in 53 matched CRC and adjacent non-tumor tissue samples by qRT-PCR . EXOSC5 Further, we detected expression levels of EXOSC5 mRNA and protein in CRC and colon cell lines by qRT-PCR and western blotting analysis. The results showed that EXOSC5 was significantly increased in HT29 and SW480 cells, and had relatively low expression in CACO2 and LOVO .In order to verify our findings, the gene expression of EXOSC5 was explored in The Cancer Genome Atlas (TCGA). The result revealed that EXOSC5 was remarkably up-regulated in Colon Adenocarcinoma (COAD) and Rectal Adenocarcinoma (READ) tissues compared to normal tissues .P = 0.001)  of CRC . Kaplan-= 0.001) . The cor= 0.001) .EXOSC5 expression in HT29 and SW480 was knocked down by two different shRNA (#1 and 2#). The shRNA suppression efficiency of EXOSC5 expression was confirmed by Western blot and qRT-PCR . Colony in vivo, subcutaneous tumorigenesis assays were performed in nude mice. Overexpression of EXOSC5 in the LOVO cells significantly augmented tumor growth in vivo  (Flow cytometry was conducted to investigate the changes in the cell cycle profile following EXOSC5 knockdown. The percentage of cells in the G1 phase was remarkably augmented for shEXOSC5-transfected HT29 and SW480 cells as compared to that of shNC-transfected cells ( < 0.05) . Whereas < 0.05) .To find the regulatory mechanism of EXOSC5 in CRC, we measured the phosphorylation and total protein levels of ERK and AKT in EXOSC5 overexpression and knockdown cell lines. Western blot demonstrated that the phosphorylated ERK and AKT decreased in HT29 and SW480 cells after EXOSC5 knockdown, but increased in CACO2 and LOVO cells after overexpression of EXOSC5 compared with negative control cells .The EXOSC5 overexpressing LOVO cells were then treated with an AKT inhibitor (MK-2206) or ERK inhibitor (GDC-0994). The result of Western blots showed that Akt and ERK phosphorylation was subsequently inhibited in the EXOSC5 overexpressing LOVO cells . MoreoveIn vitro experiments showed that overexpression of EXOSC5 promoted CRC cell proliferation and colony-forming ability. EXOSC5 also promoted tumor growth in vivo in mice. Moreover, EXOSC5 knockdown significantly suppressed the cell tumor growth and proliferation both in vitro and in vivo, and caused G1/S arrest. IHC staining indicated that overexpression of EXOSC5 was associated with worse prognosis, larger tumor size, and advanced tumor stage of CRC patients. Accordingly, EXOSC5 may play an important role in CRC development, and may be a promising biomarker for CRC.Our study demonstrated for the first time that both the mRNA and protein level of EXOSC5 were upregulated in CRC cell lines and tissue samples. in vivo and in vitro experiments.EXOSC5, also known as Rrp46p or CML28, is a non-catalytic component of the RNA exosome complex which has 3′->5′ exoribonuclease activity and participates in a multitude of cellular RNA processing and degradation events . EXOSC5 We provided the evidences in our study that EXOSC5 knockdown suppressed the phosphorylation of ERK and AKT pathways, and caused the increase in p21 and p27 expression. Flow cytometry also showed that the percentage of G1-phase cells increased significantly in EXOSC5 knockdown cells. Moreover, after treatment with the Akt inhibitor, MK-2206, and ERK inhibitor, GDC-0994, in EXOSC5 overexpressing cells, the cell growth was suppressed, the phosphorylation of Akt, and ERK was significantly decreased, and suppressed p21 and p27 expression were rescued.According to the above results, we could deduce that EXOSC5 promoting tumor cell growth and proliferation may be partly attributable to its facilitation effect of G1/S transition through ERK and Akt signaling pathways.Both PI3K/AKT and MAPK/ERK are classic intracellular signaling pathways that are correlated with the regulation of proliferation and cell cycle, and are previously reported to be activated in several kinds of cancer , 13. Actin vivo and in vitro via activating the ERK and Akt signaling pathways. Our study highlights the oncogenic role and potential mechanism of EXOSC5 in promoting CRC proliferation and tumorigenesis.In conclusion, for the first time we have revealed that EXOSC5 was overexpressed in CRC tissues and cell lines. Overexpression of EXOSC5 is significantly associated with tumor size as well as worse oncological outcomes. EXOSC5 promoted CRC cell growth All datasets generated for this study are included in the manuscript and/or the Written informed consent was obtained from all patients and the study was approved by the ethics committees of the BJH.HP and JP designed the study and wrote the draft of the manuscript. LJ revised the draft. LJ and SS conducted the data collection and analysis. HL and ZY prepared the figures and tables the manuscript.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.


