Background: Chuanxiong Rhizoma is one of the traditional Chinese medicines which have been used for years in the treatment of diabetic nephropathy (DN). However, the mechanism of Chuanxiong Rhizoma in DN has not yet been fully understood.Methods: We performed network pharmacology to construct target proteins interaction network of Chuanxiong Rhizoma. Active ingredients were acquired from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. DRUGBANK database was used to predict target proteins of Chuanxiong Rhizoma. Gene ontology (GO) biological process analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were also performed for functional prediction of the target proteins. Molecular docking was applied for evaluating the drug interactions between hub targets and active ingredients.Results: Twenty-eight target genes fished by 6 active ingredients of Chuanxiong Rhizoma were obtained in the study. The top 10 significant GO analyses and 6 KEGG pathways were enriched for genomic analysis. We also acquired 1366 differentially expressed genes associated with DN from GSE30528 dataset, including five target genes: KCNH2, NCOA1, KDR, NR3C2 and ADRB2. Molecular docking analysis successfully combined KCNH2, NCOA1, KDR and ADRB2 to Myricanone with docking scores from 4.61 to 6.28. NR3C2 also displayed good docking scores with Wallichilide and Sitosterol , revealing good binding forces to active compounds of Chuanxiong Rhizoma.Conclusions: Chuanxiong Rhizoma might take part in the treatment of DN through pathways associated with steroid hormone, estrogen, thyroid hormone and IL-17. KCNH2, NCOA1, KDR, ADRB2 and NR3C2 were proved to be the hub targets, which were closely related to corresponding active ingredients of Chuanxiong Rhizoma. Diabetic nephropathy (DN) is a common microvascular complication of diabetes mellitus (DM), which is also the most chronic complication of DM with high risk of disability and difficulty in curing Traditional Chinese medicine (TCM) has been used in the treatment of DN for years Currently, network-target and multiple-component-therapeutics have been increasingly recommended in the study of TCM Using complex network and visualization technology, network pharmacology can display multiple targets, multiple pathways and multi-target synergy of TCM in the treatment of many diseases, which could also provide new ideas and effective measures for TCM mechanism research. In addition, multi-database exploration could facilitate mechanism understanding and drug repurposing Figure Based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) The STRING database was used to analyze the target proteins interactions. The minimum required interaction score was set at 0.7 for network diagram. The interaction network of target proteins was constructed using Cytoscape 3.5.1 software Based on the interactions of target proteins and genes, we performed Gene ontology (GO) biological process analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to predict the potential function of target proteins. The clusterProfiler package in R was used for statistical analysis and visualization of functional profiles for genes and gene clusters Raw CEL data and corresponding clinical data of microarray profile from GSE30528 dataset In order to evaluate interactions between drug and potential target proteins, the selected hub proteins were evaluated by SystemsDock with the high-precision docking simulation assessing protein-ligand interaction www.rproject.org) and SPSS 13.0  were used for data analysis. P value less than 0.05 was considered significant. We also applied Cytoscape 3.5.1 software to visualize the compound-target-pathway interaction network of Chuanxiong Rhizoma in this study.R 3.6.1 (Table 1). One active ingredient (Senkyunone) without any corresponding target was excluded. Based on the reliable prediction of the target protein from the DRUGBANK database, we finally obtained 28 target proteins of Chuanxiong Rhizoma after excluding the repeated targets (Table 2). Compound-target network of Chuanxiong Rhizoma was shown in Figure Seven active ingredients of Chuanxiong Rhizoma  were acquired from TCMSP database according to the suggested screening criteria: OB ≥ 30% and DL ≥ 0.18  for further analysis. Target protein interaction network of Chuanxiong Rhizoma from STRING was shown in Figure We searched “diabetic nephropathy” in the CTD database and found 18652 DN-related genes. Ultimately, Venn diagram summarized 28 common targets both associated with DN and Chuanxiong Rhizoma , target genes were mainly enriched in response to steroid hormone (GO: 0048545); rhythmic process (GO: 0048511); steroid hormone mediated signaling pathway (GO: 0043401); cellular response to drug (GO: 0035690); hormone-mediated signaling pathway (GO: 0009755); cellular response to steroid hormone stimulus (GO: 0071383); reproductive structure development (GO: 0048608); intracellular receptor signaling pathway (GO: 0030522); transcription initiation from RNA polymerase II promoter (GO: 0006367); DNA-templated transcription, initiation (GO: 0006352). Figure  showed the enriched KEGG pathways of targets, including progesterone-mediated oocyte maturation (hsa04914), thyroid hormone signaling pathway (hsa04919), estrogen signaling pathway (hsa04915), IL-17 signaling pathway (hsa04657), small cell lung cancer (hsa05222) and prostate cancer (hsa05215).Twenty-eight targets fished by 6 active ingredients of Chuanxiong Rhizoma and DN from CTD database were further analyzed for functional prediction. The top 10 significant GO analyses and 6 KEGG pathways were enriched for genomic analysis. In GO analysis . Further analysis revealed that gene signatures of KCNH2, NCOA1, KDR and NR3C2 were significantly lower in DN glomeruli tissues when compared to controlled normal glomeruli tissues, while gene signature of ADRB2 was significantly higher in DN glomeruli tissues . The corresponding five target proteins were designed as hub proteins with ligand-protein interaction diagrams  for further molecular docking analysis.Based on GSE30528 dataset, 1366 DEGs associated with DN were identified, which were shown as green plots in Fig. ), revealing good binding forces to active compounds of Chuanxiong Rhizoma.With the help of SystemsDock, we successfully combined KCNH2, NCOA1, KDR and ADRB2 to Myricanone with docking scores from 4.61 to 6.28. What more, NR3C2 also displayed good binding scores with Wallichilide