Gout is a common disease with high incidence due to unhealthy diet and living habits. Simiao Powder, as a classic formula consisted of four common herbs, has been widely used in clinical practice since ancient times to prevent and treat gout. However, the pharmacological mechanism of Simiao Powder is still unclear.Based on network pharmacology, Simiao Powder active compounds were identified in TCMSP, ETCM and BATMAN database, used to establish a network of interaction between potential targets of Simiao Powder and known therapeutic targets of gout. Subsequently, the key potential targets are being used for protein–protein interaction, GO enrichment analysis and KEGG pathway enrichment analysis through several authoritative open databases. Molecular docking through AutoDockTools software can verify interaction between molecules. Finally, to validate the predicted results, in vivo experiments based on hyperuricemic-gout mice model were designed and treated with Simiao powder and allopurinol. Serum levels of uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN) and xanthine oxidase (XOD) were determined using a customized assay kit while the expression of PPAR-γ, PTGS1, IL-6 and Bcl2 mRNA were analyzed through qRT-PCR.Disease-target-compound network was visualized basing on the 20 bioactive compounds and the 19 potential targets using Cytoscape software. The results of PPI analysis, GO enrichment and KEGG pathway enrichment analysis indicate that the potential mechanism of Simiao Powder in treating gout may be achieved by regulating immune and inflammatory reactions, improving metabolism and endocrine. The results of molecular docking show that most of the targets and components have good binding activity. In vivo experiments revealed that Simiao powder can decreased serum UA and XOD levels in hyperuricemic-gout mice, and improved renal function. Furthermore, Simiao powder certainly regulates the expression of PPAR-γ, PTGS1, IL-6 and Bcl2 mRNA in ankle tissue in hyperuricemic-gout mice.Collectively, this research predicted a multiple compounds, targets, and pathways model mechanism of Simiao Powder in the prevention and treatment of gout, providing new ideas and methods for in-depth research, via vivo experiments. Gout is a common chronic disease with urate crystal deposition in and around the joints results from longstanding hyperuricaemia , 2. The The treatment of gout mainly includes anti-inflammatory and analgesic treatment in acute arthritis, uric acid control in chronic phase and daily personal life management . MedicatAtractylodes Lancea (Thunb.)Dc, Phellodendri Chinrnsis Cortex, Cyathulae Radix, Coicis Semen, is derived from traditional Chinese medicine and widely used in the clinical treatment of gout in China due to good efficacy and few side effects [At present, it has become the focus of pharmaceutical research to search for drugs to treat gout and reduce hyperuricemia from Chinese herbs and natural materials –17. Rece effects , 19. Mod effects –22. HoweNetwork pharmacology is a new method of drug research proposed based on multi-directional systematic pharmacology and biology –25, whichttp://lsp.nwu.edu.cn/tcmsp.php), ETCM [http://www.tcmip.cn/ETCM/index.php/Home), BATMAN [http://bionet.ncps-b.org.cn/batman-tcm) using “Atractylodes Lancea (Thunb.)Dc”, “Phellodendri Chinrnsis Cortex”, “Cyathulae Radix” and “Coicis Semen”. TCMSP, a unique systems pharmacology database for users to comprehensively study Chinese herbal medicines, contains information on all 500 TCM formulae registered in the Chinese Pharmacopoeia (2010 edition), with a total of 30,069 compounds extracted by literature mining and database integration [To identify the chemical compounds of the four herbs in Simiao Powder, we performed a search in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  , ETCM   evaluation, two ADME-related models, including the evaluation of oral bioavailability (OB) and drug-likeness (DL), were employed to identify the potential bioactive compounds of Simiao Powder. Oral bioavailability (OB) and drug-likeness (DL) are very important pharmacokinetic parameters and key indicators in ADME process . Accordihttps://www.drugbank.ca/) while the gene names were extracted from UniProt Knowledgebase [http://www.uniprot.org). The DrugBank database is a bioinformatics and cheminformatics resource that combines detailed drug data with comprehensive drug target information. The UniProtKB is a central hub for the collection of functional information on proteins, with accurate, consistent and rich annotation, includes widely accepted biological ontologies, classifications and cross-references, and clear indications of the quality of annotation in the form of evidence attribution of experimental and computational data.The protein targets of the compounds were searched from the TCMSP and Drugbank database  (https:/edgebase  (UniProthttps://www.genecards.org), the therapeutic target database [https://db.idrblab.org/ttd/), Pharmacogenomics Knowledge Base [https://www.pharmgkb.org/), and the Online Mendelian Inheritance in Man (OMIM) database [www.omim.org/). The three databases mentioned above are commonly used databases for analysis of genomic known diseases with a genetic component and linked them to the relevant genes in the human genome, and they complement each other with their own characteristics. “Gout” was used to search in all the database as the keyword.Therapeutic targets of gout were collected from four sources, include GeneCards database  and String [https://string-db.org). The two open databases covered the majority of known protein–protein interactions information for human.Based on the prediction targets between compounds and disease, protein–protein interaction (PPI) data were generated through two major existing public PPI databases with the species limited as “Homo sapiens”, including Metascape  (http://d String  (https:/https://david-d.ncifcrf.gov/) and OmicShare platform (https://www.omicshare.com/).The gene ontology (GO) enrichment analysis was conducted to further validate whether the potential targets are indeed a match for gout in three levels, contained biological process analysis, molecular function analysis, cell component analysis. The GO enrichment analysis was performed using the functional annotation tool of DAVID Bioinformatics Resources 6.7   was purchased from Liaoning Changsheng Biotechnology Co., Ltd. . Animals were housed under a normal 12-h/12-h light/dark schedule, room temperature (22 ± 2 °C), relative humidity (55 ± 5%), and given a standard chow and water ad libitum for the duration of the study. All studies were carried out in accordance with the Institutional Animal Care Committee at the Jinan University. Simiao Powder, consisting of four classical herbs: Atractylodes Lancea (Thunb.)Dc, Phellodendri Chinrnsis Cortex, Cyathulae Radix, Coicis Semen,were purchased from Huarun Sanjiu Medicine Co., Ltd. According to the literature , it was Experimental animal model of Hyperuricemic-Gout Mice induced by uricase inhibitor potassium oxonate has been used to study drug action for many years. Mice were divided into 4 groups: Normal + water(group A), Model + water(group B), Model + Simiao Powder(group C), Model + allopurinol(group D). The normal group was intraperitoneally injected with 0.5% CMC-Na solution, while the other groups were intraperitoneally injected with suspension composed of 300 mg/kg potassium oxazinate and 200 mg/kg hypoxanthine at 15:00 p.m. every day for seven consecutive days . Simiao Powder and allopurinol were orally initiated at 16:00 p.m. on the day by intragastric administration. Dosages of Simiao powder and allopurinol were determined based on the conversions from clinical adult dosages and previous studies. The dosage was as follows : allopurWhole blood samples were collected 1 h after final administration on the seventh day, and then allowed to clot for approximately 1 h at room temperature and centrifuged at 3500 rpm for 10 min to obtain serum, stored at − 80 °C until assayed. Simultaneously, ankle tissue was rapidly and carefully separated on ice-plate and stored at − 80 °C for assays, respectively.Serum levels of uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN) and xanthine oxidase (XOD) were determined using a customized assay kit. All assay kits were purchased from Jiancheng Biotech. . Uric acid concentration in serum was determined by the phosphotungstic acid method. Blood urea nitrogen was determined by rapid urease method while Creatinine was measured using sarcosine oxidase method. Xanthine Oxidase was detected by colorimetric method. The operation method of the experiment shall strictly refer to the manufacturer's instructions.Related methods are described in our previous article . BrieflyP < 0.05 was considered statistically significant. Figures were obtained by GraphPad Prism 9 .All data are statistically analyzed using one-way ANOVA analysis with the statistical software SPSS 13.0. Data were expressed as Mean ± Standard Error of the mean(S.E.M.). A value of Atractylodes Lancea (Thunb.)Dc, 4 in Phellodendri Chinrnsis Cortex, 37 in Cyathulae Radix, 9 in Coicis Semen. Among the 59 satisfied compounds, a total of 56 compounds were finally obtained after excluding the duplicates.Based on the criteria of OB ≥ 30% and DL ≥ 0.18, a total of 59 compounds of Simiao Powder were retrieved from the TCMSP, including 9 in According to the target prediction system in TCMSP and Drugbank databases, the 56 potential active components of Simiao Powder collected were input into the databases and 824 putative targets were obtained by pairing. The corresponding target proteins were searched through the UniprotKB database for their corresponding gene symbol names.A total of 756 targets associated with gout were collected from the four open databases, namely, 734 in GeneCards database, 15 in TTD, 4 in PharmGKB and 3 in OMIM database. After removing the overlapping targets, 746 associated targets were obtained.The prediction targets of compounds and disease-related targets were combined and intersected to obtain the possible targets of drug treatment for gout. Subsequently, the result showed that gout share 19 targets with 20 bioactive compounds of Simiao Powder  data were generated for the 19 common targets mentioned above (The confidence score > 0.4). The PPI network showed current interactions among the 19 common targets  enrichment analysis was performed and the results were revealed in three levels via biological process, molecular function and cell component analysis. Figure The KEGG pathway enrichment analysis was performed using the functional