Manubot adopts the contribution workflow used by many large-scale open source software projects to enable collaborative authoring of scholarly manuscripts. With Manubot, manuscripts are written in Markdown and stored in a Git repository to precisely track changes over time. By hosting manuscript repositories publicly, such as on GitHub, multiple authors can simultaneously propose and review changes. A cloud service automatically evaluates proposed changes to catch errors. Publication with Manubot is continuous: When a manuscript’s source changes, the rendered outputs are rebuilt and republished to a web page. Manubot automates bibliographic tasks by implementing citation by identifier, where users cite persistent identifiers , whose metadata is then retrieved and converted to a user-specified style. Manubot modernizes publishing to align with the ideals of open science by making it transparent, reproducible, immediate, versioned, collaborative, and free of charge.Open, collaborative research is a powerful paradigm that can immensely strengthen the scientific process by integrating broad and diverse expertise. However, traditional research and multi-author writing processes break down at scale. We present new software named Manubot, available at  Traditionally, scholarly manuscripts have been written in private by a predefined team of collaborators. But now the internet enables realtime open science, where project communication occurs online in a public venue and anyone is able to contribute. Dispersed teams of online contributors require new tools to jointly prepare manuscripts. Existing tools fail to scale beyond tens of authors and struggle to support iterative refinement of proposed changes. Therefore, we created a system called Manubot for writing manuscripts based on collaborative version control. Manubot adopts the workflow from open source software development, which has enabled hundreds of contributors to simultaneously develop complex codebases such as Python and Linux, and applies it to open collaborative writing. Manubot also addresses other shortcomings of current publishing tools. Specifically, all changes to a manuscript are tracked, enabling transparency and better attribution of credit. Manubot automates many tasks, including creating the bibliography and deploying the manuscript as a webpage. Manubot webpages preserve old versions and provide a simple yet interactive interface for reading. As such, Manubot is a suitable foundation for next-generation preprints. Manuscript readers have ample opportunity to not only provide public peer review but also to contribute improvements, before and after journal publication. PLOS Computational Biology Software paper.This is a open scholarly writing, in which anyone is able to contribute and the contribution history is preserved and public. We introduce Manubot, a new tool and infrastructure for authoring scholarly manuscripts in the open, and report how it was instrumental for the collaborative project that led to its creation.The internet enables science to be shared in real-time at a low cost to a global audience. This development has decreased the barriers to making science open, while supporting new massively collaborative models of research . Howeverhttps://github.com/greenelab/deep-review) to coordinate and manage contributions. GitHub is a platform designed for collaborative software development that is adaptable for collaborative writing. From the start, we made the GitHub repository public under a Creative Commons Attribution License (CC BY 4.0 at https://github.com/greenelab/deep-review/blob/master/LICENSE.md). We encouraged anyone interested to contribute by proposing changes or additions. Although we invited some specific experts to participate, most authors discovered the manuscript organically through conferences or social media, deciding to contribute without solicitation. In total, the Deep Review attracted 36 authors, who were not determined in advance, from 20 different institutions in less than two years.Based on our experience leading a recent open review , we discThe Deep Review and other studies that subsequently adopted the Manubot platform were unequivocal successes bolstered by the collaborative approach. However, inviting wide authorship brought many technical and social challenges such as how to fairly distribute credit, coordinate the scientific content, and collaboratively manage extensive reference lists. The manuscript writing process we developed using the Markdown language, the GitHub platform, and our new Manubot tool for automating manuscript generation addresses these challenges.Manubot supports citations by adding a persistent identifier like a Digital Object Identifier (DOI) or PubMed Identifier (PMID) directly in the text so that large groups of authors do not have to coordinate reference lists. When text is changed, Manubot automatically updates the manuscript’s web page so that all authors can read and edit from the latest version. Because manuscripts are created from GitHub repositories, Manubot supports a workflow where all edits are reviewed and discussed, ensuring that the collaborative text has a cohesive style and message and that authors receive precise credit for their work. These and other features support an open collaborative writing process that is not feasible with other writing platforms.There are many existing collaborative writing platforms  6]. In . In 6]. Existing platforms work well for editing text and are widely used for scholarly writing. However, they often lack features that are important for open collaborative writing, such as versatile version control and multiple permission levels. For example, Manubot