Engineering microorganisms to grow on alternative feedstocks is crucial not just because of the indisputable biotechnological applications but also to deepen our understanding of microbial metabolism. One-carbon (C1) substrate metabolism has been the focus of extensive research for the prominent role of C1 compounds in establishing a circular bioeconomy. Methanol in particular holds great promise as it can be produced directly from greenhouse gases methane and carbon dioxide using renewable resources. Synthetic methylotrophy, i.e. introducing a non-native methanol utilization pathway into a model host, has therefore been the focus of long-time efforts and is perhaps the pinnacle of metabolic engineering. It entails completely changing a microorganism’s lifestyle, from breaking up multi-carbon nutrients for growth to building C-C bonds from a single-carbon molecule to obtain all metabolites necessary to biomass formation as well as energy. The frontiers of synthetic methylotrophy have been pushed further than ever before and in this review, we outline the advances that paved the way for the more recent accomplishments. These include optimizing the host’s metabolism, “copy and pasting” naturally existing methylotrophic pathways, “mixing and matching” enzymes to build new pathways, and even creating novel enzymatic functions to obtain strains that are able to grow solely on methanol. Finally, new approaches are contemplated to further advance the field and succeed in obtaining a strain that efficiently grows on methanol and allows C1-based production of added-value compounds. Synthetic methylotrophy refers to the design and engineering of methanol assimilation pathways into established non-methylotrophic production hosts making use of their vast biotechnological potential  and provEscherichia coli  molecule. Furthermore, in all these implemented pathways carbon is assimilated in the form of formaldehyde, a central but highly toxic intermediate that can lead to cell death in case of imbalance between dissimilation and assimilation. In the engineering strategies based on naturally occurring methanol assimilation pathways, formaldehyde assimilation is achieved by a cyclic process and requires a C1-acceptor that enables the formation of a C-C bond . The effE. coli . This reIn this approach, existing pathways within the natural host are engineered (i.e. through gene deletion or overexpression) to improve their overall capacity towards a given function. A few studies have followed this approach to implement synthetic methylotrophy.S. cerevisiae, when grown with methanol (plus yeast extract), the final optical density increased by 39% relative to medium without methanol. In addition, 13C from labeled methanol was incorporated into intracellular metabolites (60% of the acetyl-coA pool was fully labeled). Based on these observations, Espinosa et al. decided to further boost the assimilation of methanol in S. cerevisiae by adaptive laboratory evolution (ALE). Genomic analysis of the resulting evolved strains helped in the identification of beneficial mutations allowing improvement of methanol utilization. These mutations were implemented in a wild type strain and the reconstructed strain exhibited a higher biomass yield on methanol (a 21% increase in OD600) than the parental strain. In addition, a 13C-methanol tracer analysis revealed that the reconstructed strain had a higher percentage of fully 13C-labelled intracellular metabolites  compared with the parental strain (In l strain .lipolitica (P. putida (Bacillus subtilis possesses a Ribulose Monophosphate (RuMP) pathway which may act as a detoxification system for formaldehyde  pathway, the Calvin-Benson-Bassham (CBB) cycle and the Serine cycle . In thosIn this pathway only three heterologous enzymes, i.e. a methanol dehydrogenase (Mdh), a 3-hexulose-6-phophate synthase (Hps) and a 6-phospho-3-hexuloisomerase (Phi) are needed to integrate the methylotrophic module . The chaMycobacterium and that uses mycofactocin as an in vivo electron acceptor (mdh gene from Bacillus methanolicus was the first to be used (13C-enrichment in phosphoenolpyruvate (PEP) with 1M of 13C-methanol)  but yeast extract . To further improve the kinetic parameters of Mdh, engineering strategies were applied. By using directed molecular evolution, a variant of the Mdh from C. necator with a catalytic efficiency for methanol 6-fold higher than the wild-type was obtained -dependent Mdh found in Gram-negative methylotrophs, the cytoplasmic NAD-dependent Mdh common in Gram-positive methylotrophs and the cytoplasmic NDMA -dependent Mdh found in acceptor . The PQQacceptor  and requ be used . Howeverethanol) . Scientim value . Whitake extract . Anotherobtained . In anotive Mdh2 .+ ratios are unfavourable to methanol oxidation. Results obtained after ALE experiments aiming at improving methanol assimilation in E. coli showed mutations in the nadR gene encoding for a transcriptional regulator of genes involved in NAD+ transport and de novo synthesis  and hxlB (6-phospho-3-hexuloisomerase) from B. subtilis  variant of the RuMP pathway) are key genes involved in Ru5P regeneration. Their deletion resulted in the loss of the capacity of the bacteria to grow on methanol . Authors hypothesised that by inhibiting this reaction, which connects the RuMP pathway to lower glycolysis, larger intermediate pools of the upper glycolytic and RuMP pathways could be maintained in starved cells. This strategy led to a higher methanol incorporation into central metabolites in E. coli. The 13C-enrichment in fructose-6-phosphate (F6P) reached 27.5% when iodoacetate was present. Moreover, the intracellular concentrations of F6P, Ru5P and sedoheptulose-7-phosphate (S7P) were higher . In synthetic methylotrophs, Ru5P regeneration is ensured by the non-oxidative part of the pentose phosphate pathway (PPP). In the natural methylotroph methanol . Thereforomosome . Even ife higher . Rohlhilperiment .E. coli’s genome. The first one included mdh, hps and phi genes while the second one was composed by the same mdh and phi genes plus the hps, tkt and tal genes from various organisms. Adding the tkt and tal genes in the operon helped enhancing Ru5P recycling  which activates the dissimilatory pathway to avoid any intracellular formaldehyde accumulation. Several examples of engineered Pfrm in synthetic methylotrophs have been described  is present and then to subject the strain to ALE .E. coli methanol dependent-strain  co-utilizing gluconate (E. coli strain (∆rpiAB) co-utilizing xylose and another one (∆rpe) co-utilizing ribose (rpiAB but switched from E. coli BL21 to E. coli K12 BW25113 to facilitate the genetic engineering efforts (pgi) that converts glucose-6-phosphate (G6P) to F6P, so that G6P is only used by the oxidative part of the PPP to directly fill the pool of Ru5P. The frmA gene, encoding for formaldehyde dehydrogenase, was also deleted in order to push formaldehyde towards assimilation  and another with triosephosphate isomerase (tpiA) deleted. Those strains were methanol dependent and showed a 99% fractional incorporation of methanol into RuMP cycle metabolites  for methanol assimilation by investigating the mutations and their consequences. Mutations found in evolved strains can be classified in three categories, mutations affecting: 1) the dissimilatory pathway and the recycling pathways; 2) the redox and energy balance; 3) the substrate uptake and other enzymes connected to metabolism .frm operon genes leading to the inactivation of the formaldehyde dissimilatory pathway were found in all the evolved E. coli strains  from ATP. The transcription of most of the TCA cycle enzymes is activated by cAMP. Most probably a cyaA mutation would lower the TCA cycle activity and consequently NAD(P)H production, at a beneficial cost for methanol oxidation. Moreover, inactivation of frmA and fdoG (encoding a formate dehydrogenase) for the synthetic methylotrophic strain, help limiting further the production of NADH n. They also demonstrated an improvement in methanol conversion to F6P in vitro  and a dihydroxyacetone kinase (DhaK) from Pichia pastoris in its genome  but was not enough to convert methanol into biomass , the complete pathway was tested in vivo by using 13C-methanol and xylose. Label derived from methanol was found in intracellular metabolites . However, there is no report of the in vivo functionality of the MCC pathway yet.In 2014, Bogorad et al. designed a non-natural pathway enabling methanol assimilation named methanol condensation cycle (MCC)  Bogorad. In the E. coli using an iterative combination of dry and wet lab approaches to design, implement and optimize this metabolic trait  and a dihydroxyacetone synthase (Das) from yeast was designed. To identify the best combination of enzymes to introduce into E. coli, a library of 266 pathway variants containing different combinations of Mdh and Das homologues was built and then screened using high-throughput 13C-labeling experiments. The highest level of incorporation of methanol into central metabolism intermediates (i.e. 22% into PEP) was obtained using a variant composed of a Mdh from Acinetobacter gerneri and a codon-optimized version of Pichia angusta Das. Finally, the activity of the HMD pathway was further improved by engineering strategic metabolic targets identified using omics and modelling approaches. The final synthetic strain had 1.5 to 5.9 times higher methanol assimilation in intracellular metabolites and proteinogenic amino acids than the starting strain but was still unable to grow on methanol as the sole carbon and energy source (The hybrid Mdh-Das (HMD) pathway  was builic trait . Throughy source .Y. lipolytica by introducing both the RuMP and the XumP pathways  as well as glpX from B. methanolicus that encodes for a SBPase and a FBPase. After ALE experiments, two evolved strains with improved methanol assimilation were obtained. Interestingly, in one strain, only the expression of the genes of the RuMP pathway was upregulated while in the other one the expression of both RuMP and XuMP pathways was increased. This highlights the capacity of the microbial strain to invoke several strategies in response to externally introduced genetic perturbations.A chimeric methanol assimilation pathway was engineered in pathways . In thisde novo-enzyme design, it becomes possible to create these reactions and integrate them into a synthetic pathway to perform a novel function. So far, only four studies have implemented synthetic methylotrophic pathways that involve novel catalytic transformations  . Fls is methanol .in vitro and the authors then decided to implement the pathway in E. coli. The functionality of the pathway was tested in vivo in rich media. By combining the SACA pathway with the Mdh from B. stearothermophilus, a slight improvement in the final OD was observed. When tested on minimal media by using 13C-methanol, label was also found in some proteinogenic amino acids after 26 h but no growth was observed (The synthetic acetyl-CoA (SACA) is a linear pathway that was designed and constructed by Lu et al. . This paobserved .in vitro using engineered versions of GalS and transaldolase B and reached a carbon yield of 88%. However, no proof of in vivo functionality of this pathway was shown. More recently a new GalS was engineered based on a newly discovered glyoxylate carboligase enzyme found in E. coli , 66% of the formaldehyde was converted into ethylene glycol  pathway . This pa E. coli . One of  E. coli . When the glycol .Rhodospiralleles bacterium (RuHacl) exhibiting the condensation activity of formaldehyde with formyl-CoA. They tested the pathway in vivo in an E. coli strain deleted for ∆frmA, ∆fdhF∆fdnG∆fdoG and ∆glcD to avoid any competitive reactions using formaldehyde, formate or gluconate. The strain produced 0.5 g/L of glycolate corresponding to a yield of 67%. After enzyme engineering using directed evolution, a variant of RuHacl with improved enzyme kinetics was selected. By expressing this variant, E. coli produced 1.2 g/L of glycolate. However, the pathway has not been tested yet in vitro or in vivo on methanol with the addition of a Mdh  is a synthetic pathway designed by Chou et al. where Hacl condenses formaldehyde with formyl-CoA to produce glycolyl-CoA . Glycolyof a Mdh . Later, of a Mdh . The FORE. coli, which could limit its efficiency (Methylorubrum extorquens (MeOxc) was subjected to several rounds of iterative site mutagenesis. A quadruple variant MeOxc4 was selected showing affinity towards formaldehyde similar to natural formaldehyde converting-enzymes and with an improvement for the carboligation activity of 200-fold compared to wild-type MeOxc. To test this enzyme in vivo, RuHacl was replaced with MeOxc4 in the E. coli strain previously designed for whole-cell bioconversion . It has been previously described that even if growth could not be supported on formate, the latter compound could be still assimilated in E. coli via the H4F-dependent pathway . Moreovnditions , we quicnditions , ALE helnditions . The len2 and 9 h on methanol  are far from the performance of natural methylotrophs that is 0.20 h−1 for Bacillus methanollicus at 37°C  while improving the reaction efficiencies as enzymes are in close vicinity to their substrates and intermediates  locus in their genome and that microcompartments are found in 23 different phyla  includinmediates . Furthermediates . For allChanging a lifestyle from heterotrophic to methylotrophic represents major engineering that have been partially achieved. In 4–10 years from now, it is likely that the next generation of synthetic methylotrophs will be further developed for the production of chemicals  from methanol, after which commercial production may be feasible. However, there is undoubtedly a long way ahead to achieve a synthetic strain able to use methanol for the production of both biomass and chemicals.