and Sitosterol , 10 GO processes and 6 KEGG pathways. As shown in As one of the major causes of ESRD around the world, DN is closely associated with cardiovascular and cerebrovascular diseases and increased mortality of DM patients In this study, we carried out a network pharmacology research focus on Chuanxiong Rhizoma to further explore its potential mechanisms on DN. By applying GO biological process analyses, the special targets of Chuanxiong Rhizoma were significantly enriched in hormone-mediated signaling pathway, response to steroid hormone, and other pathways related to reproductive structure development. Previous studies suggested that steroid hormone might play a vital role in the pathogenesis of DN in DM patients KEGG pathway enrichment analyses suggested that Chuanxiong Rhizoma was associated with estrogen signaling pathway, thyroid hormone signaling pathway and IL-17 signaling pathway in the treatment of DN. Estrogen has been reported to act on renal protection by up-regulating the level of endothelial NO synthase and inhibiting the synthesis of inducible NO synthase to maintain normal renal function In our study, KCNH2, NCOA1, KDR, ADRB2 and NR3C2 are closely related to corresponding active ingredients of Chuanxiong Rhizoma with favorable molecular docking scores, indicating good binding relationships between compounds and target proteins. Among the hub targets, KCNH2 is involved in ventricular cardiac muscle cell action potential repolarization, while NCOA1 is enriched in thyroid hormone signaling pathway and estrogen signaling pathway. ADRB2 regulates beta-2 adrenergic receptor (β2AR), which has been reported to act on inhibiting of macrophage function There are still some shortcomings in our study. Firstly, we just explored the potential functional mechanism of Chuanxiong Rhizoma on DN, without analyzing the mutual interferences of drug components. Secondly, this study failed to perform stratification analysis of different pathological stages of DN. Finally, the key targets and pathways we obtained have not been experimentally verified. Further molecular mechanism researches and clinical validations are still needed to be carried out in future work.Chuanxiong Rhizoma has been proven to play a role in the treatment of DN. Through network pharmacology analysis, our study revealed that Chuanxiong Rhizoma might take part in the treatment of DN through pathways associated with steroid hormone, estrogen, thyroid hormone and IL-17. KCNH2, NCOA1, KDR, ADRB2 and NR3C2 were proved to be the hub targets, which were closely related to corresponding active ingredients of Chuanxiong Rhizoma through molecular docking. However, further molecular mechanism researches and clinical validations are still needed in the future.


Salvia miltiorrhiza (SM) and Radix puerariae (RP) in the treatment of cardio-cerebral vascular diseases (CCVDs).  This study is aimed to analyze the active ingredients, drug targets, and related pathways in the combination of  The ingredients and targets of SM and RP were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the disease targets were obtained from Therapeutic Target Database (TTD), National Center for Biotechnology Information (NCBI), and Online Mendelian Inheritance in Man (OMIM) Database. The synergistic mechanisms of the SM and RP were evaluated by gene ontology (GO) enrichment analyses and Kyoto encyclopedia of genes and genomes (KEGG) path enrichment analyses.  A total of 61 active ingredients and 58 common targets were identified in this study. KEGG pathway enrichment analysis results showed that SM- and RP-regulated pathways were mainly inflammatory processes, immunosuppression, and cardiovascular systems. The component-target-pathway network indicated that SM and RP exert a synergistic mechanism for CCVDs through PTGS2 target in PI3k-Akt, TNF, and Jak-STAT signaling pathways.  In summary, this study clarified the synergistic mechanisms of SM and RP, which can provide a better understanding of effect in the treatment of CCVDs. Cardio-cerebral vascular diseases (CCVDs) seriously threaten human health and life for the high occurrence and mortality . Severe Salvia miltiorrhiza (SM), a Traditional Chinese Medicine (TCM), has been widely used in the treatment of CCVDs [Radix puerariae (RP) is one of the earliest medicinal materials used in ancient China, and the effects on CCVDs in the elderly have been reported [of CCVDs . Radix preported . The comreported , 7.SM has been extensively used in Asian countries against CCVDs. The primary active ingredients of SM are water-soluble phenolic acids represented by salvianolic acid B and fat-soluble ketones represented by tanshinone IIA. In previous studies, it has been proved that SM has improved microcirculation, inhibition of platelet aggregation, and antioxidation , 9. RP aThe network pharmacology approaches, concentrated on analyzing the network connectivity and dynamics as ingredients of drug targets and designing the optimal therapeutic strategies, can expose the underlying complex relationships between the herbal formula and the whole body . Coincidhttps://tcmspw.com/tcmsp.php), a phytochemical database, with the keywords “Salvia miltiorrhiza” and “Radix puerariae”. The OB and DL of chemical ingredients of SM and RP were collected from TCMSP. In this study, screening standard was set to OB ≥ 30% and DL ≥ 0.18, and chemical ingredients lower than this standard were eliminated from the final list of active ingredients of SM and RP.It is useful to explore the molecular mechanisms based on pharmacokinetics characteristics . SM and https://www.uniprot.org/), with limitation to “Homo sapiens.” Subsequently, protein names were transformed into official target name and duplicates were deleted.All proteins related to the active ingredients were obtained from the TCMSP databases. Protein names of SM- and RP-related protein were unified to official target name by UniProt  (https:/http://bidd.nus.edu.sg/group/cjttd/), NCBI [https://www.ncbi.nlm.nih.gov/gene) and the OMIM [http://www.omim.org/) database, and merely, “Homo sapiens” proteins connected to CCVDs were chosen. UniProt was used to unify the name of target associated with CCVDs because the target information of the disease is derived from different databases and has a nonstandardized name, and the duplicates were deleted. Active ingredients and disease targets were uploaded into the