is the only platform listed in issues for organization, opening a new issue for each discussion topic. For example, in a review manuscript like the Deep Review, this includes each primary paper under consideration. Within a paper’s issue, contributors summarize the research, discuss it , and assess its relevance to the review. In a primary research article, issues can instead track progress on specific figures or subsections of text being drafted. Issues serve as an open to-do list and a forum for debating the main messages of the manuscript.Manubot’s collaborative writing workflow adopts standard software development strategies that enable any contributor to edit any part of the manuscript but enforce discussion and review of all proposed changes. The GitHub platform supports organizing and editing the manuscript. Manubot projects use GitHub forked by individual contributors, creating a copy they can freely modify. A contributor then adds and revises files, grouping these changes into commits. When the changes are ready to be reviewed, the series of commits are submitted as a pull request through GitHub, which notifies other authors of the pending changes. GitHub’s review interface allows anyone to comment on the changes, globally or at specific lines, asking questions or requesting modifications [GitHub and the underlying Git version control system ,8 also sications . Convershttps://github.com/greenelab/deep-review/issues/575) and pull request (https://github.com/greenelab/deep-review/pull/638) on protein-protein interactions demonstrate this process in practice. A new contributor identified a relevant research topic that was missing from the review manuscript with examples of how the literature would be summarized, critiqued, and integrated into the review. A maintainer confirmed that this was a desirable topic and referred to related open issues. The contributor made the pull request, and two maintainers and another participant made recommendations. After four rounds of reviews and pull request edits, a maintainer merged the changes.The Deep Review issue (https://github.com/greenelab/deep-review/graphs/contributors) summarize all edits and commits from each author, providing aggregated information that is not available on most other collaborative writing platforms. Because the Manubot writing process tracks the complete history through Git commits, it enables detailed retrospective contribution analysis. These pull request and contribution tracking examples both come from Deep Review, the largest Manubot project to date, but illustrate the general principles of transparency and collaboration that are shared by all open Manubot manuscripts.We found that this workflow was an effective compromise between fully unrestricted editing and a more heavily-structured approach that limited the authors or the sections they could edit. In addition, authors are associated with their commits, which makes it easy for contributors to receive credit for their work. https://github.com/greenelab/meta-review/issues/124). Alternatively, a reviewer can post feedback with a pseudonymous GitHub account or have a trusted third party such as a journal editor post their comments anonymously. Authors can elect to respond to reviews in the GitHub issues or a public response letter (https://github.com/greenelab/meta-review/blob/v3.0/content/response-to-reviewers.md), creating open peer review.GitHub issues can also be used for formal peer review by independent or journal-selected reviewers. A reviewer conducting open peer review can create issues using their own GitHub account, as one reviewer did for this manuscript  and the corresponding rendered HTML (https://manubot.github.io/rootstock/v/091ca8d85c8ef2d7af16fcc8d2ed3ebcbc187f13/).With Manubot, manuscripts are written as plain-text Markdown files. The Markdown standard itself provides limited yet crucial formatting syntax, including the ability to embed images and format text via bold, italics, hyperlinks, headers, inline code, codeblocks, blockquotes, and numbered or bulleted lists. In addition, Manubot relies on extensions from Pandoc Markdown to enable citations, tables, captions, and equations specified using the popular TeX math syntax. Markdown with Pandoc extensions supports most formatting options required for scholarly writing  but currBy virtue of its readable syntax, Markdown is well suited for version control using Git. Markdown treats a single line break between text as a space and requires two-or-more consecutive line breaks to denote a new paragraph. For optimal tracking of Markdown files with Git, we recommend placing each sentence on its own line. This convention allows Git to display diffs on a per sentence basis, avoids unnecessary reflows associated with line wrapping, and supports easy rearrangement of sentences.http://hackage.haskell.org/package/pandoc-citeproc). Therefore, Manubot removes invalid fields according to the CSL Data specification (https://github.com/citation-style-language/schema). In cases where automatic retrieval of metadata fails or produces incorrect references—which is most common for URL citations—users can manually provide the correct metadata using common reference formats. Manual metadata also supports references without