annotation tool of DAVID Bioinformatics Resources 6.7 and OmicShare platform. Figure Finally, Compounds-Targets-pathways network for Simiao Powder acting on gout was constructed shown as Fig. The molecular docking software AutoDockTools can apply semi-flexible docking methods on the basis of complex networks to study the interaction between molecules, and then evaluate the protein–ligand binding potential based on its comprehensive characteristics. There were 3 targets with Degree value ≥ 6 in the disease-target-compound network diagram, and top 3 targets in the PPI network diagram. After removing duplicate targets, we obtained 4 targets in total: IL-6, PTGS1, PPARG, BCL2. These targets were searched in the literature respectively and all targets were related to gout. They were respectively molecularly docked with the potential active compounds of Simiao Powder.According to the results of comprehensive molecular docking, the minimum binding energy was -3.31 kcal/mol and the maximum is -7.72 kcal/mol. Among them, the 4 potential active compounds of Simiao Powder  and the amino acid residues of the targets IL-6, PTGS1, PPARG, BCL2 were combined by intermolecular hydrogen bonds. Most of the targets and components had good binding activity because of high binding energy. This indicates that the main characteristic chemical components of Simiao Powder have good binding ability with the targets IL-6, PTGS1, PPARG and BCL2. In the results of molecular docking, there were 31.25% of them binding energy higher than -5 kcal/mol while others were lower than -5 kcal/mol. In addition, there were two of them lower than -7 kcal/mol. Among them, the binding energy of PPARG and stigmasterol is the highest, -7.72 kcal/mol. The results of molecular docking were shown in Table As expected, intraperitoneally injected potassium oxazinate and hypoxanthine significantly elevated serum UA and XOD levels compared with normal mice Fig. a. SimiaoSerum BUN and Cr levels in Hyperuricemic-gout mice were significantly higher than those in the normal group Fig. c-d. SimiThe effects of Simiao powder and allopurinol on mRNA levels of ankle PPAR-γ, PTGS1, IL-6, and Bcl2 in hyperuricemic-gout and normal mice were shown in Fig. Traditional Chinese medicine considers gout to be arthralgia syndrome result from the dysfunction of viscera , which iThis research demonstrates that 20 active compounds and 19 potential genetic targets of Simiao Powder in the treatment of gout were found through network pharmacology. Then, specific functions and mechanisms are revealed through protein interaction network analysis, GO enrichment analysis and KEGG pathways enrichment analysis. The results showed that Simiao Powder, through multi-components-targets-pathways mode, play an important role on the immune regulation, inflammatory response, metabolism and endocrine regulation, cell signal transduction, cell growth and apoptosis, so as to achieve anti-inflammatory and analgesia, uric acid lowering and metabolism improvement. Further classification and analysis showed that the therapeutic effects of Simiao Powder on gout mainly focused on several modules, such as immune regulation and inflammatory response, metabolism and endocrine regulation, and cell function. To visually reflect the important pathway diagram and its key targets, they were compressed and drawn in Fig. Generally speaking, uric acid crystal deposition in articular cavity is the trigger of gout , 13. TheAmong the major putative targets and pathways of Simiao Powder, most of them are related to the immune regulation and inflammatory response for gout. At the gene target level, PTGS1 is the key target with the largest number of junctions, usually responds to hormone stimulation rapidly to produce prostaglandins and continuously regulate physiological processes, such as mediating responses to inflammatory stress . FurtherGout is defined as a metabolic disorder . StudiesOne mechanism of acute gout inflammation is the noninflammatory phagocytosis of macrophages to dying neutrophils , 59. TheThese modules have their own unique functions, but they are connected and affect with each other, forming a dynamic adjustment network. That reflects the characteristics of multiple compounds, multiple targets and multiple pathways of Simiao Powder in the treatment of gout. Furthermore, gout is often accompanied by other comorbidities , 68, 69 Molecular docking reveals the interaction between components and targets in the network, thereby improving the accuracy of the network . The negSimiao powder decreased serum UA and XOD levels in hyperuricemic-gout mice, and improved renal function via regulating serum BUN and Cr levels. It was noted that both Simiao powder and allopurinol can effectively reduce serum uric acid and XOD, but Simiao powder may bring few side effects and improve renal function in the treatment. Our experimental results are consistent with a previous study . In addiOur research still has some limitations. For example, the actual absorption of drugs may be different from the reality, and drug compounds may react with each other to form new unknown compounds. Furthermore, our screening criteria for drug compounds are based on oral absorption and pharmacodynamics, but some compounds may not be absorbed into the blood but act on intestinal microecology, which is conducive to the regulation of immunity and metabolism.Collectively, this research predicted a multiple compounds, targets, and pathways model mechanism of Simiao Powder in the prevention and treatment of gout, providing new ideas and methods for in-depth research, via vivo experiments.


Staphylococcus aureus, the SaeRS two-component system is essential for the bacterium’s hemolytic activity and virulence. The Newman strain of S. aureus contains a variant of SaeS sensor kinase, SaeS L18P. Previously, we showed that, in the strain Newman, SaeS L18P is degraded by the membrane-bound protease FtsH. Intriguingly, the knockout mutation of clpP, encoding the cytoplasmic protease ClpP, greatly reduces the expression of SaeS L18P. Here, we report that, in the strain Newman, the positive regulatory role of ClpP on the SaeS L18P expression is due to its destabilizing effect on FtsH and degradation of MoeA, a molybdopterin biosynthesis protein. Although the transcription of ftsH was not affected by ClpP, the expression level of FtsH was increased in the clpP mutant. The destabilizing effect appears to be indirect because ClpXP did not directly degrade FtsH in an in vitro assay. Through transposon mutagenesis, we found out that the moeA gene, encoding the molybdopterin biosynthesis protein A, suppresses the hemolytic activity of S. aureus along with the transcription and expression of SaeS L18P. In a proteolysis assay, ClpXP directly degraded MoeA, demonstrating that MoeA is a substrate of the protease. In a murine bloodstream infection model, the moeA mutant displayed reduced virulence and lower survival compared with the WT strain. Based on these results, we concluded that ClpP positively controls the expression of SaeS L18P in an FtsH and MoeA-dependent manner, and the physiological role of MoeA outweighs its suppressive effect on the SaeRS TCS during infection.In  Staphylococcus aureus is an important human pathogen causing a broad range of diseases from minor skin infections to life-threatening infections, such as pneumonia, bacteremia, and osteomyelitis [myelitis . The bacmyelitis . The promyelitis .hla] and nuclease [nuc]) ) ,6. SeverS system . IntriguS system .S. aureus, there are three ATP-dependent proteases:ClpP, HslV (ClpQ), and FtsH [S. aureus [S. aureus, it is required for stress resistance and virulence [ATP-dependent proteolysis systems maintain protein homeostasis by degrading unfolded or misfolded substrate proteins . In S. aand FtsH . The Clpand FtsH . The Clp. aureus , whereas. aureus . FtsH isirulence . In partirulence . Here, wHuman heparinized venous blood was obtained from healthy volunteers following a protocol approved by the ethics committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. The written informed consent were obtained from all individuals before donating blood.The animal experiment was approved by the ethics committee of Renji Hospital, School of Medicine, Shanghai Jiaotong University. We try our best to minimize the suffering of the animals.S. aureus strains and plasmids used in this study are listed in Supplementary Table S1. Various media including lysogeny broth (LB) and tryptic soy broth (TSB) was used for Escherichia coli and S. aureus culture, respectively. However, heart infusion broth (HIB) supplemented with 5 mM CaCl2 was used for transduction of plasmids. When necessary, antibiotics were added to the growth media at the following concentrations: ampicillin, 100 μg/ml; erythromycin, 10 μg/ml; kanamycin, 50–100 μg/ml and chloramphenicol, 10 μg/ml.The E. coli by the method of Hanahan and Meselson [S. aureus RN4220 with a gene pulser (Bio-Rad). The plasmids from RN4220 were introduced into target strains of S. aureus by transduction with ϕ 85.The restriction enzymes and DNA modification enzymes (New England Biolabs) were used for plasmid construction. The plasmid miniprep kit (Omega) was used for plasmid extraction according to the manufacturer’s instructions. Plasmid DNA was transformed Meselson  and elecclpQ deletion [clpQ were amplified by PCR using PrimSTAR (Takara). The primers used for the amplification were listed in Supplementary Table S2. After treatment with T4 DNA polymerase in the presence of dGTP (vector) or dCTP (insert DNA), the PCR products were mixed and transformed E. coli DH5α. The resulting plasmid pIMAYΔclpQ was introduced into the strain Newman as mentioned above. The deletion was acquired according to the protocol mentioned in the literature [A ligation independent cloning (LIC) method was used for deletion . Brieflyterature .moeA gene. The plasmid was constructed and introduced into the strains Newman using the same method as above. The deletion was carried out according to the protocol for pKOR1 [The plasmid pKOR1 was used for the deletion of the or pKOR1 .moeA complement plasmid, moeA was PCR-amplified using the primers PN5/6 (Supplementary Table S2). The restriction enzyme SmaΙ/BamHΙ were used for the pOS1 and amplified fragment digest [moeA were introduced into the strain NM∆moeA for the complementation test. To express MoeA proteins in vitro, moeA was amplified using the primers PN7/8 by PCR. The enzymes EcoRI/BamHI were used for pET28a-moeA construction. The protein was expressed in the strain E. coli BL21 (DE3).To construct t digest . The res600 ≈2). The Mini-Beadbeater (Biospec Products) was used for cell disruption at maximum speed for 30 s. After incubation on ice for 5 min, the suspension was centrifuged. The supernatant was used for RNA extraction according to the manufacturer’s instructions (Qiagen). Totally, 1 μg of total RNA was used for DNA removal using a TURBO DNA-freeTM kit (Ambion). The HieffTM first Strand cDNA Synthesis Super Mix for RT-PCR kit  was used for cDNA synthesis. The following real-time PCR was performed by SYBR-Green PCR reagents (Roche) using cDNA as a template on a 7500 Sequence Detector (Applied Biosystems). Primers used for RT-PCR are listed in Supplementary Table S2. All RT-PCR experiments were performed using gyrB as an internal control.The bacteria was cultured in 3 ml TSB in tubes (15 ml) with shaking (200 rpm) at 37°C and harvested at stationary phase (OD600 = 2. The cell pellets were treated with lysostaphin (50 μg/ml) for 30 min at 37°C and mixed with 2 × SDS loading buffer. Western blot analysis was carried out by following the protocol described in the literature [The test strains were diluted 1:100 into fresh TSB (3 ml) in tubes (15 ml) from overnight culture. After shaking at 37°C (200 rpm) for 8 h, the bacteria were collected and normalized to ODterature . The FtsWe collected the bacterial culture supernatants by passing through a 0.22 μm filter. After dilution with fresh TSB (1:10), the cell-free supernatants were incubated with 500 μg/ml salmon sperm DNA (Sigma) at 37°C At the different time points, samples were mixed with 6× loading buffer. 