Veeny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/) website to obtain common targets of active ingredients and disease.Disease association targets were acquired from the TTD  (http:///), NCBI  (https:/the OMIM  (http://https://david.ncifcrf.gov/), and the species was set as “Homo sapiens” for GO and KEGG enrichment analysis. Among them, GO enrichment analysis includes three parts: cellular component (CC), biological process (BP), and molecular function (MF). A histogram was drawn according to the analysis results.Common targets of ingredients and disease were uploaded to Visualization and Integrated Discovery (David)  variatiohttps://string-db.org/) database by uploading the common targets with the species limited as “Homo sapiens” (human) and “minimum required interaction score: highest confidents (0.900)”. Other parameters remain the default settings, and the interactive relationship will be obtained. PPI network was drawn by importing the interaction information in Cytoscape 3.6.1; thereafter, topological parameter was examined by using the network analyzer in the software.The protein-protein interaction (PPI) information was obtained from the Search Tool for the Retrieval of Interacting Genes   pathway plays an essential role in intracellular signal transduction involved in cell proliferation, cell survival, inflammation, and metabolism . Neurona effects ; Janus k effects  show tha effects . Thus, SIn summary, this study explored the related diseases and complex diseases from the perspective of systemic pharmacology to identify active ingredients and improve cognition of the effective mechanism of TCM. We mainly from the following aspects to study the synergistic effects of SM and RP on CCVDs, such as the active ingredients in SM and RP, related targets, signal pathways, and biological processes involved in the related targets. In general, all nodes in the PPI network directly or indirectly affect the pathological process of CCVDs. Through the ingredient-target-pathway network, it was found that SM and RP play a synergistic therapeutic role mainly through PTGS2 target and PIK3-Akt, HIF-1, Jak-STAT, and TNF signaling pathways. This study first theoretically explained the synergistic therapeutic effect of SM and RP on CCVDs and further explained the principles of compatibility of TCMSP. On the other hand, it explored the active ingredients, targets, and pathways of SM and RP in the treatment of CCVDs to illustrate the synergistic effect, and it also provided reference for further study of pharmacology experiment of CCVDs treatment and other related studies of TCM.


Owing to the increase in energy consumption, fossil fuel resources are gradually depleting which has led to the growing environmental concerns; therefore, scientists are being urged to produce sustainable and ecofriendly fuels. Thus, there is a growing interest in the generation of biofuels from renewable energy resources using microbial fermentation.Butanol is a promising biofuel that can substitute for gasoline; unfortunately, natural microorganisms pose challenges for the economical production of 1-butanol at an industrial scale. The availability of genetic and molecular tools to engineer existing native pathways or create synthetic pathways have made non-native hosts a good choice for the production of 1-butanol from renewable resources. Non-native hosts have several distinct advantages, including using of cost-efficient feedstock, solvent tolerant and reduction of contamination risk. Therefore, engineering non-native hosts to produce biofuels is a promising approach towards achieving sustainability. This paper reviews the currently employed strategies and synthetic biology approaches used to produce 1-butanol in non-native hosts over the past few years. In addition, current challenges faced in using non-native hosts and the possible solutions that can help improve 1-butanol production are also discussed.Non-native organisms have the potential to realize commercial production of 1- butanol from renewable resources. Future research should focus on substrate utilization, cofactor imbalance, and promoter selection to boost 1-butanol production in non-native hosts. Moreover, the application of robust genetic engineering approaches is required for metabolic engineering of microorganisms to make them industrially feasible for 1-butanol production. However biomass . Nowaday biomass , and wit biomass . HoweverClostridium spp. faces several challenges. First, this process produces other byproducts such as hydrogen, lactic acid and propionic acid, acetone, ethanol, and isopropanol, which increases the downstream processing cost of 1-butanol purification [Clostridium) [Clostridium spp. utilizes low-cost feedstock such as cellulose and hemicellulose and other organic wastes  directly; however, an exception is Clostridium pasteurianum that can use glycerol as a substrate [The production of 1-butanol by ABE fermentation using fication . These ntridium) , 16. Furtridium) . The curtridium) ; the maitridium) , 20. Besubstrate , 22. TheSaccharomyces cerevisiae [Escherichia coli [Lactobacillus brevis, Pseudomonas putida, and Bacillus subtilis were also used for their potentially higher solvent tolerance [Clostridium cellulovorans was engineered because of its ability to consume most effective feedstock (cellulose) as a substrate, while a cyanobacterium Synechococcus elongatus was used for the direct conversion of CO2 into 1-butanol [Pyrococcus furiosus and Thermoanaerobacterium saccharolyticum were used for 1-butanol production because of their several distinctive benefits over mesophilic strains. Until now, the most significant advantage of using thermophilic strains is the reduction in contamination risks. All non-native hosts used for 1-butanol production are summarized in Table In general, non-native hosts have comparatively simple nutrient requirements, rapid growth rate, known genetic background, and they are easy to manipulate, although these conditions can vary on case to case basis. Further, it is economically feasible to use non-native hosts for large-scale production processes –25. Recerevisiae  and Eschhia coli  owing toolerance , 30. Clo-butanol , 32. TheThe advances and challenges in the engineering of 1-butanol production strains using clostridial species, which are the native 1-butanol producers, have been extensively discussed , 33–35. C. acetobutylicum was engineered to produce 1-butanol using cellulose