standard identifiers, such as print-only newspaper articles.Manubot includes an additional layer of citation processing, currently unique to the system. All citations point to a standard identifier, for which Manubot automatically retrieves bibliographic metadata such as the title, authors, and publication date. http://editor.citationstyles.org/searchByName/) or build their own [Manubot formats bibliographies according to a CSL style specification. Styles define how references are constructed from bibliographic metadata, controlling layout details such as the maximum number of authors to list per reference. Manubot’s default style emphasizes titles and electronic (rather than print) identifiers and applies numeric-style citations . Alternaheir own . As a rehttps://pandoc.org/) to convert manuscripts from Markdown to HTML, PDF, and optionally DOCX outputs. Pandoc also supports Journal Article Tag Suite (JATS), a standard format for scholarly articles that is used by publishers, archives, and text miners [https://vega.github.io/vega-lite/), Bokeh (https://bokeh.pydata.org/), or Plotly (https://plot.ly/) [Manubot uses Pandoc (t miners –27. Pandlot.ly/) ,29.Manubot comes with several “plugins” that can be included in manuscripts exported as HTML. These plugins add special interactive features that enhance the user experience of viewing and reading manuscripts . For exaThe Manubot “front-end”  was developed in line with current best practices and user expectations of the modern web. The plugins use standard technology built in to most major web browsers, allowing them to be relatively lightweight, modular, and easy to configure.Manubot performs continuous publication: Every update to a manuscript’s source is automatically reflected in the online outputs. The approach uses continuous integration (CI) –32, spechttps://github.com/greenelab/meta-review. When this repository changes, Travis CI rebuilds the manuscript (https://travis-ci.org/greenelab/meta-review). If successful, the output is deployed back to GitHub (to dedicated output and gh-pages branches). As a result, https://greenelab.github.io/meta-review stays up to date with the latest HTML manuscript. Furthermore, versioned URLs, such as https://greenelab.github.io/meta-review/v/4b6396bcefd1b9c7ddf39c1d3f0b3eab2dd63f31/, provide access to previous manuscript versions.To illustrate, the source GitHub repository for this article is https://opentimestamps.org/) to timestamp the HTML and PDF outputs on the Bitcoin blockchain. This procedure allows one to retrospectively prove that a manuscript version existed prior to its blockchain-verifiable timestamp [The idea of the “priority of discovery” is important to science, and Vale and Hyman discuss the importance of both disclosure and validation . In theiimestamp ,34–37. Timestamp . Since t{{total_commits}} Git commits so that the commit count can be automatically updated.Manubot and its dependencies are free of charge and largely open source. It does rely on gratis services from two proprietary platforms: GitHub and Travis CI. Fortunately, lock-in to these services is minimal, and several substitutes already exist. Manubot provides a substantial step towards end-to-end document reproducibility, where every figure or piece of data in a manuscript can be traced back to its origin  and is whttps://github.com/manubot/rootstock, referred to as Rootstock, demonstrates Manubot’s features and serves as a template for users to write their own manuscripts with Manubot. The current setup process includes cloning the Rootstock repository, rebranding it to the user’s manuscript, and configuring continuous integration. The setup process is complex but must only be performed once per manuscript. Incorporating new Manubot features into an existing manuscript is also possible by pulling the latest commits from Rootstock, which sometimes involves resolving Git conflicts.An example repository at https://github.com/manubot/try-manubot.Contributing to a manuscript is less technical and can be performed entirely through GitHub’s web interface, as discussed in the contribution workflow section and demonstrated in Pacific Symposium on Biocomputing, we led a working group where 17 conference participants contributed to a different demo manuscript (https://git.dhimmel.com/psb-manuscript/). Based on this experience, we believe most computational scholars have the expertise to contribute to a Manubot manuscript. Proficiency with Manubot requires familiarity with Markdown, Git, GitHub, and continuous integration. While these tools do present a barrier to entry, they are also highly applicable outside of Manubot and increasingly part of the standard curriculum for computational scholars. For example, Markdown is used for documenting Jupyter and R Markdown notebooks.At the 2019 https://github.com/greenelab/scihub-manuscript)—performed openly on GitHub from its inception—investigated Sci-Hub’s repository of pirated articles [https://github.com/greenelab/scihub-manuscript/issues/17) the initial preprint from this study in a series of tweets, pointing out a major error in one of the analyses. Within hours, the authors used Markdown’s strikethrough formatting in Manubot to cross-out the errant sentences  at