1% agarose gel with ethidium bromide was used for DNA visualization.E. coli BL21 (DE3) strain carrying the corresponding plasmid was grown in LB with 50 μg/ml kanamycin to exponential growth phase (OD600 = 0.6). then we added 0.5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for inducing protein expression. The culture was further incubated at 16°C overnight. The His-ClpX, His-ClpP, and His-MoeA fusion proteins were purified with Ni-NTA agarose (Novagen) according to the manufacturer’s instruction. Proteins were eluted in lysis buffer  containing 500 mM imidazole. Purified proteins were concentrated and dialyzed against the dialysis buffer  glycerol, 50 mM KCl, 5 mM MgCl2), using a 10 K molecular weight dialysis cassette (Sangon Co.). The Bradford Protein Assay kit (Sangon Co.) was used for the qualification of protein concentration using bovine serum albumin as a standard.The His-ClpX, His-ClpP and His-MoeA proteins were purified as described by Ni . Briefly2, 1 mM EDTA, 5% (w/v) glycerol, and 1 mM DTT. The assay was performed at 37°C when 6 μM purified His-FtsHc, His-MoeAor 0.5 mg/ml α-Casein (Sigma C6780) was added to the mixture. At each given time point, 15 μl sample taken from the assay was mixed with 2 × SDS loading buffer. The samples were subjected to SDS-PAGE and the protein was visualized by coomassie blue staining.For protease degradation assay, 3 μM ClpX, 5 μM ClpP, 8 mM ATP, and an ATP regeneration system (80 μg/mL creatine kinase and 40 mM creatine phosphate) were mixed within reaction buffer containing 50 mM HEPES-KOH (pH 7.6), 50 mM KCl, 20 mM MgClclpP transposon mutant of Newman strain, where the transposon in clpP confers resistance to erythromycin.The transductant was inoculated into 10 ml fresh TSB containing 100 μg/ml kana and 10 μg/ml chl. After shaking overnight at 30°C (200 rpm), the culture was diluted at 1:100 into 10 ml fresh TSB containing 100 μg/ml kana and cultured overnight at 42°C with shaking. This process was repeated twice.The putative transposon insertion mutants (kanR and chlS) were selected and were used for library screening.Transposon mutagenesis was performed using plasmid pMA15. The plasmid has a chloramphenicol (chl)-resistance marker and can deliver a mariner transposon with kanamycin (kana) resistance marker. The plasmid also contains an improved temperature-sensitive replicon repC3 of pT181, and its replication is blocked at 42°C . First, After culture for 18 h, the culture filtrates were collected and incubated with human red blood cells (2% v/v in PBS) in a 96-well round-bottom plate. After incubation at 37°C for 1 h, the plate was centrifuged at 4000 rpm for 10 min, and the supernatant was transferred into another 96-well flat-bottom plate. The optical density at 540 nm was measured on a plate reader . The assay was performed in triplicate.600 ≈1.0). After washing with PBS twice, the bacteria was adjusted to different concentration in PBS and administered into eight female Balb/c mice (6 week-old) mice via retro-orbital injection.The tested strains were grown in TSB to exponential growth phase (OD8 CFU) and observed for 4 days. The survival was compared by Log-rank (Mantel-Cox) test with Prism.For survival curve, the mice were injected with 100 μl bacterial suspension .All the statistical analysis was performed by unpaired, two-tailed Student’s S. aureus Newman. As expected, the deletion of ftsH increased the protein level of SaeS  of nearby region. Nonetheless, the transcript analysis results were mirrored by Western blot and nuclease assay: The Nuc expression level and the nuclease activity were reduced by the clpP mutation and restored by the clpP complementation plasmid  ,12. To alpP gene . The rea plasmid . Based oftsH was not affected by ClpP , a known Sae target, and hemolytic activity of culture supernatant  showed increased hemolytic activity. Inverse PCR analysis showed that the transposon was inserted in moeA, encoding molybdopterin molybdenum transferase  . All the strains reached the stationary growth phase when OD600 was around 2, so the samples were collected at the stationary growth phase. We observed that the transcriptional expression of saeS was significantly decreased in clpP mutant strain, which can be reversed in clpP/moeA double mutant strain , which is essential for the activity of various metabolic enzymes. Therefore, we decided to examine the role of MoeA in the virulence of S. aureus using a murine blood infection model. When the WT, the moeA and the sae mutant of Newman were administered to mice via retro-orbital injection, the WT strain killed around 70% of mice by day 1 post-infection; while the moeA mutant strain killed 30% of mice during the same time. Obviously, the moeA mutant strain took significantly longer time to kill all the mice compared with the WT strain. However, the sae mutant strain killed only 60% mice by day 4 post-infection (moeA and sae mutants were identified, as compared with the WT strain (sae mutant compared with moeA mutant (in vivo survival of S. aureus. The suppressive effect on the Sae activity by MoeA also contribute to the virulence using bloodstream infection model.The SaeRS TCS is critical for the virulence of nfection . When thT strain . InteresA mutant . These rS. aureus, SaeRS TCS is a signaling system required for the expression of many virulence factors [In  factors . Previou factors . This stS. aureus, ATP-dependent protease ClpP and FtsH play important roles in stress resistance and virulence regulation of S. aureus [clpP/ftsH double mutant strain compared with ftsH single mutant strain (E. coli FtsH, which forms a complex with HflKC protein [S. aureus FtsH might form a complex with other proteins and is subjected to proteolysis by other proteases, likely in the membrane. So far, however, no such FtsH-controlling proteins have been reported in S. aureus and await for identification.Proteolysis is a key process for the turnover and homeostasis of proteins in bacteria. In . aureus ,27. Altht strain , indicatt strain . Consist protein , S. aureE. coli and Mycobacteria tuberculosis, MoeA is necessary for nitrate reductase activity and anaerobic metabolism [To explain the positive regulatory role of ClpP in the SaeRS TCS, initially, we hypothesized that ClpP antagonizes the FtsH activity by degrading a protein required for the stability of FtsH. However, when we tested the hypothesis by a transposon mutagenesis approach, we did not find such a protein factor; instead, we identified MoeA as a mediator for ClpP. As a molybdopterin molybdenum transferase, MoeA catalyzes the conjugation of molybdate to molybdopterin, the last step of the MoCo synthesis. MoCo forms the active site of various enzymes involved in bacterial growth, metabolism, virulence etc ,32. For tabolism ,34. Sinctabolism , MoeA watabolism . TherefosaeS was reversed in clpP/moeA double mutant strain (clpP/moeA double mutant strain than in the moeA deletion mutant (clpP/moeA double mutant strain (clpP/moeA double mutant strain compared with WT (clpP/moeA double mutant is still sufficient to activate the alpha-hemolysin synthesis and maintain the hemolytic activity.This study demonstrated that MoeA not only is directly degraded by ClpXP but also serves as a negative regulator for the SaeRS TCS in the Newman strain  Intriguit strain , SaeS L1n mutant . It is lt strain . Althoug with WT , both st with WT . It is kmoeA is a negative regulator for the virulence regulator SaeRS, its disruption did not increase the virulence of S. aureus; on the contrary, the moeA mutant showed lower virulence and survival in vivo (moeA mutant displayed higher virulence compared with the sae mutant in the bloodstream infection model (S. aureus, MoCo is a cofactor for nitrate reductase and formate hydrogenase, enzymes for anaerobic nitrate respiration [moeA mutation, the higher activity of the SaeRS TCS and expression of higher levels of virulence factors seem insufficient to support the bacterial survival. Previously, Mashruwala, et al., reported that the supplementation of nitrate to anaerobic S. aureus culture suppressed the SaeRS-dependent biofilm formation and proposed a model where the SaeRS TCS responds to the cellular respiratory status [moeA gene in SaeS expression might be due to the disruption of the anaerobic nitrate respiration. Nonetheless, how the SaeRS TCS senses the cellular respiratory status remains unknown and warrants further investigation.Although  in vivo . Howeveron model . In S. apiration . Therefoy status . TherefoS. aureus Newman, ClpP positively affects the expression of SaeS L18P by destabilizing FtsH and degrading the metabolic protein MoeA. In S. aureus, the cytoplasmic ClpP proteolytic system appears to be functionally connected to the membrane protease FtsH. The metabolic protein MoeA is also involved in the regulation of SaeS L18P expression by unknown mechanism. The ClpP-mediated degradation of MoeA indicates that MoeA might be the focal regulatory point where ClpP affects metabolism and virulence simultaneously.In conclusion, we provided evidence that, in Click here for additional data file.