Background: Curcumin (CUR) displays the capability of suppressing the proliferation and metastasis of various cancer cells. However, the effects and underline mechanisms of CUR to treat triple-negative breast cancer (TNBC) have not been systematically elucidated with an appropriate method.Methods: In the present research, a combination method of network pharmacology, molecular docking, and in vitro bio-experiment was used to investigate the pharmacological actions and underline mechanisms of CUR against TNBC. First, common targets of CUR and TNBC were screened via Venny 2.1.0 after potential CUR-related targets and targets of TNBC were got from several public databases. Then, the Gene Ontology (GO) function and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed on the Metascape website, and the network of compound-targets-pathways was constructed via Cytoscape software. Moreover, the network of protein-protein interaction was constructed by the STRING database to screen potential targets. Moreover, molecular docking was applied to affirm the interaction of CUR with the screened top 10 potential targets. Finally, in vitro experiments were used to further verify the effects and mechanisms of CUR and its nano-formulation (CUR-NPs) against TNBC.Results: Forty potential targets of CUR against TNBC were obtained. STAT3, AKT1, TNF, PTGS2, MMP9, EGFR, PPARG, NFE2L2, EP300, and GSK3B were identified as the top 10 targets of CUR against TNBC. In vitro experiment verified that CUR and CUR-NPs could not only restrain the invasion, migration, and proliferation of MDA-MB-231 cells but also induce their apoptosis. In addition, molecular docking demonstrated that CUR could bind spontaneously with the screened top 10 targeted proteins, and a real-time PCR experiment demonstrated that both CUR and CUR-NPs could downregulate the genetic expression levels of the 10 targets. Moreover, according to the CUR-targets-pathways network, PI3K-Akt, EGFR tyrosine kinase inhibitor resistance, JAK-STAT, Foxo, and HIF-1 signaling pathways were identified as the important pathways of CUR effects on TNBC. Among them, the inhibiting effects of CUR and CUR-NPs on the JAK-STAT signaling pathway were further verified by the western blot analysis.Conclusion: Taken together, the present research demonstrates that CUR and CUR-NPs have pharmacological effects against TNBC via a multi-target and multi-pathway manner. In recent years, tremendous therapeutic progress has been achieved in the treatment of non-triple-negative breast cancer (non-TNBC), but the therapeutic strategies of TNBC are still limited to conventional chemotherapeutic drugs such as paclitaxel, doxorubicin, and platinum-based agents, which have serious cytotoxicities and side effects . So, it via the combination of network pharmacology and molecular docking. So, we hypothesized that the combination of network pharmacology, molecular docking, and in vitro bio-experiment will be a powerful tool to systematically investigate the pharmacological actions of CUR against TNBC.Network pharmacology is a powerful tool for systematically exploring the complex pharmacological effects of drugs . By the via Venny 2.1.0 after potential CUR-related targets and targets of TNBC were obtained from public databases. Subsequently, the Gene Ontology (GO) function and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed on the Metascape website. The network of compound-targets-pathways was also constructed by Cytoscape software. In addition, the database of Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) was used to construct the network of the protein-protein interaction (PPI) for screening potential targets. Moreover, molecular docking was applied to affirm the interaction of CUR with the screened top 10 potential targets. Finally, in vitro bio-experiments were used to further verify the effects and mechanisms of free CUR and CUR-loaded polyethylene glycol5k-bock-poly (l-lactide)5k nanoparticles (CUR-NPs) which were prepared by encapsulating CUR with the carrier of polyethylene glycol5k-bock-poly (l-lactide)5k, (PEG5k-b-PPLA5k). The workflow of this study is exhibited in Herein, common targets of CUR and TNBC were screened 5k-b-PPLA5k was purchased from Jinan Daigang Biomaterial Co., Ltd. . MDA-MB-231 cells were obtained from the ATCC . MTT was supplied by Sigma . Rabbit anti-human anti-STAT3 (BM4052), anti-phospho-STAT3 (Tyr705) (BM4835), anti-JAK2 (BM4165), and anti-phospho-JAK2 (Tyr1007/Tyr1008) (BM4839) antibody products were obtained from Boster Biological Technology Co., Ltd. . HRP-labeled Goat Anti-Rabbit IgG (A0208) and the Annexin V-FITC apoptosis detection kit (C1062M) were purchased from Beyotime Biotechnology . 24-well transwell chambers (Cat. No. 3422) and polymerized Matrigel (Cat. No. 356234) were bought from Corning .CUR was purchased from Shanghai D&B biological science and technology Co., Ltd. . PEGhttp://www.swisstargetprediction.ch/) and the Encyclopedia of Traditional Chinese Medicine (ETCM) database (http://www.tcmip.cn/ETCM/index.php/Home/). In the Swiss Target Prediction database, potential CUR-related targets were retrieved by the structure of CUR (both keto and enol forms) in Homo sapiens”. In the ETCM database, potential CUR-related targets were searched directly using the keyword of “CUR”. All targets obtained in both the Swiss Target Prediction database and ETCM were selected as potential CUR-related targets.Potential CUR-related targets were obtained from the Swiss Target Prediction database  (http://database  (http://https://omim.org/), the Therapeutic Target Database (http://db.idrblab.net/ttd/), and the DisGeNET database (https://www.disgenet.org/) using keywords of “triple negative breast cancer/carcinoma”. Subsequently, Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was applied to screen the common targets of CUR and TNBC which were defined as the potential targets of CUR against TNBC in the present research.First, TNBC-related targets were identified in the Online Mendelian Inheritance in Man database . Molecular function (MF), cell component (CC), and biological process (BP) were included in the GO analysis. The analyses were carried out using a p value of less than 0.01. The top 10 items of GO and the top 20 items of the KEGG pathway were selected and visualized on the Bioinformatics website (http://www.bioinformatics.com.cn/).GO and KEGG pathway enrichment of the common targets of CUR and TNBC were analyzed on the Metascape website (https://cytoscape.org/download.html) was applied to construct and analyze the network of CUR, targets, and pathways based on KEGG pathway enrichment analysis for understanding the complex relationships among them.The software of Cytoscape 3.7.1 (https://string-db.org/), using common targets of CUR and TNBC with minimum required interaction score ≥0.4, and species limited to “Homo sapiens”. After download, the TSV format of the PPI network was imported into the Cystoscope software to screen the top 10 targets of CUR against TNBC by using the maximal clique centrality (MCC) algorithm of the CytoHuba plug-in.The PPI network of potential targets of CUR against TNBC was constructed by the database of STRING  (https:/https://www.rcsb.org/), respectively. Autodock Vina (v1.1.2.0) was used for molecular docking in our study  with a Malvern zetasizer (MALVERN zetasizer nano ZEN3700). The morphology of the prepared CUR-NPs was observed by a transmission electron microscope (JEOL JEM-2100).A standard calibration of CUR  was prepared with dimethylsulfoxide (DMSO). Then, the CUR concentration of CUR-NPs after dilution with DMSO was quantified using the prepared standard calibration of CUR by a SPARK 10M plate reader  at 470 nm.in vitro was studied in PBS (pH7.4). Briefly, 1 ml of the prepared CUR-NPs solution was added to a dialysis bag . Then, the dialysis bag containing CUR-NPs solution was immersed in 30 ml of PBS (pH7.4) with 0.2% tween-80 at 37°C. The external buffer solution was substituted with fresh PBS (pH7.4) with 0.2% tween-80 at predetermined time intervals. Concentrations of CUR in the external butter solution were measured using the method described in the “Quantification of CUR”.The CUR release from the prepared CUR-NPs 5k-b-PLLA5k on MDA-MB-231 cells were determined by the MTT assay. MDA-MB-231 cells were seeded into 96-well plates at a density of 3 × 103 cells/well and cultured overnight. Afterward, free CUR (in DMSO) or CUR-NPs was added into each well with concentrations of CUR from CUR from 0.027 to 271.46 µM for 48 h; PEG5k-b-PLLA5k was added into each well with concentrations from 0.1 to 100 µM for 48 h. Thereafter, the culture media were replaced with 10 µL of the MTT solution (the concentration of the MTT solution was 12 mM) after washing with PBS three times and incubated for another 4 h. At the end of incubation, 100 µL of DMSO was added into each well after the culture medium was discarded. The optical density was measured by the Spark M10 multimode plate reader  at 570 nm. The half-inhibitory concentrations (IC50) of different groups were calculated using the software GraphPad Prism 7.The cytotoxic effects of free CUR, CUR-NPs, and PEG5 cells/well) and cultured overnight. Afterward, free CUR, CUR-NPs, or PEG5k-b-PLLA5k was added into each well with a CUR concentration of 27.15 µM or PEG5k-b-PLLA5k concentration of 10 µM for 48 h. Then, cells were harvested after washing with PBS three times. 