https://greenelab.github.io/scihub-manuscript/v/8fcd0cd665f6fb5f39bed7e26b940aa27d4770ba/, thereby alerting readers to the mistake and preventing further propagation of misinformation. One month later, a larger set of revisions (https://github.com/greenelab/scihub-manuscript/pull/19) explained the error in more detail and was included in a second version of the preprint. As such, continuous publication via Manubot helped the authors address the error without delay, while retaining a public version history of the process. This Sci-Hub Coverage Study preprint was the most viewed (http://web.archive.org/web/20171221221858/http://www.prepubmed.org/top_preprints/) 2017 PeerJ Preprint, while the Deep Review was the most viewed 2017 bioRxiv preprint [Since its creation to facilitate the Deep Review, Manubot has been used to write a variety of scholarly documents. The Sci-Hub Coverage Study  [https://zach-hensel.github.io/low-noise-manuscript/) [https://openclimatedata.github.io/global-emissions/), visual perception (https://laurentperrinet.github.io/2019-05_illusions-visuelles/) [https://trang1618.github.io/tpot-fss-ms/) [https://jmonlong.github.io/manu-vgsv/) [https://yt-project.github.io/yt-3.0-paper/). Manubot is also being used for documents beyond traditional journal publications, such as research tips (https://benjamin-lee.github.io/deep-rules/), quality standards (https://indigo-dc.github.io/sqa-baseline/) [https://greenelab.github.io/manufund-2018/), progress reports (https://greenelab.github.io/czi-hca-report/), undergraduate research reports (https://zietzm.github.io/Vagelos2017/) [https://slochower.github.io/synthetic-motor-literature/), and lab notebooks. Finally, manuscripts written with other authoring systems have been successfully ported to Manubot, including the Bitcoin Whitepaper (https://git.dhimmel.com/bitcoin-whitepaper/) [https://git.dhimmel.com/rephetio-manuscript/) manuscript [Additional research studies are being authored using Manubot, spanning the fields of regulatory genomics (script/) , synthetscript/) , climateuelles/) , machinefss-ms/) , computau-vgsv/) , and datseline/) , grant pos2017/) , literatepaper/)  and Projnuscript .https://kipoi.org/) model zoo for genomics [PeerJ:The manubot Python package provides easy access to Manubot’s citation-by-identifier infrastructure, whose functionality extends beyond just Manubot manuscripts. For example, the Kipoi , environment management (e.g. from Conda to Docker), and repository hosting (e.g. from GitHub to GitLab). In addition to simplifying setup, such migrations may also present the opportunity to decrease dependency on proprietary services and address other Manubot shortcomings, such as the current inability to view rendered manuscripts produced by pull request builds.pro bono assistance to upgrade or troubleshoot manuscripts. Users can open an issue (https://github.com/manubot/rootstock/issues) at the Rootstock repository for help.Upgrading a Manubot instance is an opt-in procedure. Therefore, when we introduce fundamental changes, existing manuscripts continue to function. However, large Rootstock changes can make upgrading existing manuscripts difficult. We are happy to provide users Journal of Open Source Software [One strategy to grow Manubot usage is to identify a specific user group or use case for which Manubot can be widely adopted. For example, a journal may decide to build their publishing workflow around Manubot, such that submissions would consist of a Manubot repository. This application would be most suitable for journals that currently use GitHub for submissions and publishing, such as the Software . Manubothttps://github.com/Benjamin-Lee/deep-rules/blob/cfb7f744573ca0532a19ca1a8e9473a555cf8eb2/contributors.md). This provides a transparent way to record contributions such as experimental research that generated data for the manuscript and discuss whether they meet that project’s authorship criteria. Contribution transparency helps prevent ghostwriting [Manubot does not impose any restrictions on authorship. It allows authors to adhere to the author inclusion and ordering conventions of their field, which vary considerably across disciplines . Some Matwriting  and is eTo determine authorship for the Deep Review, we followed the International Committee of Medical Journal Editors (ICMJE) guidelines and used GitHub to track contributions. ICMJE recommends authors substantially contribute to, draft, approve, and agree to be accountable for the manuscript. We acknowledged other contributors who did not meet all four criteria, including contributors who provided text but did not review and approve the complete manuscript. Although these criteria provided a straightforward, equitable way to determine who would be an author, they did not produce a traditionally ordered author list. In biomedical journals, the convention is that the first and last authors made the most substantial contributions to the manuscript. This convention can be difficult to reconcile in a collaborative effort. Using Git, we could quantify the number of commits each author made or the number of sentences an author wrote or edited, but these metrics discount intellectual contributions such as discussing primary literature and reviewing pull requests. Therefore, we concluded that it is