Abdominal aortic aneurysm (AAA) is a fatal disease characterized by high morbidity and mortality in old population. Globally, effective drugs for AAA are still limited. Si-Miao-Yong-An decoction (SMYAD), a traditional Chinese medicine (TCM) formula with a high medical value, was reported to be successfully used in an old AAA patient. Thus, we reason that SMYAD may serve as a potential anti-AAA regime.  The exact effects and detailed mechanisms of SMYAD on AAA were explored by using the experimental study and bioinformatics analysis.  Firstly, C57BL/6N mice induced by Bap and Ang II were utilized to reproduce the AAA model, and the effects of SMYAD were systematically assessed according to histology, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA). Then, network pharmacology was applied to identify the biological processes, pathways, and hub targets of SMYAD against AAA; moreover, molecular docking was utilized to identify the binding ability and action targets.  In an animal experiment, SMYAD was found to effectively alleviate the degree of pathological expansion of abdominal aorta and reduce the incidence of Bap/Ang II-induced AAA, along with reducing the damage to elastic lamella, attenuating infiltration of macrophage, and lowering the circulating IL-6 level corresponding to the animal study, and network pharmacology revealed the detailed mechanisms of SMYAD on AAA that were related to pathways of inflammatory response, defense response, apoptotic, cell migration and adhesion, and reactive oxygen species metabolic process. Then, seven targets, IL-6, TNF, HSP90AA1, RELA, PTGS2, ESR1, and MMP9, were identified as hub targets of SMYAD against AAA. Furthermore, molecular docking verification revealed that the active compounds of SMYAD had good binding ability and clear binding site with core targets related to AAA formation.  SMYAD can suppress AAA development through multicompound, multitarget, and multipathway, which provides a research direction for further study. Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease, mainly associated with the risk factors that include male gender, smoking, and old age . AAA devFlos Lonicerae (Chinese name Jin-Yin-Hua), as the principal drug, has the efficacy of clearing away heat and detoxifying; Radix Scrophulariae (Chinese name Xuan-Shen) and Radix Angelica Sinensis (Chinese name Dang-Gui) are both adept in invigorating blood circulation and eliminating blood stasis; and Radix Glycyrrhizae (Chinese name Gan-Cao), as the mediator drug, coordinates the drug actions. Consequently, SMYAD can be used to promote poor blood circulation and remove detoxify, which means that it has the corresponding theoretical basis of TCM in the treatment of AAA. Moreover, SMYAD has been attempted to use in an old AAA patient and the clinic effect was fine [According to the dialectical treatment of TCM, AAA was primarily related to the blood stasis. Si-Miao-Yong-An decoction (SMYAD) is a traditional Chinese medicine (TCM) that has been used safely for hundreds of years. This classic ancient recipe was traditionally used for gangrene  and in twas fine . HoweverChinese materia medica is a complicated system of multicompounds, multitargets, and synergistic effect among compounds. Articulating the relationship among these elements is a difficult but fascinating challenge. Encouragingly, bioinformatics analysis provides us an up-to-date perspective to understand the interactions of compounds, pathways, and targets with disease . It can Male C57BL/6N mice, 6-7 months old, weighing 28–32 g were supplied by Charles River  and maintained on a 12:12-hour light-dark cycle with free ad libitum access to food and water for a one-week acclimatization period. Animal experiments were conducted in accordance with the guidelines for animal experiments of Wenzhou Medical University and approved by the animal ethics committee.Yanfangxinpian” (Qing dynasty) were Flos Lonicerae (90 g), Radix Scrophulariae (90 g), Radix Angelica Sinensis (60 g), and Radix Glycyrrhizae (30 g). The abovementioned raw herbs were all obtained from Tongrentang pharmacy. According to the procedures of automatic decocting machine (HYDY-5), two copies of daily raw herbs (270 g × 2) were washed thoroughly and put into the decocting pot. The raw herbs were soaked in distilled water (1.5 L × 2) for 1h and then decocted. Extracted solution (0.5 L × 2) was obtained after secondary filtered, and the concentration of the collected solution was equivalent to 0.54 g raw herbs/ml. The experimental dosage of SMYAD was determined according to the body surface conversion between human and mouse. Finally, the above solution was evaporated to the concentration of 13.2 g raw herbs/ml (defined as SMYAD-low dose) and 26.4 g raw herbs/ml (defined as SYMAD-high dose) by a rotary evaporator (LC-RE-5000). The above operations were repeated every day to prepare SMYAD.Daily doses of SMYAD recorded in “n = 6, control for short) and model group (n = 36). From the Day 1 to Day 42, the 6 mice in control received no Bap/Ang II and other treatments; the 36 mice in the model group were injected intraperitoneally with Bap  at a dose of 10 mg/kg body weight weekly and a dose (0.72 mg/kg/day) of Ang II  using subcutaneous osmotic mini-pumps . Current guideline for use of bio-hazardous materials was followed when using Bap. At Day 15, 36 mice in the model group were randomly and equally divided into three groups, namely, Bap-/Ang II-treated + saline group , Bap-/Ang II-treated + SMYAD-low-dose group , and Bap-/Ang II-treated + SMYAD-high-dose group . From Day 15 to Day 42, mice in Bap + Ang II + SL and Bap + Ang II + SH were fed intragastrically 1 ml of SMYAD-low dose or SMYAD-high dose once daily, respectively; meanwhile, mice in Bap + Ang II were fed intragastrically with 1 ml of saline solution once daily. On Day 43, all survival mice were weighed and then euthanized.After one-week acclimatization, 42 mice were weighed and then randomly divided into two groups (count this day as Day 1): normal control group (μm thick) were prepared and stained with haematoxylin-eosin (HE) stain. HE stain was used to assess the morphology and structure of abdominal aorta. Due to the irregular shapes of slices, the perimeter, instead of diameter, was measured with ImageJ for comparison. For each animal, two slices of abdominal aorta were selected and measured, and then, the average perimeter was calculated and recorded. The abdominal aorta with perimeter increases equaled or greater than 50% of average perimeter of mice in control was considered AAA formation. The hearts of mice were also collected and weighed.After euthanasia, the thoracic and abdominal cavities were probed, and the aorta was sequentially irrigated with PBS and 4% formaldehyde through the left ventricle. Macroscopic examination of aortas was performed and carefully cleaned under a dissection microscope. The suprarenal segments or obvious lesions of abdominal aorta were dissociated and fixed in 10% paraformaldehyde and then embedded in paraffin wax. The paraffin slices of the abdominal aortic sections (4 μm thick) were examined for macrophage infiltration by immunohistochemistry staining with the monoclonal CD68 antibody  against mouse macrophage according to the streptavidin-peroxidase (SP) method. Brown or dark brown staining in cytoplasm was defined as positive expression. The extensity of macrophage infiltration within the abdominal aorta was quantified with Image-Pro Plus (IPP) 6.0 software by the number of positively stained cells per 0.01 mm2. For each animal, two slices of stained abdominal aortic sections were selected and the average number of positive cells was recorded by an investigator who was unaware of sample's identity.The paraffin slices of the abdominal aortic sections -ELISA by commercially available ELISA kit . Testing was performed independently by a researcher who was unaware of sample's identity.t test; differences among groups were analyzed by one-way ANOVA, followed by Dunnett's T3 test. For the incidence of AAA, counts and percentages are presented. Differences among groups