as carbon source; however, the engineered strain was unable to utilize cellulose because cellulases are complex enzymes that are difficult to be expressed heterologously [C. cellulovorans and Clostridium cellulolyticum, which efficiently converts it into cost-effective advanced biofuels such as 1-butanol [The cost of the substrate is one of the most significant factors in ABE fermentation, which constitutes approximately 60% of the total cost of the process . Productlogously . Therefo-butanol , 52.C. acetobutylicum, C. beijerinckii, and C. saccharobutylicum are unable to used glycerol as a sole carbon source [C. pasteurianum can utilize glycerol as a carbon source to produce 1-butanol, but due to its complex physiology and limited availability as well as success on the application of genetic tools on it [E. coli. It grows faster than any Clostridium species and can use glycerol as a carbon source; therefore, it was engineered for 1-butanol production by transferring the CoA-dependent pathway [S. cerevisiae, P. putida, L. brevis, and B. subtilis were also engineered for the production of 1-butanol, using glucose and glycerol as carbon sources. Moreover, another bacterial strain K. pneumonia was engineered for the production of 1-butanol [E. coli and utilizes glycerol as the sole carbon source.Glycerol is a by-product of the biodiesel industry, and the production of 20 kg of biodiesel yields approximately 2 kg of crude glycerol . High abn source . On the ls on it , researc pathway . Additio-butanol , which e2, CO, and H2). It can be obtained from different sources like coal, biomass, and natural gas, which are also found in some industrial waste gases like in steel industries [Clostridium ljungdahlii and Clostridium autoethanogenum are two acetogenic bacteria that can metabolize such gases via the Wood–Ljungdahl pathway , but the concentration of CO2 in the industrial zone is significantly higher. Sometimes in the industrial zone, the level of CO2 reached 600 to 700 ppm. Moreover, every year, the concentration of CO2 in the atmosphere increases by 2–3 ppm because of the burning of fossil fuels [2 to useful products. Additionally, cyanobacteria can grow rapidly, and its genetic manipulation is easy [Synechocystis PCC 6803; the engineered strain was able to accumulate the highest titer of 4.8 g/L for 1-butanol with a maximal rate of 302 mg/L/day. It represented the highest 1-butanol titer produced from CO2 in cyanobacteria [Sunlight and COil fuels . Cyanoba is easy . Owing tbacteria . Thus, cB. subtilis GRSW2-B1, L. brevis (up to 3%), S. cerevisiae YPS128 (up to 4%) and P. putida (up to 6%) [L. brevis, P. putida, and B. subtilis, which can grow in the presence of a high amount of 1-butanol [High-value chemicals and biofuels are toxic to native-hosts and as well as to non-native hosts. The toxicity of the solvent is a significant limiting factor that hinders the production of 1-butanol during the fermentation process. Developing a microbial strain with the ability of a solvent tolerance is a difficult task for the researchers . The curp to 6%) , 86–88. -butanol , 30.Clostridium sp. and Zymomonas mobilis) cannot metabolize two sugars simultaneously due to the mechanism called carbon catabolite repression (CCR) [T. saccharolyticum JW/SL-YS485, Thermoanaerobacter sp. X514) can utilize two sugars (pentose and hexose) simultaneously in an unbiased manner for the production of biofuels [T. saccharolyticum JW/SL-YS485 and P. furiosus) were successfully engineered with the synthetic pathway of 1-butanol [Thermophilic organisms are a fascinating and promising group of microorganisms due to their several distinct advantages over mesophilic organisms when used as a production host. For example, their ability to thrive in extreme conditions such as high temperature lowers the contamination risk and reduces the production cost due to the elimination of the need for sterilization. Additionally, thermophilic conditions provide the opportunity for continuous removal of volatile compounds . Moreoveon (CCR) , 91. Intbiofuels , 93. Owi-butanol , 59.thl). Acetoacetyl-CoA then undergoes a series of dehydration and reduction to form butyryl-CoA. The genes involved in these enzymatic steps are hbd, crt and bcd-etf complex. The final step of clostridial 1-butanol pathway from butyryl-CoA to 1-butanol, is catalyzed by a bifunctional aldehyde/alcohol dehydrogenase, encoded by either adhE1 or adhE2.The clostridial CoA-dependent 1-butanol pathway has been heterologously expressed to produce 1-butanol Fig. . The clobcd-etf complex enzyme, which expressed poorly in non-native hosts, and this enzyme is also oxygen-sensitive. Second, the initial step of acetyl-CoA condensation is thermodynamically unfavorable. To avoid these problems, alternative enzymes were employed [L. brevis, P. putida, and B. subtilis were also engineered for 1-butanol production; however, the engineered strains accumulated lower titers of 1-butanol [C. tyrobutyricum was engineered to produce 1.1 g/L of 1-butanol by the overexpression of adhE2 gene  and an alcohol dehydrogenase (adh). 2-ketovalerate can be produced from 2-ketobuturate by ketoacid chain elongation process catalyzed by the endogenous enzymes (LeuABCD), overexpression of the endogenous enzymes can enhance 1-butanol production. 2-ketobutyrate is an intermediate of amino acid biosynthesis pathway that can be produced via native threonine biosynthetic pathway or the heterologous citramalate pathway  using two reaction steps, by the successive action of a 2-ketoacid decarboxylase  gene from B. subtilis, to produce 1-butanol (92 mg/L) using glycine as the substrate via Ehrlich pathway  mediated pathway  pathway; the EMC pathway is a central metabolic pathway in M. extorquens AM1 that converts acetyl-CoA to glyoxylate for reincorporation into the serine cycle during C1 assimilation and replenishes metabolites that either leave the cycle for biosynthetic purposes or are consumed by the tricarboxylic acid (TCA) cycle during C2 assimilation. The EMC pathway provides a continuous supply of crotonyl-CoA, which can be used as a direct precursor for 1-butanol production [M. extorquens AM1 for 1-butanol production. Carbon flux was directed from its native EMC pathway towards the 1-butanol pathway by expressing the trans-enoyl-CoA reductase gene from T. denticola and the alcohol dehydrogenase gene from C. acetobutylicum  more 1-butanol than the base strain BHB9 after