5 μL of Annexin V-FITC and 10 μL of PI were added to the harvested cells after the cells were resuspended in 195 μL 1×binding buffer. Flow cytometric analyses were performed on a CytoFLEX  utilizing 10,000 events after the cells were incubated for another 10–20 min.The apoptotic rates of MDA-MB-231 cells after different treatments were detected using the method of Annexin V-FITC/PI double staining. Briefly, MDA-MB-231 cells were seeded into 6-well plates (1 × 105 cells/well) with 10% FBS until they reached confluence, and a 10 μL pipette tip was used to create wounds. Free CUR, CUR-NPs, or PEG5k-b-PLLA5k was added into each well with a CUR concentration of 13.57 µM or a PEG5k-b-PLLA5k concentration of 5 µM. The images of migration were captured with a ZEISS Observer A1 inverted microscope (×10 objective) .MDA-MB-231 cells were seeded into 6-well plates .Transwell chambers in 24-well plates were coated with 100 μL of polymerized matrigel (30 μg), followed by seeding of MDA-MB-231 cells at a density of 1 × 105 cells/well), and then free CUR, CUR-NPs, or PEG5k-b-PLLA5k were added into each well with a CUR concentration of 13.57 µM or a PEG5k-b-PLLA5k concentration of 5 µM for 48 h. Afterward, total RNA was isolated from MDA-MB-231 cells with the TRIzol® reagent, and cDNA synthesis was conducted with the RevertAid First Stand cDNA Synthesis Kit. Real-time PCR (RT-PCR) was then conducted using Hieff® qPCR SYBR® Green Master Mix (Yeasen Biotechology). Primers used for RT-PCR were STAT3 , AKT1 , TNF , PTGS2 , MMP9 , EGFR , PPARG , NFE2L2 , EP300 , GSK3B , β-ACTIN .MDA-MB-231 cells were seeded into 6-well plates (1 × 105 cells/well), and then free CUR, CUR-NPs, or PEG5k-b-PLLA5k were added into each well with a CUR concentration of13.57 µM or a PEG5k-b-PLLA5k concentration of 5 µM for 48 h. Proteins were extracted using a radio-immuno-precipitation assay buffer, and the extracted protein samples were transferred to nitrocellulose membranes after boiling with a 5×loading buffer and separating by 10% SDS-polyacrylamide gel electrophoresis. The membranes were then blocked with 5% skim milk for 1 h at room temperature, followed by incubating with one of the following antibodies at indicated dilution for about 12 h at 4°C: anti-STAT3 , anti-phospho-STAT3 (1:750), anti-JAK2 , anti-phospho-JAK2 (1:750), and anti-GAPDH . Then, the membranes were washed with 1×TBST buffer three or four times, followed by incubating with secondary HRP-conjugated goat anti-Rabbit IgG  for 1 h at room temperature. After washing with TBST buffer, the membranes were visualized using the Ultra-sensitive ECL chemiluminescence kit (Beyotime Biotechnology), and the gray scales of the bands were semi-quantitated by VisionWorks software .MDA-MB-231 cells were seeded into 6-well plates  by the IBM SPSS Statistics 23 software . The difference was considered statistically significant for all tests when p < 0.05.The data were analyzed To illustrate the anti-TNBC mechanisms of CUR, the GO and KEGG pathway enrichment of the 40 common targets of CUR and TNBC were investigated. As shown in Based on the top 20 signaling pathways and the genes enriched in these signaling pathways, a network of CUR-targets-pathways was established. As shown in p value is less than 1.0 e−16, and the local clustering coefficient is 0.642  from the results of PPI network analysis were chosen for further validation via a method of molecular docking by the Autodock Vina software (v1.1.2.0). CUR creates hydrogen bonds in the blinding pockets of the aforementioned 10 proteins  effect because the renal clearance threshold is 8 nm and the average gap of leaking blood vessels in cancer tumors is about 200 nm . In cont 200 nm . The bipt 200 nm  preparedt 200 nm  prepared50 value of free CUR against MDA-MB-231 cells was 14.29 μM which was lower than the treatment of CUR-NPs (20.15 μM). Moreover, the cytotoxicity of the carrier (PEG5k-b-PLLA5k) against MDA-MB-231 cells was also explored, and the result showed that PEG5k-b-PLLA5k has negligible cytotoxicity against MDA-MB-231 cells at the concentration used to prepare CUR-NPs (5k-b-PLLA5k) to induce the apoptosis of MDA-MB-231 cells were investigated by Annexin V-FITC/PI double staining. As depicted in 5k-b-PLLA5k or the negative control. These results demonstrated that both free CUR and CUR-NPs have the ability to restrain cell proliferation of MDA-MB-231 cells and induce their apoptosis in vitro. Compared with free CUR, the lower anti-proliferation and induced-apoptosis ability of CUR-NPs in vitro may be partly attributed to that only 30% of CUR was released from CUR-NPs to have effects on cancer cells after 48 h of incubation. Moreover, there are many other factors that affect the anti-proliferation and induced-apoptosis of CUR such as cellular uptake and metabolism. It was reported that the cellular uptake and metabolism of CUR play an important role in the cytotoxicities of CUR against hormone-dependent ZR-75-1 and hormone-independent MDA-MB-231 breast cancer cells  and JAK2 (Tyr1007/1008) residue were determined by immunoblotting. It was found that both the levels of phosphorylated STAT3 and JAK2 were obviously suppressed, however, there were no significant changes in the total levels of STAT3 and JAK2 proteins . The ratTNBC still lacks effective targeted therapy due to the negative expression of human epidermal growth factor receptor 2. Treatments with chemotherapeutic drugs such as taxanes, anthracyclines, and platinum-based agents are the major choice for patients with TNBC. Unfortunately, there are many problems with chemotherapy in the clinic, for example, the chemotherapeutic drugs have serious cytotoxicities and side effects, and patients develop resistance to the chemotherapeutic drugs. Therefore, it is worth developing a new drug with diminished toxicity for the treatment of TNBC. CUR extracted from turmeric exhibits a lot of pharmacological effects such as antioxidant, anti-aging, and anticancer activities. To comprehensively and systematically elucidate the effects and mechanisms of CUR on TNBC, network pharmacology which is a powerful tool to study the complex mechanisms of drug formulations was used in the present study.via inhibiting MMP-9 and MMP-2  which exhibited a significantly higher bioavailability for oral consumption was demonstrated to suppress breast cancer cells via inhibiting the expression of cyclinD1 (in vivo as compared with free CUR. in vivo.Although CUR exhibits the potential capability of preventing the proliferation, invasion, and metastasis of TNBC, its clinical translation is restricted by the poor solubility and instability in physiological conditions. So, it is urgent to solve these problems of CUR. Nanoscale drug delivery systems such as a nanoparticle, liposome, and nano-emulsion have been reported to have the capabilities of addressing these needs. A nano-emulsion of CUR was reported to improve the solubility, bioavailability, and antioxidant activity of CUR . Nano-focyclinD1 . Li et a5k-b-PPLA5k as the carrier to encapsulate CUR, and then the in vitro experiments were used to further verify the effect and mechanism of CUR in the formulation of nanoparticles. PEG5k-b-PLLA5k is an amphiphilic block copolymer, which can form core-shell nanoparticles with PEG as the shell (5k-b-PLLA5k. Moreover, it was found from western blot results of our research that PEG5k-b-PLLA5k has little effect on the expression levels of phosphorylated STAT3 and JAK2, which also indirectly proves that the nanoparticles prepared with PEG5k-b-PLLA5k have a low possibility of interacting with targeted proteins. Overall, our results demonstrated that CUR-NPs could obviously enhance the water solubility of CUR, but have little effect on the mechanism of CUR against TNBC.Some proteins have the ability to bind drugs and nanoparticles and affect the targeted ability of nanoparticles , and PEGhe shell . So, it in vitro bio-experiment validation, we demonstrate that CUR and CUR-NPs have the capabilities to suppress the migration and invasion, restrain the proliferation, and induce the apoptosis of TNBC cells, mainly by downregulating the genetic expression of STAT3, AKT1, TNF, PTGS2, MMP9, EGFR, PPARG, NFE2L2, EP300, and GSK3B and inhibiting JAK-STAT signaling pathway.Taken together, using the combination of network pharmacology, molecular docking, and