were analyzed by Fisher's exact test, followed by the Bonferroni test. The relationship of two independent normality quantitative samples was analyzed by linear correlation. All analyses were conducted with statistical SPSS software 25.0 . The level of significant difference was set as a 2-sided P < 0.05.Exploratory data analysis and Shapiro–Wilk tests were performed to determine the normality of the data distribution. Continuous variables are expressed as mean ± SD unless otherwise noted. The self differences analyzed by paired-https://tcmsp-e.com/,Ver.2.3) [https://pubchem.ncbi.nlm.nih.gov/) [https://targetnet.scbdd.com) [https://www.uniprot.org) [The compounds of SMYAD were screened out from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database (Ver.2.3) . Then, tih.gov/) . Then, tbdd.com) . Finallyrot.org) .Excel files containing the information of SMYAD-matched targets were established. Next, they were imported into Cytoscape 3.7.2 software . Finallyhttps://www.genecards.org/) [https://ctdbase.org/) [https://www.disgenet.org/home/) [https://www.omim.org/geneMap) [Homo sapiens” targets linked to AAA were selected and verified by their unique UniProtKB ID and target names in the UniProt database.“Abdominal aortic aneurysm” and “aortic aneurysm” were selected and set as searching keywords, and then, AAA-related targets were respectively screened from four sources: (1) GeneCards database (ds.org/)  with relse.org/)  with infg/home/)  with a sgeneMap) . Then, ahttps://www.bioinformatics.com.cn/) to obtain a Venn diagram.The SMYAD-matched targets and AAA-related targets were imported into the Bioinformatics platform (https://metascape.org) [P value cutoff = 0.01, and Min. enrichment = 1.5. The entries of GO were chosen and listed. The BP and KEGG pathways were selected out and visualized by Cytoscape.Two parts were included in enrichment analysis: Gene Ontology (GO) functional enrichment  and Kyotape.org) . The GO https://www.string-db.org) [PPI network provides information regarding the predicted and experimental interactions of proteins. The STRING database (-db.org)  was util-db.org) .https://www.rcsb.org) [Z-score)—row, clustering direction—bidirectional, clustering method—complete, and distance method—correlation. Moreover, the binding affinity, sites, and interactions between active compounds and potential targets were achieved and analyzed by classical molecular dynamics using Auto Dock Tools-1.5.6, Pymol 2.3, and Discovery Studio 4.5 Client.The 3D chemical structural formulas of chosen compounds were obtained from PubChem and energy minimizing employed to ChemBioDraw 3D. The crystal structures of selected targets were collected from the protein data bank (PDB) (csb.org)  for moleμm) than that in control (872 ± 31 μm), which indicated the successful establishment of the AAA model. The perimeter of abdominal aorta was significantly decreased, in both Bap + Ang II + SL (1251 ± 93 μm) and Bap + Ang II + SH (1208 ± 114 μm), compared to Bap + Ang II. Similarly, in comparison with control (0/6), the incidence of AAA was significantly increased to 66.7% (8/12) in Bap + Ang II (P < 0.05). The incidence of AAA was presented at 25.0% (3/12) and 16.7% (2/12) in Bap + Ang II + SL and Bap + Ang II + SH, respectively, which was significantly decreased compared to Bap + Ang II. Meanwhile, there was no significant difference in these indexes between Bap + Ang II + SL and Bap + Ang II + SH.The timeline of Bap-/Ang II-treated AAA model and SMYAD treatment is shown in HE staining was used to evaluate the histological structure of abdominal aorta, especially for elastic lamella. Elastic lamella was known to closely related to the stability of vascular structure . As show2 . The results shown in IL-6 was a clear and important mediator of inflammation, and increased circulating concentration of IL-6 had been found in patients with AAA . TherefoFlos Lonicerae, 4 in Radix Scrophulariae, 3 in Radix Angelica Sinensis, and 78 in Radix Glycyrrhizae. Among all compounds, quercetin (C2), beta-sitosterol (C3), kaempferol (C4), and stigmasterol (C5) were the common compounds. A total of 199 SMYAD-matched targets were identified and selected from TargetNet. In order to elucidate the inner relationship between the active compounds and SMYAD-matched targets clearly, the Cytoscape software was used to establish the “compounds-targets” network  –39, wereSimilarly, C1-C22 compounds of SMYAD were chosen as ligands. A total of 264 times were docked, and the total binding energy of each receptor and ligand was Figures . AccordiIn the present study, SMYAD reduced the pathological dilation of abdominal aorta and the incidence of AAA in Bap-/Ang II-treated mice. Decreased AAA formation occurred concomitantly with a reduction of elastin fiber destruction, macrophage infiltration, and expression of IL-6. These above results clearly demonstrated that SMYAD alleviated over-activated inflammation and ameliorated the damage to abdominal aortic. Thus, SMYAD was a potentially protective agent for Bap/Ang II-induced AAA.Smoking has long been considered a key risk factor for AAA. Bap, one of the major components of cigarettes, has been verified to contribute to AAA development. Furthermore, Bap could work synergistically with Ang II to induce AAA formation in mice by promoting macrophage infiltration and disruption of elastic lamella . Bap/AngT and B cells are observed in AAA tissue samples and might play roles in AAA. Moreover, the migration and infiltration of macrophage may play a prominent role in the development of AAA [An emerging concept is that AAA development is due to the inflammatory response, whereby many inflammatory cells and mediators have played important roles in regulating the activation of matrix-degrading proteins and smooth muscle cell apoptosis, resulting in the loss of medial elastic lamella and thinning of the tunica media , 43. Thet of AAA –49. Thert of AAA . Meanwhit of AAA –54, whicOur experimental study results suggested that SMYAD has a protective effect on the deterioration of AAA. We propose the pharmacological effects of SMYAD are as follows: Firstly, SMYAD contains anti-inflammatory factors. Besides quercetin, kaempferol, and beta-sitosterol, SMYAD also contains glycyrrhizin, which is well known for its anti-inflammatory activity . These pNext, the network pharmacology was performed to predict the active compounds, related pathways, and core targets involved in the SMYAD-provided protection on AAA. The enrichment results disclose the pivotal participation of inflammatory response, defense response, apoptotic, cell migration and adhesion, and reactive oxygen species metabolic in the SMYAD against AAA, as well as the collaborative involvement of the TNF signalling pathway, NF-kappa B signalling pathway, PI3K-Akt signalling pathway, and IL-17 signalling pathway –69. MoreThere are some limitations in our study: first, the intervention dosage of SMYAD was not reasonable enough, which leads to the effect of SMYAD high-dose group, was not better than that of the SMYAD-low-dose group. So, setting another drug such as statins may better reflect the effect of SMYAD. Second, we only test for the levels of IL-6 in circulating blood, not in the tissues of abdominal aortic, making it impossible to compare the differences of IL-6 in different tissues and get more information. Third, except for pathogenic target-IL-6, we do not know for sure whether there are protective targets in those core targets. Next, we intend to design a clinical study that focuses on the effect of SMYAD in AAA patients and screen for differential genes, to further clarify the mechanism of action.Taken together, the experimental results and bioinformatics findings highlight the protective effect of SMYAD on AAA. Furthermore, SMYAD may be used in clinical to treat AAA, based on the identified pharmacological functions and clear signalling pathways. Moreover, the potent compounds of SMYAD against AAA were identified, supporting it as an effective medicine with clear targets for action. Thus, it may be a safe and promising drug candidate for AAA.