introduction of a plasmid harboring the 1-butanol synthetic pathway [A facultative methylotrophic α-proteobacterium ompounds –99. It hoduction , 101. Thcum Fig. . The rec pathway .E. coli K12, the engineered strain accumulated the highest 1-butanol titer of 85 mg/L by cultivating the cells in bioreactors using glucose as substrate [In the 2-oxoglutarate pathway, seven enzymatic steps are involved in producing 1-butanol. The resulting set of genes catalyzing seven enzymatic steps are derived from diverse microorganisms. The 2-oxoglutarate pathway uses 2-oxoglutarate intermediate of TCA (try-carboxylic acid) cycle as a starting substrate for 1-butanol production. In the 2-oxoglutarate pathway, first 2-oxoglutarate is converted into 2-hydroxyglutarate by 2-oxoglutarate reductase. In the second reaction, the CoA is transferred from acetyl-CoA to 2-hydroxyglutarate to make 2-hydroxyglutaryl-CoA; this reaction is catalyzed by glutaconate-CoA transferase. In the third reaction, 2-hydroxyglutaryl-CoA is dehydrated into glutaconyl-CoA; this enzymatic step is catalyzed by 2-hydroxyglutaryl-CoA dehydratase. In the fourth reaction, glutaconyl-CoA is decarboxylated into crotonyl-CoA by glutaconyl-CoA decarboxylase. The remaining steps are similar to clostridial pathway. The crotonyl-CoA is reduced to butyryl-CoA by butyryl-CoA dehydrogenase. Finally, the last two steps are successive reductions of butyryl-CoA to butanal and butanal to 1-butanol; the reactions are catalyzed by aldehyde dehydrogenase and alcohol dehydrogenase, respectively . The novubstrate . The titE. coli, alternative carbon consuming pathways were deleted by the deletion of the adhE, ldhA and frdBC genes, which shifted the carbon flux towards 1-butanol and resulted in twofold increase in 1-butanol production [E. coli was engineered by deleting ldhA, adhE, frdA, and pta genes and inserting native atoDA, acs and Clostridium adhE2 genes. The recombinant strain (BuT-3EA) was able to accumulate a titer of 6.2 g/L of 1-butanol using glucose and butyrate as a carbon sources [The strategy of elimination of the competitive pathways has been employed in different non-native hosts, which has successfully increased the titers of 1-butanol by securing the key precursor (like acetyl-CoA) and reducing factor used for 1-butanol synthesis. For example, to increase the flux towards 1-butanol production in oduction . In anot sources , the tit sources .ack (encoding acetate kinase) in C. tyrobutyricum to reduce the acetyl-CoA conversion to acetate. The deletion of ack led to a ninefold increase in the titer of 1-butanol [C. acetobutylicum adhE2 gene in C. tyrobutyricum. The recombinant strain (without any gene deletion) was able to produce the highest titers of 6.8 g/L and 20.5 g/L of 1-butanol, using glucose and mannitol as the carbon sources, respectively; while the ACKKO-adhE2 mutant (∆ack-adhE2) accumulated 16.68 g/L of 1-butanol (2.5-fold increase) using glucose as the carbon source [cat1 (butyrate: acetate coenzyme A transferase) with adhE2, the mutant Δcat1::adhE2 strain produced 26.2 g/L 1-butanol in batch fermentation. This is the highest 1-butanol production that has ever been reported in batch fermentation [Similarly, Yu et al. deleted -butanol . In anotn source , 47. Recentation .GPD1 and GPD2 (encoding glycerol- 3-phosphate dehydrogenases) genes in S. cerevisiae that compete with 1-butanol production by utilizing the same intermediate metabolites, the deletion of the GPD1 and GPD2 genes resulted in approximately 1.3-fold increase in 1-butanol production [ADH1, ADH4, GPD1, and GPD2 genes involved in ethanol and glycerol biosynthesis in S. cerevisiae, redirected the carbon flux towards acetyl-CoA, resulting in a fourfold increase in 1-butanol production [CIT2 (encoding citrate synthase) and overexpression of aldehyde dehydrogenase (Ald6), alcohol dehydrogenase (Adh2), and acetyl-CoA synthetase mutant (AcsL641P) to enhance the carbon flux towards cytosolic acetyl-CoA, also led to a 2.5-fold increase in 1-butanol titer [Sakuragi et al. deleted oduction . Additiooduction . Moreoveol titer .PduP) is a possible rate-limiting step in the 1-butanol pathway in Synechococcus elongatus [PduP and inserting the gene encoding the subunit of the ACCase from Yarrowia lipolytica into the aldA  site, S. elongatus recombinant strain DC11 was able to accumulate 418.7 mg/L of 1-butanol, which is almost 1.4-fold more than its parent strain BUOHSE after 6 days of fermentation [Metabolomics is a comprehensive study of metabolites in a biological sample. In metabolic engineering, a metabolomics approach is an excellent strategy to search for the rate-limiting step in a biosynthetic pathway. Using metabolomics strategy, Noguchi et al. revealed that the reduction reaction of butanoyl-CoA to butanal catalyzed by acylating aldehyde dehydrogenase (longatus . Therefoentation .pta gene (encoding phosphate acetyltransferase) resulted in pathway imbalance as it prevents the release of CoA; they identified the adhE2-mediated reaction (conversion of butyryl-CoA to butyryl-dehyde) as the rate-limiting step. Thus, a simultaneous balancing of the CoA and enhancing the activity of adhE2 increased the titer of 1-butanol up to 1.3-fold (E. coli JCL299FT strain) compared with its parent strain JCL299F under anaerobic conditions [Similarly, Ohtake et al. also used a metabolomics approach to determine the bottleneck in the engineered pathway of 1-butanol. They discovered that the deletion of the nditions . These rDifferent synthetic pathways aforementioned were used for the production of 1-butanol. In these engineered pathways, clostridial CoA-dependent 1-butanol pathway is one of the most well-known pathway used for 1-butanol production in the heterologous hosts. One of the major advantages could be that the clostridial CoA-dependent pathway is a natural pathway, therefore there is no need to design new reaction or to mine for novel enzymes like 2-oxoglutarate pathway. However, the major limitation of the clostridial CoA-dependent 1-butanol pathway; is the length (six enzymatic steps) of the pathway, which needs more enzymes to be expressed and thus, the cell takes a greater metabolic burden compared with Ehrlich pathway. Another bottleneck in the reconstruction of the clostridial CoA-dependent pathway in the heterologous hosts is the poor expression of oxygen susceptible nature of enzymes .E. coli strain. The key advantage of 2-oxoglutarate