Some inherited or somatically-acquired gene variants are observed significantly more frequently in the genome of cancer cells. Although many of these cannot be confidently classified as driver mutations, they may contribute to shaping a cell environment that favours cancer onset and development. Understanding how these gene variants causally affect cancer phenotypes may help developing strategies for reverting the disease phenotype. Here we focus on variants of genes whose products have the potential to modulate metabolism to support uncontrolled cell growth. Over recent months our team of expert curators has undertaken an effort to annotate in the database SIGNOR 1) metabolic pathways that are deregulated in cancer and 2) interactions connecting oncogenes and tumour suppressors to metabolic enzymes. In addition, we refined a recently developed graph analysis tool that permits users to infer causal paths leading from any human gene to modulation of metabolic pathways. The tool grounds on a human signed and directed network that connects ∼8400 biological entities such as proteins and protein complexes via causal relationships. The network, which is based on more than 30,000 published causal links, can be downloaded from the SIGNOR website. In addition, as SIGNOR stores information on drugs or other chemicals targeting the activity of many of the genes in the network, the identification of likely functional paths offers a rational framework for exploring new therapeutic strategies that revert the disease phenotype. The cell responses to environmental stimuli or genetic perturbations are modulated by the signalling network and by the cell metabolic landscape. In growing cells, the metabolite composition of the cell milieu not only supports anabolic processes but also affects signal transduction and, as a consequence, cell fate and function. Signalling and metabolism crosstalk as activation of canonical cellular pathways often regulate metabolic-enzyme concentration or activity . On the These considerations are particularly relevant in cancer biology where alterations in oncogenes, onco-suppressors (TSG) or additional modifier genes play important roles in redirecting cell metabolism to fulfil the biosynthetic demands associated with proliferation . In addiA complete and holistic understanding of the mechanisms that lead to cell transformation is essential to devise pharmacological regimens that revert the disease phenotype by targeting metabolic vulnerabilities .A wide range of computational strategies have been proposed to characterise the capabilities of biological systems through analysis of metabolic network models . These iAll the methods described leverage on prior knowledge models extracted from the literature or from existing repositories. Over recent years several resources have endeavoured to represent published experimental information on signalling or metabolism in a computer readable format   metabGeneNet) .Activity-flow (AF) models offer a simple, albeit powerful, framework to represent the relationships governing the crosstalk between biological entities . They arSIGNOR was originally conceived as a resource to link signalling proteins via causal relationships . It lateOver the past couple of years cancer genes have been a target of the curation effort by the SIGNOR team. In 2019 a related resource CancerGeneNet  was deve could noRecently, two consortia from different institutes have reported CRISPR-Cas 9 screening that yielded lists of prioritized therapeutic-gene targets . The resSome cancer genes, when mutated, modify cell metabolism to facilitate the biosynthesis of the macromolecules and organelles required for assembling new cells. However, in many cases, the underlying molecular mechanisms are poorly understood.The integration of metabolic reactions into the human signalling network permits to connect, via causal paths, the activity of cancer genes to metabolic pathways. To this end we have built, in the resource CancerGeneNet, a graph tool that searches for short weighted-signed-directed paths linking two nodes in the human causal network. The tool allows one to infer how likely it is that a query protein (or a list of proteins) modulates the activity or expression of key rate-limiting enzymes in nine metabolic pathways.p-value <0.05) were considered to be likely to have an impact on the pathway.Briefly, the tool is based on a selection of approx. 13,000 paths that significantly link genes in SIGNOR to key rate-limiting metabolic enzymes. To obtain this dataset we filtered the entire SIGNOR interactome by applying the three following steps: 1) we identified paths connecting, with four causal steps or fewer, any gene in SIGNOR to metabolic pathways; 2) As each path is associated with a distance score (see Methods), for each considered metabolic pathway we could draw a distance distribution and calculate a Z score for each gene distance; 3) genes with a Z score < −1.96  were considered to be likely to have an impact on the pathway. Next, we have asked whether the list of genes annotated as oncogenes or tumour suppressor genes by the CGC resource were enriched for genes that affect metabolism. The result of this analysis showed that 40% (97/245) of the genes annotated as oncogenes are significantly close to metabolic pathways. This is significantly  higher than observed in 1000 collections of randomly sampled genes (average 21%) and supports the notion that genes that are found preferentially mutated in tumours are more likely to affect metabolism than random genes.Genes with a Z score < −1.96  that, when activated by the ITD oncogenic mutation, promotes aerobic glycolysis is predicted by our algorithm to do so via AKT-mediated upregulation of hexokinase. This prediction is consistent with previously reported experimental findings . A detaiMetabolic changes in tumours have long been acknowledged  and metaAn in-depth understanding of the functional interactions underlying cancer metabolic reprogramming is essential to identify cellular vulnerabilities that can be exploited for therapeutic opportunities . Here wehttps://signor.uniroma2.it/CancerGeneNet) where it is possible to visualize the paths connecting genes to activity of rate limiting enzymes. The resource can be interrogated with a query protein or a list of proteins and results are displayed either graphically or in a tabular format.The results of this approach are made available via interrogation of the CanceGeneNet resource  by offering a framework that combines signaling and metabolism. The resource has been primarily conceived to model how the signal transduction cascades, triggered by oncogenes and TSG, modulate the metabolic profile of malignant cells. However, it is not limited to this scope, as the interactome that is available for download in the CancerGeneNet resource contains causal paths linking any gene in SIGNOR to metabolic pathways, thus supporting the integration of omic data in a variety of biological contexts. To this end, it would be of interest to expand the landscape of metabolic pathways integrated in the resource.When examining the paths by the approach described here, users should consider that the SIGNOR human interactome is assembled from observations in a variety of experimental systems. Not all molecular connections that are annotated in the resource are active in all cell contexts because either one of the proteins is not present or not active in the specific cell type. Thus, each proposed molecular path should be critically examined by domain experts. In principle gene expression evidence could be exploited to filter out paths that pass over nodes representing proteins that are not expressed in the biological system of interest. Nevertheless, given that a number of possible paths are suggested by interrogating the resource, domain-experts are in the position to identify paths that are worth testing experimentally.The cancer-driver gene list (v95) was downloaded from the Cancer Gene Census (CGC) web site . The CGCThe strategy to find functional paths connecting cancer genes to metabolic enzymes builds on a human causal network assembled from the causal interactions captured from the literature and annotated in the SIGNOR resource . SIGNOR Interactions in SIGNOR are assigned a significance score, ranging from 0.1 to 1. Interactions between proteins, protein families and complexes are assigned a significance score calculated by using a principal component regression (PCR) approach  which yihttps://signor.uniroma2.it/documentation/.OR and score values extracted from the STRING database . The modp-value <0.05) The Z-score is computed over the distribution of path distance scores considering the distance between every protein in SIGNOR to the RLEs of any given pathway. Nodes that cannot connect with four steps or fewer to RLEs were arbitrarily assigned the highest path distance score in the distribution.To investigate the regulatory impact of a protein (or a list of proteins) on a metabolic pathway, we make use of the graph representation of the human causal network annotated in SIGNOR 2.0. We developed a tool that, for each query node and metabolic pathway, “navigates” the network graph and retrieves the shortest paths connecting the query node to a rate-limiting enzyme of the end pathway. Shortest paths are identified using a strategy adapted from The paths connecting the query protein to a metabolic pathway are characterized by a distance and by a sign that specifies whether the protein is inferred to have a positive or a negative effect on pathway activity. Proteins connected by paths formed by an odd number of inhibitory steps are defined as inhibitors, otherwise are considered as activators.https://signor.uniroma2.it/CancerGeneNet/)  .To identify the impact of oncogenes and tumour suppressor genes (TSG) on the modulation of metabolic pathways we used the strategy outlined in the previous section to link to the up- or down-regulation of 10 metabolic pathways, 245 oncogenes or 245 TSGs as annotated in the Cancer Gene Census (CGC) resource. We used the two-sided t-test to assess whether the proportion of paths starting from oncogenes/TSGs and leading to the up-/down-regulation of a key metabolic pathway is significantly greater or smaller than the mean extracted from a randomized dataset. Briefly, we generated lists of 700 genes that were randomly chosen among the genes annotated in SIGNOR. 700 corresponds to the number of genes in the CGC resource. We generated 1000 of such random genes lists and, for each, we evaluated the fraction of short paths (<= 4 steps) starting from the input genes and impacting the up- or down-regulation of the key metabolic pathways. We eventually charted the distribution of these fractions and performed a t-test.To facilitate the interpretation of the results, the SIGNOR pathway “glycolysis and gluconeogenesis” was demerged into two sub-pathways: “Glycolysis” and “Gluconeogenesis.”