G6PD, MT3, CBX2, CDKN2B, CCNA2, MAPT, EZH2, and SLC7A11. The risk score of each patient, which was determined by the multigene signature, was identified as an independent prognostic marker. The immune cell infiltration patterns, response rates to ICI therapy, and the estimated sensitivity of 89 chemotherapeutic drugs were associated with risk scores. Individual prognostic genes were also associated with susceptibility to various FDA-approved drugs. Our study indicates that a comprehensive transcriptomic analysis of OSRGs can provide a reliable molecular model to predict prognosis and therapeutic response in patients with HCC.Hepatocellular carcinoma (HCC) is a highly heterogeneous malignancy with poor outcomes, and the assessment of its prognosis as well as its response to therapy is still challenging. In this study, we aimed to construct an oxidative stress response–related genes–(OSRGs–) based gene signature for predicting prognosis and estimating treatment response in patients with HCC. We integrated the transcriptomic data and clinicopathological information of HCC patients from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) databases. LASSO Cox regression analysis was utilized to establish an integrated multigene signature in the TCGA cohort, and its prediction performance was validated in the ICGC cohort. The CIBERSORT algorithm was employed to evaluate immune cell infiltration. The response rate to immune checkpoint inhibition (ICI) therapy was assessed using a TIDE platform. Drug activity data from the Cancer Genome Project and NCI-60 human cancer cell lines were used to predict sensitivity to chemotherapy. We successfully established a gene signature comprising  Hepatocellular carcinoma (HCC) ranks fifth among all malignancies worldwide and is the second most common cause of cancer-associated deaths . DespiteMGST1/3, G6PD, GSR, and GPX2/4 play vital roles in glutathione synthesis, which serves as an antioxidant defender in cancer cells [It is well known that cancer cells are generally under stressful biological conditions, among which oxidative stress is one the most representative . Increaser cells . Besideser cells . GeneralEven though OSRGs are essential for the survival of HCC cells, the prognosis prediction and therapy recommendation value of OSRGs have not yet been sufficiently elucidated. Therefore, in this study, our aim was to investigate the prognosis prediction and therapy recommendation value of OSRGs. Thus, we successfully developed a novel eight OSRGs-based gene signature for HCC. First, we integrated transcriptomic profiling data and clinicopathological information related to HCC from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) databases. Next, we filtered the OSRGs related to overall survival (OS) in the TCGA cohort and established an eight-gene signature, whose robustness was then validated using the ICGC cohort. We also analyzed the correlation between tumor-infiltrating immune cells and risk scores. Further, immune checkpoint expression, immune checkpoint inhibition (ICI) therapy response rate, and tumor mutation burden (TMB) scores were evaluated to identify potentially valid immunotherapies. Finally, chemotherapeutic susceptibility was estimated according to the risk scores obtained and the expression levels of the individual prognostic genes. In summary, our work not only developed a practical prognostic tool but also provided a novel and reliable gene signature for selecting appropriate therapeutic methods for HCC. These innovative discoveries offer promising prospects for enhancing antitumor effects as each patient can receive individualized treatment guidance through our developed gene signature.https://www.gsea-msigdb.org/gsea/msigdb/) (Table https://www.proteinatlas.org/).We downloaded the transcriptomic and clinical data of HCC from the Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset in the TCGA database and the LIRI-JP cohort in the ICGC database. Both TCGA and ICGC databases are publicly available; hence, an ethical review is not required for their use. OSRGs were retrieved from the MSigDB database v7.4 (/) Table . The expWe identified the differentially expressed genes (DEGs) between the normal liver tissue and HCC tissues in the TCGA cohort using the “limma” package in R software Version 4.0.2 with the following threshold values: |log2FC| ≥ 1 and adj.p < 0.05. The shared candidate genes of DEGs and OSRGs were analyzed using univariate Cox regression analysis, genes with adj.p < 0.001 were preserved. Then, LASSO Cox regression analysis was adopted to construct a prognostic gene signature, using R software with “glmnet” package. The risk score of each patient was determined as follows: Risk score = ∑(Coef(i)∗Expression of gene(i)). The Kaplan-Meier (K-M) curve was plotted using the “survminer” and “survival” packages in R software. We delineated the receiver operating characteristic (ROC) curves with the “timeROC” package in R software to assess the accuracy of the prognostic gene signature.A forecast nomogram was developed by incorporating risk scores, age, gender, stage, and TNM classification using the R package “RMS.” The one-, two-, and three-year OS probabilities were estimated using the total points. Calibration curves were used to evaluate the accuracy.We used the CIBERSORT algorithm to quantify the subsets of infiltrating immune cells in the tumor environment of HCC samples. CIBERSORT is a deconvolution algorithm that uses the gene expression matrix to calculate the proportion of specific types of immune cells .http://tide.dfci.harvard.edu/) [The tumor immune dysfunction exclusion (TIDE) analysis platform was utilized to generate the TIDE score of each patient, which could serve as a surrogate biomarker to predict the responses to ICI therapy, mainly including anti-PD1 and anti-CTLA4 therapy (rd.edu/) . A high TMB is a measure of gene mutation frequency in a cancer cell. We obtained the VarScan processed somatic mutation data of HCC from TCGA database. Strawberry-Perl version 5.30.1 based on the JAVA8 platform was used to determine the TMB score depending on the genome mutation information of each HCC sample.https://discover.nci.nih.gov/cellminer).The R package “pRRophetic” was adopted to calculate the half-maximal inhibitory concentration  of widely used chemotherapy drugs for each patient. This package was constructed based on the chemical screening data from the Cancer Genome Project (CGP) dataset . To explP value < 0.05 was considered statistically significant.We identified DEGs among HCC and normal liver tissues using Wilcoxon signed-rank test. The K-M curve and the log-rank test were used to assess the difference in OS between groups. The correlation analysis was performed using the Pearson correlation coefficient. GraphPad Prism Version 7.0.4 and R software Version 4.0.2 were utilized to generate diagrams. G6PD + 0.177∗expression level of MT3 + 0.206∗expression level of CBX2 + 0.063∗expression level of CDKN2B + 0.078∗expression level of CCNA2 + 0.164∗expression level of MAPT + 0.248∗expression level of EZH2 + 0.213∗expression level of SLC7A11 , CTNNB1 (24%), TTN (23%), MUC16 (18%), and APOB (11%) in the high-risk group, while CTNNB1 (27%), TTN (23%), ALB (13%), TP53 (12%), and MUC16 (11%) were the top five in the low-risk group. In addition, the overall genome mutation occurrence rate was 86.71% and 82.49% in the high- and low-risk group, respectively. As displayed in Further, we investigated the associations of TMB with the risk score. As mentioned before, we stratified HCC patients into high- or low-risk groups in the TCGA cohort. The comprehensive mutation data for each group were represented using a waterfall plot . The fivG6PD, MT3, CBX2, CDKN2B, CCNA2, MAPT, and EZH2 was associated with increased resistance to mitomycin, teniposide, 6-thioguanine, and fulvestrant, etc. By contrast, elevated CBX2 and SLC7A11 expression is a hallmark of increased sensitivity to dasatinib, ixazomib citrate, and arsenic trioxide.First, we evaluated the chemotherapeutic response using the drug activity and transcriptomic data from the CGP cell lines. In the high-risk group, HCC patients were sensitive to 45 chemotherapy and targeted therapy drugs and were resistant to other 44 drugs, as indicated by the variation of the IC50 Figures . For eacSurgery is the first choice therapeutic strategy for HCC; however, most patients lose out on the opportunity to undergo tumor resection due to late diagnosis and large tumor sizes at the advanced stage. To overcome such challenges, in some studies, solutions, such as the use of novel synthetic materials to promote liver cell regeneration, have been proposed . HoweverG6PD, MT3, CBX2, CDKN2B, CCNA2, MAPT, EZH2, and SLC7A11. Compared with normal tissue, these genes are highly expressed in HCC tissue and are associated with an unfavorable prognosis. G6PD encodes the protein glucose-6-phosphate dehydrogenase, which can resist damage by oxidative stress in cells [MT3 encodes the protein metallothionein3, which displays a solid ROS-scavenging capacity in oxidative stress-conditioned cells [CBX2 is strongly associated with genome-scale DNA methylation in various types of cancer cells, which is a response to micro-environmental stresses, particularly oxidative stress [CDKN2B expression and causes cell cycle arrest in HCC cells [CDKN2B expression levels and prognosis is still unclear [CCNA2 encodes Cyclin A2, which is a suppressor of intracellular oxidative stress [MAPT encodes the protein Taus, which plays a significant role in the antioxidative response in neurons [EZH2 is an oncogene that is upregulated under oxidative stress induced by H2O2 [In this study, we first identified DEGs between HCC tissues and normal liver tissues in the TCGA cohort. Then, the 55 genes shared by OSRGs and DEGs were analyzed using univariate Cox regression, which revealed that 13 of these genes were related to OS. An eight-gene signature was then established using the LASSO Cox regression analysis. The gene signature achieved excellent performance in predicting the OS rate in the TCGA and ICGC cohorts. In addition, the nomograms constructed with risk scores and clinicopathological factors showed good predictive ability for OS, and multivariate Cox analysis identified the risk score as an independent predictor of prognosis. The eight OSRGs contained in this signature are in cells . Abnormain cells . MT3 enced cells . Howevered cells , 21. As ed cells , 23, CBXe stress . RecentlCC cells . Nonethe unclear , 27. CCNe stress  that is e stress , 30. The neurons . Elevate neurons . EZH2 is by H2O2 , 34. Fin by H2O2 . In addi by H2O2 . The aboTP53 mutation rate in the high-risk group was much higher than that in the low-risk group (44% vs. 27%). Some research findings suggested that the mutant p53 protein usually loses antioxidant function and increases intracellular ROS, which drives a function switch from a cancer suppressor protein to a cancer promoter protein [For unresectable HCC, immunotherapy plays a vital role in improving clinical outcomes. Unfortunately, current statistics show that response rates to immunotherapy remain low . It is wsk group % vs. 27%Likewise, traditional cytotoxic chemotherapeutic drugs and targeted therapy drugs can still bring benefits for improving the prognosis of advanced HCC. In general, HCC cells are resistant to intravenous cytotoxic chemotherapy. Instead, transarterial chemoembolization and oral administration of sorafenib/lenvatinib are the globally accepted standards for treating advanced HCC. Doxorubicin and cisplatin are the frequently-used drugs in transarterial chemoembolization . In the Presently, an increasing number of studies involving the use of multigene signatures for predicting the prognosis of HCC have been reported. In these studies, the researchers used m6A-related genes, energy metabolism-related genes, inflammation-related genes, immune response-related genes, and ferroptosis-related genes to construct their prognostic models –58. SimiThere is no denying that some limitations exist in this study. Firstly, our gene signature was suited to the patients with surgical or biopsy specimens because this gene signature was based on gene expression data. Secondly, we used Cox regression analysis to screen the prognostic genes, which may lead to the overlook of some genes with biological significance. Thirdly, although we used a variety of methods to ensure the accuracy of our gene signature, the reliability of this model needs to be iteratively improved in long-term clinical applications with larger sample size.By integrating the expression matrix of OSRGs as well as survival statistics based on two independent HCC cohorts, we established and validated a novel eight OSRGs-related gene signature for patients with HCC. This gene signature showed significant correlation between the TMB and immune cell infiltration in the TME. More importantly, its application could facilitate the estimation of the prognosis as well as therapeutic responses to immunotherapy and chemotherapy for patients with HCC. Notwithstanding, even though our gene signature showed good application prospects, it still needs further verification in clinical application to improve its stability. Taken together, our findings can help clinicians estimate the outcome of patients and select the appropriate therapeutic strategy for HCC to improve its prognosis. Our work also provides new insights into the diagnostic and therapeutic value of oxidative stress and OSRGs, laying a preliminary foundation for future mechanistic studies on OSRGs in HCC.