pathway is the thermodynamically favorable first step to avoid unfavorable condensation of two molecules of acetyl-CoA in comparison to the clostridial pathway [Recently, 2-oxoglutarate pathway of 1-butanol was employed for the production of 1-butanol in  pathway , 103. Hokivd gene, and thus reduces the overall amount of 1-butanol production [Another pathway producing 1-butanol is the Ehrlich pathway. The advantages of the Ehrlich pathway is the requirement of only two heterologous enzymatic steps in comparison with 2-oxoglutarate pathway and clostridial pathway, which consist of seven and six enzymatic steps, respectively. Additional advantage of Ehrlich pathway is that it does not need high amount of reducing factor like 2-oxoglutarate and clostridial pathways. Another major advantage of the Ehrlich pathway over CoA-dependent pathway is that it circumvents the production of toxic metabolites, especially CoA-dependent intermediates . Howeveroduction . HoweverC. tyrobutyricum (Table E. coli produced 1-butanol to a titer (20 g/L) with yield of 34%  in batch fermentation [C. acetobutylicum strain JB200 [In summary, the results above demonstrate that the clostridial CoA-dependent pathway performs better among all of the 1-butanol pathways expressed in non-native hosts. Recently, a record high 1-butanol production was achieved by the recombinant strain of um Table  via the entation , which iin JB200 . HoweverE. coli, through which 1-butanol production was improved to a level comparable to that produced by the Clostridium species, with a 1-butanol titer of 30 g/L and increasing the yield to 70% of the theoretical level [E. coli by activating pdh gene anaerobically that encodes pyruvate dehydrogenase complex and with the expression of S. cerevisiae NAD+-dependent formate dehydrogenase (fdh1), which efficiently enhanced the titer of 1-butanol [The introduction of a non-native pathway into a microorganism can cause a cofactor imbalance, which results in a metabolic burden on the cells. Therefore, various cofactor engineering strategies have been developed to regulate the redox balance and improve the yield and efficiency of microbial cell factories . To mainal level . Howeveral level . In anot-butanol . These rbcd-etf) in non-native hosts and its need for ferredoxin as an additional redox partner. These factors hampered the enzyme activity in the non-native hosts, and thus the expression of bcd-etf complex led towards the production of a very minute quantity of 1-butanol in the non-native hosts. To circumvent the problem of poor expression of enzymes, alternative enzymes were proposed to improve the titers. For example, bcd-etf complex was replaced by ter (encoding trans-enoyl-CoA reductase) gene from Treponema denticola, this gene replacement led to an 18-fold increase in titer of 1-butanol in E. coli after 24 h of fermentation [thl gene with E. coli atoB gene due to its higher specific activity for acetyl-CoA than thl [S. cerevisiae, 1-butanol production was improved by replacing ccr (S. collinus) with ter gene [S. elongatus PCC 7942, the bldh was substituted by oxygen-tolerant CoA-acylating aldehyde dehydrogenase (encoded by pdup) from Salmonella enterica in the 1-butanol synthetic pathway. The modified pathway resulted in 1-butanol production to a cumulative titer of 404 mg/L, exceeding the parent strain expressing bldh by 20-fold [Various recombinant pathways have been constructed for the production of 1-butanol in different microbes. However, the major hurdle facing the non-native hosts is the poor expression of heterologous enzymes. For example, the clostridial 1-butanol pathway is the most employed pathway in the non-native hosts. As aforementioned that the major problem of the clostridial 1-butanol pathway is the poor expression of the oxygen-sensitive enzyme complex  [Heterologous pathways are usually constructed by the combination of multiple genes from different species. However, transferring the enzymatic pathways from one organism to another often cause a loss of the regulation, and thus imbalances occur in gene expression as well as enzymatic activity . Therefotration) . These s2 utilizing microbes are much difficult to engineer than E. coli; therefore, new genetic tools are required to make them industrially feasible.Compared with ethanol, 1-butanol is a superior biofuel and is gaining global research attention in several industrial fields. Native microorganisms are difficult to manipulate for 1-butanol production; however, in recent years, the metabolic engineering of a heterologous host for 1-butanol production has received an enormous amount of research interest with the rapid progress in gene editing and synthetic biology technologies. Various non-native organisms are being equipped with metabolic pathways for converting renewable resources into 1-butanol. Approaches including the use of exogenous enzymes with higher activity, and the removal of competitive pathways have helped in achieving further improvements in 1-butanol production. However, these non-native hosts have limitations such as issues with feedstock, contamination, and toxicity. To overcome these limitations, various microorganisms such as cellulolytic and photosynthetic bacteria, thermophiles, and solvent-tolerant organisms were engineered to produce 1-butanol. However, despite the high potential of these microorganisms, 1-butanol production using these microorganisms still has a far lower titer that is required for commercial production. Therefore, future research should focus on cofactor imbalance, proper promoter and gene selection, product toxicity, and enhancing substrate utilization in order to boost 1-butanol production. Additionally, improving the activity of key metabolic enzymes involved in a 1-butanol pathway by protein engineering is also a promising approach to enhance 1-butanol production. Moreover, lignocellulose or CO