Formularies of the Bureau of People's Welfare Pharmacies in the Song Dynasty and consists of two herbs, Puhuang (PH) and Wulingzhi (WLZ). PH-WLZ is a commonly used drug pair for the treatment of coronary heart disease (CHD), and its clinical effect is remarkable. However, our understanding of the mechanism of treatment of CHD is still unclear. In this study, the method of network pharmacology was used to explore the mechanism of PH-WLZ in the treatment of CHD. A total of 56 active ingredients were identified from PH-WLZ, of which 93 targets of 41 active ingredients overlapped with those of CHD. By performing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, we obtained the main pathways associated with CHD and those associated with the mechanism of PH-WLZ in the treatment of CHD. By constructing the protein-protein interaction (PPI) network of common targets, 10 hub genes were identified. Based on the number of hub genes contained in the enrichment analysis, we obtained the key pathways of PH-WLZ in the treatment of CHD. The key KEGG pathway in the treatment of CHD by PH-WLZ is mainly enriched in atherosclerosis, inflammation, immunity, oxidative stress, and infection-related pathways. Moreover, the results of molecular docking showed that the active ingredients of PH-WLZ had a good affinity with the hub genes. The results indicate that the mechanism of PH-WLZ in the treatment of CHD may be related to regulation of lipid metabolism, regulation of immune and inflammatory responses, regulation of downstream genes of fluid shear stress, antiaging and oxidative stress, and virus inhibition.Shixiao powder comes from the  The report on the global burden of ischaemic heart disease from 1990 to 2019 shows that, in 2019, an estimated 9.14 million people worldwide died from coronary heart disease (CHD) and an estimated 197 million people worldwide suffered from CHD. Globally, the age-standardized prevalence of CHD and the absolute number of deaths have declined over the past 30 years, but CHD mortality has declined significantly in high-income countries and remains high in low- and middle-income countries . We exprhttps://herb.ac.cn/) is a high-throughput database established by Professor Zhao Yi and his team, integrating multiple TCM databases  of traditional Chinese medicine and ingredients and determining the relationship between them. Therefore, we screened the chemical constituents of PH-WLZ in HERB with the keywords of “Puhuang” and “Wulingzhi.” For the obtained results, we normalized in PubChem (https://pubchem.ncbi.nlm.nih.gov/).HERB (https://www.swissadme.ch/) is a website that uses computer prediction models to reveal the physicochemical properties of small-molecule compounds and to assess pharmacokinetics, drug similarity, and medicinal chemical closeness. Oral bioavailability (OB) and drug likeness (DL), two ADME-related models, are the main variables affecting drug absorption from the gastrointestinal tract. This website evaluates the gastrointestinal absorption of compounds according to the BOILED-Egg method and evaluates the drug-like properties of compounds according to five methods: Lipinski, Ghose, Veber, Egan, and Muegge. Therefore, we screened the active ingredients of PH-WLZ based on the criteria of “high” for GI absorption and “Yes” for at least three of the five drug-like properties.SwissADME (https://bionet.ncpsb.org.cn/batman-tcm/), SwissTargetPrediction (https://swisstargetprediction.ch/), and STITCH (https://stitch.embl.de/), retrieval results as active ingredient-related targets. BATMEN-TCM is an online bioinformatics analysis tool to study the molecular mechanism of traditional Chinese medicine, and we set the retrieval parameters with score ≥40 and adjusted p value ≤0.05. SwissTargetPrediction is an online tool designed to predict molecular targets based on the 2D and 3D structural similarity of compounds. In the SwissTargetPrediction, the species is restricted to “Homo sapiens” and the probability is set as no less than 0.6. STITCH 5.0 is a database of predicted interactions between known chemical molecules and proteins. The species “Homo sapiens” and a confidence score ≥0.6 were set as the parameters of STITCH 5.0. Finally, to make the results concise and convenient, we canonicalize gene names using the UniProt database (https://www.uniprot.org/).We retrieved all active ingredients on 3 target prediction web servers: BATMEN-TCM (https://www.genecards.org/), and the screening criterion is relevance score ≥ 40; Comparative Toxicogenomics Database (CTD) (https://ctdbase.org/), and the screening criterion is direct evidence or inference score ≥ 60; DisGeNET (https://www.disgenet.org/home/), and the screening standard is gene-disease association score ≥ 0.1; and NCBI Gene (https://www.ncbi.nlm.nih.gov/gene), and the screening criterion is species belonging to Homo species. The data obtained in each database were merged to obtain the final disease targets of CHD. These targets are also canonical gene names in the UniProt database.To obtain disease targets, we used “coronary heart disease” as the keyword to search for the corresponding genes of CHD in different databases. The four databases and search criteria are as follows: GeneCards (https://cn.string-db.org/) is a database of known and predicted protein-protein interactions (PPIs), currently containing about 24.6 million proteins from 5,090 microorganisms. In this study, potential target interactions were analyzed using STRING, setting the species as “Homo sapiens” with a confidence score ≥ 0.7.STRING . Interactive network topology analysis was performed using the Network Analyzer plugin. For each node in an interactive network, the degree value is an important parameter to evaluate its topological characteristics, and it measures the number of connections with other nodes and reflects the importance of the node.Cluster analysis aims to screen out identical or similar nodes and protein complexes from complex PPI networks. Cluster analysis of disease PPI networks was performed using the MCODE plugin (version 3.9.0) in Cytoscape. A node score cutoff = 0.2, k-core = 2, maximum depth = 100, and degree cutoff = 2 were set as the filter conditions.https://bioinfogp.cnb.csic.es/tools/venny/), we intersected the active target of PH-WLZ with the target of CHD and obtained a direct target (common genes) of the active component of PH-WLZ for the treatment of CHD.Using Venny 2.1  to construct a visual common target PPI network and a common target active ingredient network.To understand the interaction between common genes, the STRING database was used to study the interaction network between proteins and to mine the core regulatory genes. In this study, potential target interactions were analyzed using STRING, the species was set to “The hub gene is the bottleneck gene of node connection in the network and plays an important role in the PPI network. We used the CytoHubba plugin of Cytoscape (version 3.9.0) to identify the hub genes, and the top 10 genes generated by the maximum neighborhood component (MCC) method were regarded as hub genes. A visualized TCM-active ingredient-hub gene-disease network was constructed using Cytoscape (version 3.9.0).P ≤ 0.05. GO and KEGG pathway enrichment analysis was performed on the genes and common genes of the top 5 clusters of disease-gene cluster analysis, respectively.Gene Ontology (GO) annotation was used to define and describe the function of gene products from three aspects, namely, biological processes (BPs), cellular components (CCs), and molecular functions (MFs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database integrating genomic, chemical, and systemic functional information. To obtain more accurate GO and KEGG functional enrichment information, the ClusterProfiler software package of the R platform was used for GO and KEGG functional enrichment analysis, and the screening criterion was adjusted The results of the disease enrichment analysis and the results of the common gene enrichment analysis were combined to obtain the GO and KEGG enrichment analysis results related to the treatment of CHD by PH-WLZ. The intersection entries contain entries with more hub genes as key results.https://www.rcsb.org/) database, and the structures of the active ingredients were obtained from the PubChem database. Before molecular docking, we use AutoDock (version 4.2.6) software to remove ligands, remove water molecular structure, and add hydrogen atoms and charges to the protein crystal structure. Molecular docking was performed using AutoDock Vina (version 1.1.2) software to evaluate the interaction between the target and the active ingredient. Results were analyzed and plotted using PyMOL (version 2.5.2).The crystal structures of the hub targets were downloaded from the RCSB PDB . The top 10 meaningful terms in BPs, MFs, and CCs and the top 20 meaningful KEGG pathways are shown in Figures The PPI network of CHD targets was then clustered using the MCODE plugin in Cytoscape. Thus, 31 clusters were obtained . The top 10 meaningful terms in BPs, MFs, and CCs and 20 meaningful KEGG pathways are shown in To explore the mechanism of PH-WLZ in the treatment of CHD, we used the ClusterProfiler package of the R platform to perform KEGG and GO functional enrichment analysis on 93 common targets. Therefore, we obtained 1829 BPs, 175 MFs, 107 CCs, and 159 KEGG pathways , 4 key molecular functions (number of hub genes ≥3), and 2 key cellular components (number of hub genes ≥3). The key biological process of PH-WLZ treatment of CHD: response to a molecule of bacterial origin (GO:0002237), response to lipopolysaccharide (GO:0032496), and response to tumor necrosis factor (GO:0034612). Key molecular functions: cytokine receptor binding (GO:0005126), signalling receptor activator activity (GO:0030546), receptor ligand activity (GO:0048018), and cytokine activity (GO:0005125). Key cellular components are in membrane rafts (GO:0045121) and membrane microdomains (GO:0098857).We summarized the KEGG pathways containing hub genes ≥6 and obtained 6 key KEGG pathways. Although the number of hub genes enriched in fluid shear stress and atherosclerosis and MAPK signalling pathways is less than 6, they are important CHD-related pathways . Eight kA TCM-active ingredient-hub gene-disease network was constructed based on the active ingredients related to the hub genes . This neAccording to the TCM-active ingredient-hub gene-disease network, we treat the connected hub targets and key active components as receptors and ligands, respectively. The stability of receptor and ligand binding depends on the binding energy. The lower the binding energy, the more stable the binding conformation of the receptor and ligand. We set a binding energy of −5.0 kcal/mol as the