In children, obstructive sleep apnea (OSA) can cause cognitive dysfunctions. Amyloid-beta and tau are elevated in OSA. Neurofilament light (NfL) is a marker of neuro-axonal damage, but there are no reports of NfL for OSA. The objective was to investigate the serum levels of NfL and tau in children with or without OSA and explore their relationship with cognitive dysfunctions caused by OSA.This retrospective case–control study included children diagnosed with adenoid tonsil hypertrophy from July 2017 to September 2019 at the Second Affiliated Hospital of Xi’an Jiaotong University. Correlations between cognitive scores and tau and NfL were examined.P < 0.001). Moreover, NfL was correlated with the course of the disease, apnea–hypopnea index (AHI), obstructive apnea index (OAI), obstructive apnea–hypopnea index (OAHI), average oxygen saturation (SaO2), respiratory arousal index (RAI), and cognitive dysfunctions evaluated by the Chinese Wechsler Intelligence Scale for Children (C-WISC) . The area under the receiver operating characteristics curve (AUC) of NfL was 0.816 (95%CI: 0.736–0.897). Multiple regression analysis revealed that NfL was significantly associated with verbal intelligence quotient (VIQ), performance intelligence quotient (PIQ) and full-scale intelligence quotient (FIQ) .Fifty-six OSA and 49 non-OSA children were included. The serum NfL levels were higher in the OSA group (31.68 (27.29–36.07) pg/ml) than in the non-OSA group (19.13 (17.32–20.95) pg/ml) (Serum NfL levels are associated with the severity of cognitive dysfunctions in children diagnosed with adenoid tonsil hypertrophy and might be a candidate noninvasive, objective marker to identify cognitive dysfunctions in children with OSA. Obstructive sleep apnea (OSA) is a common sleep disorder caused by repetitive episodes of upper airway obstruction due to an obstruction or increased airway resistance , with anCognitive dysfunctions, including irritability, impaired attention, emotional instability, decreased intelligence, and learning disabilities, is a major complication of OSA in children and can usually be measured with tests such as the Wechsler Adult Intelligence Scale-Revised, the Psychomotor Vigilance Task, and the Steer Clear Performance Test . Zhao etAs a neuroprotective biomarker, insulin-like growth factors 1 (IGF-1) levels were higher in children with OSA and no neurocognitive deficits. Besides, IGF-1 levels were lower in children with OSA and cognitive deficits . As for 18F-florbetaben. Aβ40 and Aβ42 levels in cerebrospinal fluid (CSF) were found to be lower in OSA patients than in controls, and the apnea–hypopnea index (AHI) was correlated to the Aβ42/Aβ40 ratio [Recently, some biomarkers of brain injury have been detected in adults with OSA. In a study of 119 male Vietnam War veterans, Elias et al.  found th40 ratio . Total t40 ratio .Neurofilament light (NfL) is a marker of neuro-axonal damage and can be measured in CSF and serum . As an iTherefore, this study aimed to investigate the serum levels of NfL and tau in children with or without OSA and explore their relationship with cognitive dysfunctions potentially caused by OSA. The results of this study could suggest objective blood diagnostic biomarkers of cognitive dysfunctions in children with OSA.This retrospective case–control study included children diagnosed with adenotonsillar hypertrophy from July 2017 to September 2019 at the Department of Otolaryngology-Head and Neck Surgery of the Second Affiliated Hospital of Xi’an Jiaotong University.The inclusion criteria were 1) ≤ 14 years of age, 2) diagnosed with adenoid tonsil hypertrophy , 30, 3) The procedures used in this study adhered to the tenets of the Declaration of Helsinki. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University . The requirement for individual informed consent was waived by the committee because of the retrospective nature of the study.Age, course of snoring, course of mouth breathing, course of choking, father’s education level, maternal education level, and body mass index (BMI) were recorded. The tonsils were graded according to the criteria of Brodsky : Grade 02), and respiratory arousal index (RAI). AHI was defined as the number of apneas and hypopneas per hour of total sleeping time. OAI was defined as the number of obstructive apnea events per hour of total sleep time. OAHI was defined as the total number of obstructive apneas, mixed apneas and obstructive hypopneas per hour of total sleep time. OSA diagnosis was based on OAHI ≥ 1 event per hour during PSG [The results of PSG were collected, including AHI, obstructive apnea index (OAI), obstructive apnea–hypopnea index (OAHI), average oxygen saturation  and stored at − 80 °C until batch assays were undertaken. NfL and total tau concentrations in serum were measured by commercially ELISA assay (detection range: 6—1000 pg/ml) and single-molecule array (detection range: 0–400 pg/mL). Each reaction was performed in triplicate.www.r-project.org) and GraphPad Prism 7 . The distribution of the continuous data was tested using the Kolmogorov–Smirnov test. The continuous variables with a normal distribution were presented as means ± standard deviation and analyzed using Student’s t-test. The continuous variables with a skewed distribution were presented as medians (quantiles) and analyzed using non-parametric tests. Categorical variables were presented as n (%) and analyzed using the chi-square test. Spearman’s correlation analysis was performed to assess the associations between biomarkers serum concentrations (NfL and tau) and parameters of PSG and cognitive dysfunctions . The diagnostic value of NfL was assessed using receiver operating characteristic (ROC). The least absolute shrinkage and selection operator (LASSO) regression analyses were used to filter independent variables and avoid multicollinearity [P-values < 0.05 were considered statistically significant.The data were analyzed using SPSS 21.0 , R language . The children in the OSA group had a larger A/N ratio and tonsil size than the non-OSA group (P < 0.001 and P < 0.001). The AHI, OAI, OAHI, and RAI in the OSA group were significantly elevated compared with the non-OSA group . SaO2 in the OSA group was lower compared with the non-OSA group (P = 0.001). VIQ and PIQ in the OSA group were lower than in the non-OSA group (P = 0.022 and P = 0.016).The characteristics of the patients are shown in Table P < 0.001) .The serum NfL levels were high in the OSA group (31.68 (27.29–36.07) pg/ml) compared with the non-OSA group (19.13 (17.32–20.95) pg/ml) , A/N ratio (P < 0.001), tonsils size (P = 0.007), AHI (P < 0.001), OAI (P < 0.001), OAHI (P < 0.001), SaO2 (P < 0.001), RAI (P < 0.001), VIQ (P < 0.001), PIQ (P < 0.001) and FIQ (P < 0.001)  and a maximal Youden index of 0.505  , VIQ, PIQ, and FIQ were separately analyzed as dependent variables  in the LASSO model used the minimum criterion of tenfold cross-validation. Partial likelihood deviation curves were plotted by lambda. Two dotted vertical lines were drawn at the optimal values at the minimum criterion and the 1-SE criterion. LASSO coefficient profiles of 17 features , course of snoring (months), course of mouth breathing (months), course of choking (months), father’s education level, maternal education level, BMI (kg/m2), A/N ratio, tonsil size, AHI, OAI, OAHI, minimum SaOles Fig. .Fig. 5LeP-values lass 0.05 in LASSO models. It revealed that VIQ, PIQ and FIQ were significantly associated with NfL . Furthermore, VIQ and PIQ were also significantly associated with minimum SaO2  and RAI   Table .Table 4M2, and RAI) and C-WISC  parameters. Since the eligibility criteria excluded the influence from comorbidities such as the genetic syndromes, history of sedatives and, neurological abnormalities, the most likely etiology of increased concentrations of NfL was OSA. Therefore, the results suggest that serum NfL levels are associated with the severity of cognitive dysfunctions in children diagnosed with adenotonsillar hypertrophy and might be a candidate noninvasive, objective marker to identify cognitive dysfunctions in children with OSA.OSA in children can potentially cause cognitive dysfunctions –11, but Recently, some other approaches were used to assess OSA with cognitive dysfunctions such as imaging and serologic examinations . Some reNfL, a novel marker of neuronal and axonal injuries, has attracted widespread attention and has been studied in a wide range of neurologic disorders , 25, 28.In the present study, the serum levels of NfL were measured in children diagnosed with adenotonsillar hypertrophy, and the NfL levels in OSA were increased compared with non-OSA, which strongly suggests that OSA can indeed lead to brain injury. Our results also showed that the NfL levels were correlated with VIQ, PIQ and FIQ, indicating that the NfL levels represented the cognitive status evaluated by the C-WISC. NfL levels might reflect the severity of cognitive dysfunctions in children with OSA. Together with previous reports , 17, 39,2 and RAI. It is noteworthy that AHI and OAHI, which are used as the objective indicator for the diagnosis of OSA, were not selected as related features in LASSO models. These analyses show that the cognitive deficits associated with pediatric OSA may be more closely related to the drop in blood oxygen levels and sleep fragmentation, rather than sleep apnea/hypopnea.Chronic intermittent hypoxia, sleep fragmentation, and inflammatory activation are the main pathophysiological mechanisms of OSA. In multiple regression models, VIQ, PIQ and FIQ were significantly associated with NfL. VIQ and PIQ were also significantly associated with minimum SaOPSG is the golden diagnostic golden standard of OSA , 32, butSerologic examinations are relatively cheap, fast, and accessible, and the development of new serologic biomarkers is essential for diagnosing OSA. Here, the results showed that the AUC of NfL was 0.816 (0.736–0.897), indicating that NfL had relatively good sensitivity and specificity as an accessible diagnostic and screening marker of cognitive dysfunction in OSA patients.A few studies have reported increased serum and cerebrospinal fluid levels of tau in patients with OSA compared to control subjects , 41, whiIt is the first study investigating the association between NfL and pediatric OSA. Considering that this study was a retrospective case–control study and the sample size was relatively small, prospective multicenter randomized controlled trials with a larger sample size are needed to confirm the NfL cutoff levels for the diagnosis of OSA and evaluate whether NfL levels are associated with the severity of OSA in children. This study only included patients formally diagnosed with adenotonsillar hypertrophy and who underwent PSG, but whether NfL could be used for OSA-related cognitive dysfunctions screening in the general pediatric population remains to be determined. Future studies will also be necessary to explore the relationships between serum NfL levels and other cognitive evaluation tests in children diagnosed with adenotonsillar hypertrophy and OSA.The serum NfL levels were elevated in children with adenotonsillar hypertrophy and OSA compared with children with adenotonsillar hypertrophy but no OSA. The serum levels of NfL were correlated with VIQ, PIQ and FIQ. NfL levels might reflect the severity of cognitive dysfunctions in children with OSA.