This paper presents the results of numerical simulations on three main factors and their influence on the buckling resistance of reinforcing bars and on their behaviour in the range of postcritical deformations. These three factors are the shape of initial deformation, the amplitude of geometric imperfection and the slenderness of bars. The analysis was made of bars fixed on both sides for three initial shapes of deformation between adjacent stirrups, four amplitudes of geometric imperfections and eight bar slendernesses. The results of the numerical analyses carried out showed that the factors analysed have a very high influence on the inelastic buckling of the bars. The initial deformation shape, the radius of curvature and the slenderness of the bars have a significant influence on the buckling resistance of these bars and their longitudinal and transverse deformations. The research demonstrates that bars which are bent or compressed initially have a smaller resistance to buckling compared to straight bars, as the amplitude of geometric imperfections increases and the slenderness of the members increases. However, for the deformation shape of the bars, which is accompanied by shear forces, the drop in the buckling resistance of the members is small, and resistance to buckling for items with a small slenderness was higher than that of straight bars. The impact of seismic and exceptional loads on reinforced concrete structures may lead to the occurrence of postcritical states in structural elements. It is, therefore, necessary to ensure that the load-bearing capacity of the structure is maintained as long as possible after yielding. Load-bearing structures, should, as a consequence, have an adequate level of ductility. During an earthquake in reinforced concrete structures, buckling of the longitudinal reinforcing bars often occurs. The buckling of bars preceded by spalling concrete cover is accompanied by a sudden and progressive decrease in the load-bearing capacity of the reinforced concrete element. In order to avoid too-early buckling of the longitudinal reinforcement bars, the longitudinal and transverse reinforcement must be adequately arranged in the plastic hinge. In the case of the analysis of reinforced concrete elements in a state of postcritical deformations, when the limit of deformations of the cover concrete are exceeded and at the same time, the deformations of the reinforcement are large, it should be considered that the compressive reinforcement works differently than the tensile reinforcement. This difference requires the use of a corresponding reinforcement model and appropriate constitutive laws for compressed bars, which are different from those for tensile bars. The inelastic buckling of the reinforcing bar is determined by many factors. The most important of these being the slenderness of the bar and the mechanical properties of the reinforcing steel. So far, little attention has been paid to the initial deformations of the bars caused by the acting loads that accompany the compressive forces transmitted to the longitudinal reinforcement bars. This lack of attention includes deformations of the bars due to bending moment, compression forces or shear force. The aim of this paper, therefore, is to examine the influence of the initial deformation shape of the bars, the amplitude of these deformations and the bars’ slenderness on the buckling resistance of the reinforcement bars.In typical situations of the design and analysis of reinforced concrete elements, according to Eurocode 2 , it is aAccording to Scribner , three bAnalysis of the damage to reinforced concrete wall structures during the Chilean earthquake in 2010  showed to,m, which depends on the radius of curvature of the column.o,n. The difference in this strain depends on the ties’ spacing, their rigidity and load level. The shear forces induce deformations in the columns  s and bar diameter s/ϕ = 5, 6, 7, 8, 9, 10, 12 and 15.The analysis was made of bars fixed on both sides for three initial shapes of deformation between adjacent ties a–c, fourThe Mander’s steel model  has beensy = 400 MPa, tensile strength fsu = 600 MPa, hardening strain εsh = 0.010, ultimate strain εsu = 0.10, Young’s modulus Es = 200 GPa and initial hardening stiffness Esh = 5 GPa.The strength parameters of steels are described as follows: yield strength fIn the numerical analysis of large elastic-plastic deformations of compressed bars, the COSMOS/M system will be used . In the o/ϕ = 0.s = F/As normalized at yield strength fsy (σs/fsy) and the average strain of the bar ∆s/s  standardized with respect to the yield strength of fsy and its average strain Δs/s. o/ϕ. The initial curvature of the bar makes the yield plateau disappear. The bars work elastically to a stress level below the yield point (σs < fsy). When the slenderness of the bars s/ϕ < 10 is exceeded, the load-bearing capacity of the bars increases very little regardless of the initial eccentricity of the longitudinal force eo/ϕ. If the imperfection amplitude eo/ϕ ≤ 0.10 and the slenderness of the bars s/ϕ ≤ 6, the resistance of the bars is greater than the yield strength. An increase in imperfection amplitude eo/ϕ reduces the resistance of bars with comparable slenderness.o/ϕ and different slendernesses s/ϕ. The behaviour of bars for compression deformation is very similar to that of bars with bending deformations. The behaviour of these bars also depends very much on their s/ϕ and imperfection amplitude eo/ϕ. The initial curvature of the bars causes the plastic flange to disappear and the bars work elastically to a certain load level below the yield point (σs < fsy). When the proportionality limit is exceeded, the resistance of the bars increases very little regardless of the initial longitudinal force eccentricity eo/ϕ if the bars slenderness s/ϕ < 10.o/ϕ ≤ 0.10 and the slenderness s/ϕ ≤ 6. An increase in the initial curvature of the bars eo/ϕ causes a decrease in the bars’ resistance regardless of its slenderness. The results of analyses of behaviour of ND bars are similar to those of experimental tests [The resistance of the bars is greater than the yield strength if the initial eccentricity eal tests ,27. The al tests . This beal tests , which so/ϕ than for the two previous forms of deformation. The initial transverse displacement of the supports has little effect on the resistance of the bars, and the behaviour of the bars is almost the same as that of straight bars. The resistance of the deformed bars is comparable to that of the straight bars . Regardless of the amplitude of the imperfections eo/ϕ, the strength of the bars is higher than the yield strength if the slenderness s/ϕ < 9.As can be seen, the behaviour of bars with shear deformations differs significantly from that of bars with bending and compression deformation shapes. The resistance of the bars in this case is much higher for all values of slenderness s/ϕ and initial curvature ebars see . It is oWhile