not possible to construct an objective system to compare and weight the different types of contributions and produce an ordered author list .To address this issue, we generalized the concept of “co-first” authorship, in which two or more authors are denoted as making equal contributions to a paper. We defined four types of contributions , from maDrosophila genome [Papers with hundreds or thousands of authors are on the rise, such as the article describing the experiments and data that led to the discovery of the Higgs Boson   and Mastering Ethereum (https://github.com/ethereumbook/ethereumbook)—written on GitHub in AsciiDoc format have engaged hundreds of contributors. Both Homotopy Type Theory and Mastering Bitcoin continue to be maintained years after their initial publication. A 2017 perspective on the future of peer review was written collaboratively on Overleaf, with contributions from 32 authors [The Deep Review was not the first scholarly manuscript written online via an open collaborative process. In 2013, two dozen mathematicians created the 600-page Homotopy Type Theory book, writing collaboratively in LaTeX on GitHub ,66. Two  authors . While d authors ,69. The Open writing also presents new opportunities for distributing scholarly communication. Though it is still valuable to have versioned drafts of a manuscript with digital identifiers, journal publication may not be the terminal endpoint for collaborative manuscripts. After releasing the first version of the Deep Review , 14 new https://github.com/cboettig/knitcitations) package enables citation by DOI or URL in R Markdown documents. Examples of continuous integration to automate manuscript generation include gh-publisher (https://github.com/ewanmellor/gh-publisher) and jekyll-travis (https://github.com/mfenner/jekyll-travis), which was used to produce a continuously published webpage (http://book.openingscience.org/) for the Opening Science book [Concepts for the future of scholarly publishing extend beyond collaborative writing –74. Pandnce book ,78. Bindnce book , Distillnce book , Idyll [nce book , and Stence book ,82 suppoJournal of Open Source Software [Several other open science efforts are GitHub-based like our collaborative writing process. ReScience  as well Software , rely onSoftware . GitHub Software , and colThere are potential limitations of our GitHub-based approach. Because the Deep Review pertained to a computational topic, most of the authors had computational backgrounds, including previous experience with version control workflows and GitHub. In other disciplines, collaborative writing via GitHub and Manubot could present a steeper barrier to entry and deter participants. In addition, Git carefully tracks all revisions to the manuscript text but not the surrounding conversations that take place through GitHub issues and pull requests. These discussions must be archived to ensure that important decisions about the manuscript are preserved and authors receive credit for intellectual contributions that are not directly reflected in the manuscript’s text. GitHub supports programmatic access to issues, pull requests, and reviews so tracking these conversations is feasible in the future.https://github.com/greenelab/deep-review/blob/v1.0/CONTRIBUTING.md) that discussed norms in the areas of text contribution, peer review, and authorship, which we identified in advance as potential areas of disagreement. Our contributor guidelines required verifiable participation for authorship: either directly attributable changes to the text or participation in the discussion on GitHub. These guidelines did not discuss broader community norms that may have improved inclusiveness. It is also important to consider how the move to an open contribution model affects under-represented minority members of the scientific community [https://www.contributor-covenant.org/) [In the Deep Review, we established contributor guidelines (ommunity . Recent ommunity . Conferent.org/) . We woulScience is undergoing a transition towards openness. The internet provides a global information commons, where scholarship can be publicly shared at a minimal cost. For example, open access publishing provides an economic model that encourages maximal dissemination and reuse of scholarly articles ,88,89. MGreater openness in both research methods and publishing creates an opportunity to redefine peer review and the role journals play in communicating science . At the Manubot is an ideal authoring protocol for real-time open science, especially for projects that are already using an open source software workflow to manage their research. While Manubot does require technical expertise, the benefits are manyfold. Specifically, Manubot demonstrates a system for publishing that is transparent, reproducible, immediate, permissionless, versioned, automated, collaborative, open, linked, provenanced, decentralized, hackable, interactive, annotated, and free of charge. These attributes empower integrating Manubot with an ecosystem of other community-driven tools to make science as open and collaborative as possible.S1 VideoThis screen recording demonstrates how to propose edits to a Manubot manuscript via GitHub. In the video , a contr(MP4)Click here for additional data file.