To explore the relationship between vitamins levels and disease-related indicators in children with acute leukemia (AL). n = 52) and noninfected group (n = 55); treatment remission group (n = 56) and nonremission group (n = 51); high-risk (HR) group (n = 44), intermediate risk (IR) group (n = 53), and slight risk (SR) group (n = 8); cyclophosphamide + cytosine arabinoside+6-mercaptopurine + pegaspargase group ; methotrexate group ; and vindesine + daunomycin + L-asparaginasum + prednisone . Hematological and serological parameters, hepatic and renal function, and changes in vitamins A, B1, B2, B6, B9, B12, C, D, and E serum content in children with AL were analyzed to investigate their relationship with AL disease-related factors.  A total of 107 hospitalized children with AL were enrolled in this study and assigned to one group in each of the following categories: infected group (P < 0.05). Compared with the nonremission group, the level of vitamin B1 in the treatment remission group was significantly higher, while the levels of vitamin B6 and B12 were notably lower (P < 0.05). The levels of vitamins B6 and B12 were notably different among the treatment groups. Multivariate analysis showed that hemoglobin (Hb) and C-reactive protein (CRP) were predisposing factors of AL in children. The disease type (acute lymphoblastic leukemia/acute myelogenous leukemia) was the factor affecting remission in AL children. Abnormal kidney function and the occurrence of icterus were the influencing factors for the risk degree in AL children. Platelet (PLT) count, activated partial thromboplastin time (APTT), neutrophils (N), and immunophenotype were shown to affect the choice of therapeutic regimens.  The vitamin D level was significantly higher in the noninfected group than in the infected group ( There are notable vitamins imbalances in children with AL. The imbalances influence disease-related factors and therefore provide some references for the prognosis and treatment of AL. Leukemia, a general term for malignant clonal disease of hematopoietic stem cells, is divided into myeloid and lymphoid according to cell types. Acute leukemia (AL) is the most common childhood hematological malignancy and is the leading cause of malignancy-related mortality in children . Approxin = 52) and noninfected group (n = 55), according to the presence of infection. Bone marrow examination was conducted to evaluate remission and residual disease, and according to the examination results, the children were divided into the treatment remission group  and nonremission group . According to clinical risks and treatment responses, the children were divided into the high-risk (HR) group (n = 44), intermediate risk (IR) group (n = 53), and slight risk (SR) group (n = 8). According to different therapeutic regimens, the children were divided into three groups: cyclophosphamide + cytosine arabinoside + 6-mercaptopurine + pegaspargase group , methotrexate group , and vindesine + daunomycin + L-asparaginasum + prednisone . Some other treatment groups (n = 45) were also set. The results in all the different groups were analyzed.A total of 107 hospitalized children with AL (from June 2018 to December 2020) were enrolled in this study. The children were divided into the infected group . Venous whole blood was collected for the routine blood analysis.The disease-related indicators were as follows. (1) General information: age, body mass index (BMI), gender, diagnostic result type, immunophenotype, abnormal liver function, abnormal kidney function, and the occurrence of icterus. (2) Biochemical indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LD), and hydroxybutyric dehydrogenase (HBDH). (3) Serological and hematological parameters: white blood count (WBC), neutrophils (N), lymphocyte (L), hemoglobin (Hb), platelet count (PLT), C-reactive protein (CRP), activated partial thromboplastin time (APTT), and troponin (Tn). (4) Vitamin (V) indicators: vitamin A (VA), VB1, VB2, VB6, VB9, VB12, VC, VD, and VE levels.t-test or repeated measures analysis of variance was applied; otherwise, a nonparametric test was utilized. Multivariate analysis was performed using binary logistic regression. P < 0.05 was considered statistically significant.Statistical analysis was done using SPSS 21.0 software. Data were expressed as mean ± standard deviation (SD). If the data obeyed normal distribution and homogeneity of variance, the P > 0.05). LD and HBDH blood levels were significantly higher in the infected group than in the noninfected group (P < 0.05). Among the serological and hematological disease indicators, WBC, L, and CRP were significantly higher, while Hb and PLT were significantly lower in the infected group than in the noninfected group (P < 0.05). For various serum vitamins level, only VD was significantly lower in the infected group than that in the noninfected group (P < 0.05). Furthermore, multivariate regression analysis . However, there were statistically significant differences in the diagnostic results, immunophenotype, liver function, and infection incidence (P < 0.05). The level of LD in the treatment remission group was significantly lower than that in the nonremission group (P < 0.05). Among the serological and hematological disease indicators, the levels of Hb, PLT, APTT, and Tn were significantly higher, while the levels of WBC and L were significantly lower in the treatment remission group than in the nonremission group (P < 0.05). For the serum vitamin levels, there was a statistically significant difference (P < 0.05) in VB1, VB6, and VB12 levels between the two groups . However, there were statistically significant differences in the occurrence of renal function and icterus (P < 0.05). There was a statistically significant difference in PLT among the three groups (P < 0.05). There were no statistically significant differences in serological and hematological parameters and vitamins levels (P < 0.05). The results of the multivariate regression analysis confirmed that abnormal renal function and icterus incidence were the influencing factors for risk degree in children with AL  (P < 0.05). For serological and hematological parameters, there were significant differences in N, PLT, and APTT among the three groups (P < 0.05). For serum vitamin levels, there was a statistically significant difference between VB6 and VB12 (P < 0.05). Furthermore, results from multivariate regression analysis showed that PLT was significantly higher and N was significantly lower in the CAML group than in the other regimen groups. The immunophenotype of the MTX group was statistically different from the other regimens. The VDLP group had lower APTT and higher VB6 compared to other regimens (P < 0.05). These results confirmed that PLT, N, immunophenotype, APTT, and VB6 were the factors that influenced the choice of AML treatment regimen in children  . Howeverchildren .In this study, a total of 107 AL children were included, and their vitamin content and clinical parameters were analyzed in relation to infection, disease status, and clinical risk and treatment regimens. According to the analysis results, the level of serum VD in the infected group was significantly lower than that in the noninfected group. Compared with the nonremission group, the serum levels of VB6 and VB12 in the treatment remission group were significantly decreased, while the level of VB1 was notably increased. Additionally, there were notable differences in the levels of VB6 and VB12 among treatment regimen groups.Recently, it has been shown that vitamins have nonclassical effects such as regulating body immunity, inhibiting tumor cell proliferation, and participating in the development and progression of tumor cells , 4. VD dGiovannucci' study  showed tPLT is the smallest cell fragment produced by megakaryocytes in the bone marrow. Under physiological conditions, PLT exerts the functions of adhesion, aggregation, release, and contraction to ensure smooth progress of the normal hemostatic process. PLT is also associated with pathological thrombosis, tumor metastasis, inflammation, and immune response . AL is aCRP is commonly applied in the diagnosis of multiple systemic inflammatory diseases . It has In this study, multivariate regression analysis results showed that abnormal renal function and icterus were effective prediction factors of different risk degrees in AL children, and L count, immunophenotype, and APTT were the influencing factors for the choice of therapeutic regimen. Overall, close attention to vitamin levels in AL children at different stages of chemotherapy, improvement of nutritional status, minimized complications, and especially reduction of the incidence of infection are important means to improve the survival rate and quality of life of AL children. However, due to the small sample size included in this study, some indicators had no significant changes at different stages of disease development in children with AL. Therefore, further studies with larger sample sizes are needed.To sum up, this study has found imbalances in serum vitamins levels in AL children, which provide some references for AL diagnosis and treatment. The results of this study suggest that close attention to vitamins levels changes and maintaining vitamin level balance (through timely supplementation or cessation of vitamin intake) can contribute to successful treatment outcomes in children with AL. In addition, Hb and CRP were found to be the main factors influencing infection in children with AL. Since low HB is likely in AL patients, the risk of infections is high, and therefore, antiinfective treatment should be considered in the treatment of AL.