threshold for determining whether receptor and ligand binding is good. The interaction of receptor and ligand is shown in CHD is characterized by atherosclerosis in the coronary arteries, leading to blockage of the vascular lumen and subsequent myocardial ischaemia, hypoxia, and necrosis. The formation of atherosclerotic plaque (As) is the pathological basis of CHD, and its occurrence and development are a chronic inflammatory process involving multiple steps and factors. AS is a multifactorial disease involving chronic inflammation, lipid metabolism and accumulation, oxidative stress, genetic predisposition, immune disorders, epigenetics, and multiple nongenetic risk factors . The “multiple hits” hypothesis suggests that all these injuries work together to cause atherosclerosis.Through constructing PPI network of PH-WLZ and CHD, common targets, AKT1, CASP3, CCL2, CXCL8, INS, JUN, MMP9, PTGS2, SERPINE1, and TNF found to be the hub genes of PH-WLZ in the treatment of CHD. These genes are mainly associated with atherosclerosis, inflammation, immunity, oxidative stress, and infection. Based on the analysis of hub genes, key active components, and major prominent KEGG pathways, the underlying mechanism of PH-WLZ in the treatment of CHD may be attributed to the following aspects.Arterial narrowing by lipid-rich plaques in the arterial vessel wall is a major feature of atherosclerosis, and elevated low-density lipoprotein (LDL) cholesterol levels are a major risk factor for atherosclerosis. LDL can accumulate in blood vessel walls and be modified by oxidation to form oxidized LDL (oxLDL). OxLDL causes endothelial cell dysfunction, inducing the expression of adhesion molecules and the recruitment of monocytes in the subendothelial space. Monocytes proliferate, differentiate into macrophages, and take up lipoproteins to form cholesterol-rich “foam cells.” Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) mediates the recognition and internalization of oxLDL, acting on multiple cells, such as endothelial cells, macrophages, platelets, fibroblasts, and smooth muscle cells; it plays a central role in atherosclerosis . PIK3CA In recent years, atherosclerosis has been regarded as a chronic inflammatory disease, which is an abnormal response of the vascular wall to various injuries, and has the characteristics of classic inflammatory degeneration, exudation, and proliferation. Inflammatory response runs through all stages of atherosclerosis and may be a common link or pathway in the pathogenesis of various atherosclerotic factors. The immune and inflammation-related pathways in the results included IL-17 signalling pathway, TNF signalling pathway, and MAPK signalling pathway.The interleukin 17 (IL-17) family is a subgroup of cytokines composed of IL-17A-F, which plays an important role in the inflammatory response of autoimmune diseases, as well as injury, physiological stress, and infection . The IL-Tumor necrosis factor (TNF) is a key cytokine that induces a wide range of intracellular signalling pathways, including apoptosis and cell survival as well as inflammation and immunity. Activated TNF assembles into homopolymers and binds to its receptors . TNFR1 signalling induces the activation of many genes, including the NF-kappa B pathway and the MAPK pathway; and TNFR2 signalling activates the NF-kappa B pathway, including the PI3K-dependent NF-kappa B pathway and the JNK pathway . StudiesThe MAPK signalling pathway is a cascade reaction composed of a series of protein kinases and their phosphorylation. The activation of MAPK is involved in the regulation of the inflammatory response in AS. In general, ligands as inflammatory substances first activate the corresponding receptors, which in turn activate the MAPK molecules, which subsequently lead to the activation of the transcription factor NF-KAPPA B, thereby regulating the expression of inflammatory cytokines . ExperimAtherosclerosis preferentially occurs in tortuous regions, branch points, and bifurcations of the arterial tree due to endothelial dysfunction caused by the action of nonlaminar characteristic fluid shear stress, which activates the expression of endothelial-related genes. Endothelial dysfunction leads to the activation of downstream signalling pathways including AP-1, NF-kappa B, Akt-PI3K, and MAPK, and the expression of these genes promotes the oxidative and inflammatory state in the arterial wall, which leads to the occurrence of atherosclerosis . The AP-α) and various atherosclerosis-related genes  [Atherosclerosis is considered a distinct disease of biological and cellular aging that promote premature or accelerated vascular aging under cardiovascular risk factors . The AGEnd RAGE) . Among tChlamydia pneumoniae, Helicobacter pylori, etc. The inflammation and immune response brought about by infection activate or accelerate the process of atherosclerosis. Human cytomegalovirus infection is significantly associated with CHD and coronary atherosclerosis [Many infections are thought to be associated with CHD, such as HIV, human, cytomegalovirus, clerosis . In the clerosis . Althougclerosis . TherefoGallic acid is a phenolic acid widely distributed in many different families of higher plants with high antioxidant properties. Gallic acid can improve high glucose levels, insulin, inflammation, and oxidative stress in diabetic complications by activating multiple effective pathways such as NO/iNOS generation, AGE/ALE, and JNK/ERK . The antα, IL-6, IFN-γ, and signalling pathways NF-kappa B and MAPK and has a relieving effect on atherosclerosis [γ, LXRα, and ABCA1 [Isorhamnetin, naringenin, kaempferol, and quercetin are all flavonoids, and many studies have shown that these flavonoids have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antibacterial, anticancer, cardioprotective, neuroprotective, antidiabetic, etc. These flavonoids also have extensive preventive and therapeutic effects on cardiovascular and cerebrovascular diseases, such as antiatherosclerosis, protection of endothelial cells, antimyocardial ischaemia, antihypotension, antihypoglycemia, and antithrombosis. Isorhamnetin can protect cardiovascular cells from inflammation, oxidative damage, and apoptosis by affecting PI3K/AKT, NF-kappa B, and MAPK signalling pathways . Naringeclerosis . Kaempfeclerosis . Quercetnd ABCA1 .We obtained the key active ingredients of PH-WLZ in the treatment of CHD, namely, gallic acid, isorhamnetin, quercetin, kaempferol, and naringenin, and these active ingredients are mainly derived from PH. By constructing a compound-target-disease network, 10 hub genes were screened, namely, AKT1, CASP3, CCL2, CXCL8, INS, JUN, MMP9, PTGS2, SERPINE1, and TNF. We obtained meaningful biological processes, molecular functions, cellular components, and KEGG pathways by performing GO and KEGG pathway analysis. The results of key active components, hub genes, and enrichment analysis indicated that the mechanism of PH-WLZ in the treatment of CHD may be related to the regulation of lipid metabolism, regulation of immune and inflammatory responses, regulation of downstream gene activation caused by fluid shear stress, and antiaging and oxidative stress, and virus inhibition.


The sesquiterpenoid hormone juvenile hormone (JH) controls development, reproduction, and metamorphosis in insects, and has long been thought to be confined to the Insecta. While it remains true that juvenile hormone is specifically synthesized in insects, other types or forms of sesquiterpenoids have also been discovered in distantly related animals, such as the jellyfish. Here, we combine the latest literature and annotate the sesquiterpenoid biosynthetic pathway genes in different animal genomes. We hypothesize that the sesquiterpenoid hormonal system is an ancestral system established in an animal ancestor and remains widespread in many animals. Different animal lineages have adapted different enzymatic routes from a common pathway, with cnidarians producing farnesoic acid (FA); non-insect protostomes and non-vertebrate deuterostomes such as cephalochordate and echinoderm synthesizing FA and methyl farnesoate (MF); and insects producing FA, MF, and JH. Our hypothesis revolutionizes the current view on the sesquiterpenoids in the metazoans, and forms a foundation for a re-investigation of the roles of this important and yet neglected type of hormone in different animals. Kruppel homolog 1 (Kr-h1), that regulate development and metamorphosis in insects [Hormones are important regulators of animal development and play important roles in both physiology and body plan diversification during animal evolution ,2. Sesqu insects ,6,7  pathway in animals ,6,7,8. TO-methyltransferase (JHAMT). Different lineages of insects utilize different end products in this sesquiterpenoid pathway [Drosophila, further conversion of MF into JH III is possible, under the reaction of an epoxidase, CYP15 [3 is produced by a methyl farnesoate epoxidase (EPOX) from MF [3 in D. melanogaster and JHAMT is also suggested to be involved in the methylation of JHB3 acid to JHB3 [Insects and other arthropods, due to the absence of squalene synthase, do not synthesize cholesterol ,10. Alte pathway . In certe, CYP15 ,18,19. I from MF . In othe from MF ,22,23. I to JHB3 ,25.Apart from the conventional JH-specific synthesis pathway, another side branch called the isoprenylation pathway was also discovered to produce farnesal . Thus, t3) [Drosophila, have also been shown to produce JH bisepoxide (JHB3) in addition to JH III [Diploptera punctata nymphs and fly Drosophila melanogaster [The biosynthesis of JH has long known to be specific to insects since its discovery in the 1930s by Vincent Wigglesworth ,23,27. D3) . Some dio JH III ,30. Furto JH III ,31. Methnogaster ,33. In gLibinia emarginata [Despite the lack of juvenile hormone in other arthropods, including crustaceans , chelicerates , and myriapods (centipedes and millipedes), other types of functional sesquiterpenoids are present ,44,45,46arginata  and was arginata . MF, in arginata ,6. Tetranychus urticae. The presence of MF in T. urticae was subsequently confirmed by GC-MS and is likely the end-product of the pathway as the epoxidase to convert MF further was missing in the genome [Parasteadtoda tepidariorum [Sesquiterpenoids in non-insect/crustacean arthropods such as the chelicerates, on the other hand, are relatively understudied. Enzymes of the mevalonate (MVA) pathway and the downstream pathway leading to the production of MF were identified in the spider mite e genome . Throughe genome ,41,42,43dariorum .Strigamia maritima, genes involved in the biosynthetic pathway leading to sesquiterpenoid production were identified [Lithobius niger, Rhysida immarginata, Thereuonema tuberculata, Anaulaciulus tonginus, Glomeris maerens, Niponia nodulosa) also applied a similar strategy and sesquiterpenoid pathway genes were also found in the genomes. JHAMT was found to be absent in the investigated millipede genomes, suggesting FA as the final product of the pathway [In 2014, with the sequencing of the first myriapod genome, the centipede entified . Recentl pathway .Capitella [Platynereis dumerilii. MF application suppressed the expression of the yolk protein gene Vitellogenin (Vg) and arrested reproductive maturation [Syllis magdolena, MF, along with dopamine and serotonin, was found to trigger the formation of a posterior gamete containing a structure known as a stolon, which upon maturation releases gametes [Biomphalaria straminea [In the 1990s, exogenous applications of MF and JH induced the settlement and metamorphosis of polychaete apitella ,48. In 2turation . Later,  gametes . Recent traminea .In a recent analysis of jellyfish genomes, the existence of genes involved in the biosynthetic pathway of sesquiterpenoids was also unexpectedly discovered . This stAmphimedon queenslandica (sponge), Hormiphora californensis (ctenophore), Trichoplax adhaerens (placozoan), Nematostella vectensis (seas anemone), Acropora digitifera , Sanderia malayensis (jellyfish), Rhopilema esculentum (jellyfish), Hydra vulgaris (hydra), Helobdella robusta (leech), Caenorhabditis elegans (nematode), Branchiostoma lanceolatum (amphioxus), Branchiostoma floridae (amphioxus), Branchiostoma belcheri (amphioxus), Ciona intestinalis (tunicate), and Strongylocentrotus purpuratus (sea urchin). The identity of each retrieved gene was subsequently submitted to the NCBI online nr database for reciprocal BLAST using the BLASTP and BLASTX algorithm. The conserved functional domain of the protein was further examined using the NCBI CD search.Gene family sequences were first retrieved from the NCBI public database. Each gene was blasted to the published animal genomes, including For phylogenetic analyses, sequences were first retrieved from the respective animal genomes and aligned to other known animal reference sequences, which were retrieved from NCBI, using ClustalW in MEGA 7.0 software . The besw/v) sodium chloride solution, and 10 ng JH III-D3 as an internal standard; ultrasonicated for 1 min; and then vortexed and extracted twice with 2 mL hexane. The hexane phase (upper layer) was removed and transferred to a new glass vial, and then dried under nitrogen flow. The residue was dissolved in 1 mL acetonitrile. The measurement of FA was determined using the liquid chromatography method coupled with electrospray tandem mass spectrometry (LC-MS/MS) reported by Ramirez et al. [Freeze-dried jellyfish samples were placed in stainless steel grinding jars with balls of Restch MM400. The grinding jars were chilled in liquid nitrogen for 30 min, and then homogenized at a frequency of 20 Hz for 0.5 min . The homz et al. .To systematically identify sesquiterpenoid pathway genes in genomes at key phylogenetic positions, we need to first understand the current animal phylogeny. Cnidaria includes jellyfish, sea anemones, and corals and is the closest relative of bilaterally symmetrical animals, Bilateria. Bilateria comprises three major clades: Deuterostomia ; Protostomia, which can be further separated into Ecdysozoa ; and Lophotrochozoa (annelids and molluscs). In all the animal genomes searched in this study, all the genes involved in the MVA pathway and isoprenylation pathway could be identified , suggestStrigamia maritima, genome sequencing identified the MVA pathway genes and the downstream pathway leading to MF as was found in chelicerates and crustaceans [In chelicerates and crustaceans, the majority of the sesquiterpenoid biosynthetic pathway genes could be identified except the epoxidase genes leading to the synthesis of juvenile hormone ,58,59 FA. MF, thstaceans ,45. Recestaceans   could be produced in these animals (O-methyltransferase (JHAMT) could also be identified in the genomes of amphioxus and echinoderms, suggesting that sesquiterpenoids MF may be well produced  could be identified in the genomes of  animals . In addiproduced . AlthougCyanea capillata, and 1.04 ± 0.48 ng/g FA in the medusa of edible jellyfish Rhopilema esculentum. Previous study revealed sesquiterpenoid biosynthetic pathway genes in jellyfish and suggested that the pathway would terminate with the production of FA  Figure . Here, wSanderia malayensis and Rhopilema esculentum, the sea anemone Nematostella vectensis, corals Acropora digitifera, Stylophora pistillata and Pocillopora damicornis, and hydroid Hydra vulgaris, and found the presence of MVA, isoprenylation, and ALDHIII genes in all these cnidarian genomes (We further searched for the sesquiterpenoid biosynthetic pathway genes in other cnidarian genomes, including the jellyfish  genomes . This fiAmphimedon queenslandica and ctenophore Hormiphora californensis. Both A. queenslandica and H. californensis possess the complete set of genes involved in the terpenoid backbone synthesis and isoprenylation pathways, suggesting the potential presence of farnesal in these animals (To explore whether the sesquiterpenoid system could be dated back to the metazoan ancestor, we also investigated the genomes of poriferan  animals . In addi animals A. FutureHere, we hypothesize that the sesquiterpenoid hormonal system is an ancestral system established in an animal ancestor and remains widespread in many animals. Different animal lineages have adapted different enzymatic routes from a common pathway that produces different sesquiterpenoid hormones. Much work needs to be conducted to test the function of these sesquiterpenoid compounds and discover their various roles in different animal lineages.


Heart failure (HF) and diabetes mellitus (DM) are life-threatening diseases. However, existing clinical drugs to treat HF complicated with DM are relatively limited. In this study, we performed a viable bioinformatics strategy combining network pharmacology and molecular docking to identify potential anti-HF and -DM targets and therapeutic mechanisms of calycosin, a functional phytoestrogen.Web-based databases were used to collect candidate genes/targets of calycosin and HF/DM and then identify the hub bio-targets of calycosin against HF/DM. Using the online-available database, all functional processes and signaling pathways of calycosin against HF/DM were screened and identified before further visualization.All potential bio-targets of calycosin and HF/DM were collected, and 20 hub targets of calycosin against HF/DM were identified. Interestingly, molecular docking findings indicated that mitogen-activated protein kinase-1 (MAPK1), β-arrestin 1 (ARRB1), and homologue-1 (ABL1) may be potent pharmacological targets of calycosin against HF/DM. In addition, all primary molecular functions of calycosin against HF/DM were identified, including regulating protein binding, ubiquitination, and the metabolic process. Furthermore, the top molecular pathways of calycosin against HF/DM were revealed, including cardiomyocyte and chemokine signaling pathways.in silico findings revealed that the identified hub targets may be used to screen and treat HF/DM.Our bioinformatics analysis uncovered the network targets and therapeutic mechanisms of calycosin against HF/DM. For the first time, the current  Radix Astragali, has potent pharmacological actions, such as anticancer, neuroprotection, cytoprotection, and anti-diabetes activity (Heart failure (HF) refers to the inability of the heart to circulate sufficient blood to sustain normal functions in the body . As an eactivity –13. Howeactivity , 15. Intactivity , 17. As http://stitch.embl.de/), Swiss Target Prediction, SuperPred webserver, Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine , ChemMapper, and Drug Target Prediction System were collectively used to screen the functional genes/targets of calycosin. Furthermore, the HF- and DM-driven genes/targets were acquired from the detection databases DisGeNET, GeneCard, DrugBank (www.drugbank.ca), PharmDB-K, and Therapeutic Target Database (TTD). After mapping the genes/targets of calycosin and HF/DM using an online website (http://bioinformatics.psb.ugent.be/webtools/Venn/), the predictive targets of calycosin against HF were determined  terms. In addition, literatures were restricted to the journal original paper and English language. The databases Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Chemical-Protein Interaction Networks  network using TSV data from the STRING database. The final hub targets of calycosin against HF/DM were obtained using the Network Analyzer tool in Cytoscape (v3.7.1) software, calculating from the degree values of topology parameters , 21.https://pubchem.ncbi.nlm.nih.gov/). The protein structures of HF/DM targets were obtained from the Protein Data Bank database (https://www.rcsb.org/). Molecular mechanics-2 force field optimization was conducted using the three-dimensional structure of the compound downloaded using the ChemBio3D Draw module in the ChemBioOffice software (version 2010). The pdbqt structure file necessary for virtual screening was created using the Raccoon software. Next, the docking active center  was set using the grid box function in the software, according to the root mean square deviation (RMSD) between the docked ligand and original ligand molecules with appropriate docking parameters. RMSD ≤4 is generally believed to be the threshold for the ligand conformation after docking to match the original ligand conformation  were used to perform a gene ontology (GO)–based biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of hub targets. Finally, the bubble charts, graphs, and histograms were obtained and visualized , 25.https://cytoscape.org/), the data of hub targets in the biological processes and pathways of calycosin against HF/DM were used to construct drug–target–GO and function–pathway–disease visualization graphics . As shown in Following the bioinformatics analysis, the accessible data revealed 1,499 and 2,359 HF- and DM-related genes, respectively. Furthermore, 113 genes of calycosin were found after screening and removing duplicates using the UniProt database , ARRB1, ABL1, CDK1, MAP3K3, STUB1, PPP1CA, STAT3, PML, IQGAP1, HSPA9, CAV1, KPNB1, SQSTM1, RUVBL1, SFPQ, PPP2CA, PCBP1, ANXA2, and PAK1 Figure 2P < 0.05). The primary signaling pathways comprised proteoglycans in cancer, oocyte meiosis, acute myeloid leukemia, the chemokine signaling pathway, focal adhesion, regulation of actin cytoskeleton, Epidermal Growth Factor Receptor (ErbB) signaling pathway, MAPK signaling pathway, neurotrophin signaling pathway, axon guidance, hepatitis C, adrenergic signaling in cardiomyocytes, pathways in cancer, herpes simplex infection, Cyclic Adenosine Monophosphate (cAMP) signaling pathway, and viral carcinogenesis -Signal Transducer and Activator of Transcription (STAT) cascade involved in the growth hormone signaling pathway, and positive regulation of dendrite development. The cellular components (CCs) of hub targets were related to cytosol, cytoplasm, nucleoplasm, extracellular exosome, ruffle, focal adhesion, cell–cell junction, nuclear membrane, cell–cell adherens junction, nuclear matrix, midbody, nuclear chromosome, telomeric region, protein complex, microtubule cytoskeleton, nucleus, basolateral plasma membrane, pseudopodium, membrane raft, membrane, and axon. The molecular functions (MFs) of the hub targets included protein binding, ubiquitin–protein ligase binding, protein kinase binding, cadherin binding involved in cell–cell adhesion, ATP binding, protein kinase activity, protein serine/threonine kinase activity, S100 protein binding, RNA polymerase II carboxy-terminal domain kinase activity, mitogen-activated protein kinase binding, transcription regulatory region DNA binding, Hsp90 protein binding, poly(A) RNA binding, Hsp70 protein binding, transcription factor binding, Rac Guanosine Triphosphatase (GTPase) binding, Smad binding, phosphoprotein phosphatase activity, identical protein binding, and protein kinase C binding .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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This review addresses the longstanding debate over whether amyotrophic lateral sclerosis (ALS) is a ‘dying back’ or ‘dying forward’ disorder in the light of new gene identifications and the increased understanding of mechanisms of action for previously identified ALS genes. While the topological pattern of pathology in animal models, and more anecdotally in patients is indeed ‘dying back’, this review discusses how this fits with the fact that many of the major initiating events are thought to occur within the soma. It also discusses how widely varying ALS risk factors, including some impacting axons directly, may combine to drive a common pathway involving TAR DNA binding protein 43 (TDP-43) and neuromuscular junction (NMJ) denervation. The emerging association between sterile alpha and TIR motif-containing 1 (SARM1), a protein so far mostly associated with axon degeneration, and sporadic ALS is another major theme. The strengths and limitations of the current evidence supporting an association are considered, along with ways in which SARM1 could become activated in ALS. The final section addresses SARM1-based therapies along with the prospects for targeting other axonal steps in ALS pathogenesis.The online version contains supplementary material available at 10.1007/s13311-022-01297-6. A plant with insufficient water wilts from its leaves, but it is the roots that have to be watered. All parts of the plant may eventually be lost if the situation persists, but the leaves are the hardest to maintain and the easiest to sacrifice and regrow. Letting these go first maximizes the chance of overall survival.One central theme of this review is the extent to which the longstanding debate over ‘dying back’ or ‘dying forward’ models of ALS , 2 relatA second theme, which is closely connected with the first, is the emerging role of SARM1 in ALS. SARM1 is particularly well known for its profound effects in killing axons , 4. AlthThe third set of questions relate to whether there is a single mechanism of axon loss in ALS or several. How might the involvement of TDP-43 in most cases relate to axon and synapse loss, and what other risk factors, including ageing, may interact with this to make axons particularly vulnerable?The final area of focus is therapy. Do we need to target axon survival specifically or might it be sufficient to address underlying issues in other compartments? What treatment or prevention strategies are being pursued and with what success? Could axon regeneration help, and what future options may come available?It is important to clarify what we understand by ‘dying back’ in neurological disorders. The term has several meanings and using them interchangeably can lead to confusion. Each meaning is considered below, along with a critical assessment of whether this applies in ALS.The first use of the term ‘dying back’ in toxic neuropathies , 13 refeThe extent to which axon loss precedes soma loss in ALS is complicated by the different methods used to assess survival or death in different compartments. The most accurate measure of axon loss in peripheral nerve or ventral roots is electron microscopy, where a dying axon is defined by granular disintegration of the cytoskeleton and mitochondrial swelling . Once thNeuronal survival on the other hand is assessed using different methods altogether. DAPI or Nissl staining are commonly used to count live neurons, while propidium iodide or TUNEL staining give a measure of neurons that are dying or have been recently lost . QuantifNevertheless, from the best estimates using these imperfect comparison methods of the timing of axon and lower motor neuron cell death, it does indeed appear that axons are lost before the cell body, even in structures as proximal as the ventral roots . MoreoveA separate question is whether more distal regions of an axon die before its more proximal regions. This can be very hard to ascertain. There is some evidence for it in some conditions, obtained by measuring axon numbers at different proximal and distal sites relative to those in control nerves, or from longitudinal axon imaging . It is lG93A transgenic mice with sparsely labelled axons, which demonstrate beyond any doubt that NMJ loss is an early event ., 56.3, 5Nmnat2−/− mice are completely rescued by the simultaneous removal of SARM1 [SARM1 has a low level of basal activity but all of its enzyme activities are strongly activated by nicotinamide mononucleotide (NMN), the substrate of its upstream regulator, NMNAT , 59. Higof SARM1  indicatiNmnat1−/− mouse model suggest this mechanism is SARM1-dependent [While SARM1 activation due to NMNAT2 loss appears to be axon-specific, there are at least three ways it can be activated in, and kill, the neuronal soma too. First, mutation of NMNAT1 in Leber’s congenital amaurosis type 9 (LCA9) causes retinal ganglion cell death . Findingependent . Thus, jependent , kills tSecond, SARM1 can be directly activated by toxic metabolites of several nicotinamide analogues , 8, and SARM1 gene with sporadic ALS [The SARM1 activation mechanism most directly related to ALS is its direct hyperactivation by rare genetic variants that disrupt its N-terminal, inhibitory ARM domain. Mutations within this region that strongly enhance basal SARM1 NADase activity are associated with sporadic ALS and they are sufficient to enhance neuronal vulnerability to other stresses , 6. Thisadic ALS . The genFinally, the recent finding that zika virus causes SARM1-dependent neuronal death , along wALS results from varying combinations of a large number of risk factors. Twin studies show sporadic ALS has up to 61% heritability with the remaining ca. 39% presumably reflecting a range of environmental risk factors . With upThis wide range of genetic and environmental risk factors  suggestsThe model in Fig. In other circumstances when axons lack sufficient resources from the soma, it is often the supply of the labile, but essential axonal protein NMNAT2 that limits their survival , 63. ThiS) level or the SARM1 level [The extent to which this pathway contributes to ALS remains unknown but GWAS association with the SARM1 chromosomal locus  indicateM1 level , 89 despM1 level . NMJ denM1 level , and gliM1 level .Part 1(b) introduced the concept that even if many of the primary pathogenic steps in ALS occur in the soma, some patients have risk factors that strongly suggest axon-intrinsic mechanisms. These are discussed here in more detail. According to the model proposed in Fig. We have previously proposed that there is a spectrum of intrinsic axon vulnerability in the human population, based on expression and activity of SARM1 and its regulator NMNAT2 . This isAmong the other most convincing axon-intrinsic mechanisms are the many causes of cytoskeletal, and particularly axonal transport dysfunction . Among tWhile axonal transport defects may contribute to disease in KIF5A cases, and potentially in others influencing cytoskeleton or motor proteins such as dynactin subunit 1 (DCTN1), neurofilament proteins , spastin (SPAST) and tubulin alpha 4a (TUBA4A) , the widLocal protein synthesis in axons is another vital mechanism that may be disrupted in ALS by an axon-intrinsic mechanism. One reflection of this could be that the appearance of aggregates of RNA binding protein TDP-43 in axons precedes their degeneration in patients . TDP-43 In view of the above discussion of whether ALS pathogenesis is driven by soma and/or intrinsic axonal events, and how the balance may differ between different patients according to the presence or absence of axon-specific risk factors, it is important to ask whether axons need to be targeted directly for an effective therapy, or whether it is sufficient to remove primary causes that may lie elsewhere.Axon-based strategies are likely to play important roles in combinatorial therapies for ALS but their potential for use in isolation may be limited to patients where the predominant risk factor is an axon-intrinsic one. For example, when an axonal role of KIF5A or TDP-43, or a deficit in local translation, makes a substantial contribution, targeting these consequences in axons could be particularly useful. Possible methods include boosting fast axonal transport by inhibition of p38 MAPK , a stratIn cases where a weakened soma fails to support the large, and potentially expanded axonal arbor Fig. , leadingIn connection with this, there is now a substantial literature and wider public discussion of therapy and prevention of neurological disorders using precursors of NAD, such as nicotinamide (Nam) and its riboside (NR) or mononucleotide (NMN) , 116. ThFinally, there are many proposed strategies to boost compensatory sprouting , 118 or In summary, there are multiple risk factors for ALS, some acting in the soma, some in axons and some potentially in glia or elsewhere. These are all likely to contribute to pathogenesis in varying degrees in different patients. However, there are common features that suggest convergence in most cases onto a mechanism involving TDP-43, probably reflected in its near ubiquitous aggregation, and ‘dying back’ of axons particularly as defined by NMJ denervation. The emerging links to SARM1, including the GWAS association of its chromosomal locus , suggestSupplementary file1 (PDF 491 KB)Supplementary file2 (PDF 109 KB)Below is the link to the electronic supplementary material.