the results of numerical analyses of ND bars are confirmed by experimental studies ,27, unfosb/fsy and the slenderness of the bars s/ϕ. The buckling resistance of the bars corresponds to the maximum average stress in the bars fsb = σs,max. The buckling resistance fsf of straight bars eo/ϕ = 0 is greater than the yield strength fsy if their slenderness s/ϕ < 8. For slenderness s/ϕ = 5, the buckling resistance is 20% greater than the yield strength fsy. For slenderness s/ϕ ≥ 8, the yield strength of straight rebar is fsb = fsy.o/ϕ ≤ 0.10 and the slenderness s/ϕ ≤ 6. In other cases, the bars resistance is less than the yield strength. In extreme cases, the deviation from the straight form of the bars (eo/ϕ = 0.50) results in a nearly 50% decrease in the resistance compared to the straight form of the bars. For imperfection amplitude eo/ϕ = 0.25, this decrease is about 30%; for imperfection amplitude eo/ϕ = 0.10, this decrease is about 15% and for imperfection amplitude eo/ϕ = 0.05, this decrease is about 10%.Geometric imperfections for the bending a and comThe behaviour of bars with initial shear deformations is surprising and completely different from the behaviour of bars with bending and compression deformations c. In thio/ϕ = 0.5, the resistance of the bars is between 50% and 70% of the yield strength depending on the slenderness of the rebar. For the remaining imperfection amplitudes, the buckling resistance of the bars is proportionally higher.o/ϕ = 0. If the slenderness of the bars s/ϕ = 8, then the load capacity of deformed bars is equal to the yield limit and is the same as the load capacity of straight bars. If the slenderness of the bars s/ϕ > 8, then their load capacity decreases slightly and remains at a level not exceeding 0.9 fsy.The behaviour of bars with shear deformations is different c. The loThe previously described behaviour of predeformed compression bars can be explained by analysing the mode of their buckling. This is illustrated in o + ∆t increasing with the shortening of the bar ∆s. For greater slenderness s/ϕ ≥ 8, horizontal displacements ∆t was the opposite sign, so the eccentricity e = eo − ∆t decreased to zero and then after changing the sign, the eccentricity increased forcing the first mode of buckling. Therefore, the bending effect caused by the eccentric action of the longitudinal force was smaller than in the case of the initial deformation of bending and compression. Hence, the higher buckling resistance of bars with shear deformations was observed.As mentioned above, the buckling resistance of bar with bending deformations a is sligThe longitudinal reinforcement bars in reinforced concrete columns are deformed by the acting loads. This has a significant impact on their behaviour under compressive forces. The numerical analyses carried out showed that the following three factors have an influence on the inelastic buckling of bars: the shape of initial deformation, the amplitude of geometric imperfection and the slenderness of the bars. The buckling resistance of the bars and their ductility both depend on these three parameters.o/ϕ = 0.50. In contrast, the loads capacity of MD and ND bars is high: the load capacity of these bars is lower than the load capacity of bars with a typical VD buckling from 5% to 40% depending on the size of the imperfection.The behaviour of compression bars with bending MD and compression ND shape of initial deformations is almost identical, but it differs significantly from the behaviour of bars with shear shape of deformation VD. The load capacity and ductility of VD bars are similar to the capacity and ductility of straight bars. The decrease in the load capacity of bars with the form of VD buckling for the entire range of analysed imperfections is very small compared to the load capacity of bars without imperfections. This amounts to a maximum of 5% for imperfections eThe influence of imperfection amplitude on the buckling resistance of bars also depends on the shape of the initial deformations. The load capacity of bars with bending and compressed shapes of initial deformations decreases with increasing imperfection amplitudes. Regardless of the slenderness of the bars and the amplitude of imperfection, their load capacity is 50–60% of the load capacity of straight bars. In the case of bars with a shear form of deformation, the load capacity of these bars for small slenderness s/ϕ = 5–7 increases with the amplitude of imperfection and is greater than the load capacity of straight bars by 5–10%. For bars with a greater slenderness s/ϕ > 8, the load capacity of the bars decreases slightly and does not exceed 10% of the load capacity of straight bars.As the slenderness of the bars increases, the buckling capacity of the bars decreases: bars with a smaller slenderness (s/ϕ < 8–10) lose more of their load capacity. Bars with a larger slenderness, however, have a smaller decrease in the loss of load capacity. The load capacity of straight bars is at least equal to the yield strength, and if the slenderness of the bars is s/ϕ < 8, the load capacity of the bars is greater than the yield strength.The stirrup/tie spacing in beams and columns in seismic-resistant frames shall not exceed eight diameters of longitudinal reinforcement bars, and in order to take account of geometric imperfections, the load capacity of the bars shall be reduced by 40%.


Diabetic kidney disease (DKD) poses a major public-health burden globally. Tripterygium wilfordii Hook F (TwHF) is a widely employed herbal medicine in decreasing albuminuria among diabetic patients. However, a holistic network pharmacology strategy to investigate the active components and therapeutic mechanism underlying DKD is still unavailable.  We collected TwHF ingredients and their targets by traditional Chinese Medicine databases (TCMSP). Then, we obtained DKD targets from GeneCards and OMIM and collected and analyzed TwHF-DKD common targets using the STRING database. Protein-protein interaction (PPI) network was established by Cytoscape and analyzed by MCODE plugin to get clusters. In addition, the cytoHubba software was used to identify hub genes. Finally, all the targets of clusters were subjected for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses via DAVID. P < 0.05).  A total of 51 active ingredients in TwHF were identified and hit by 88 potential targets related to DKD. Compounds correspond to more targets include kaempferol, beta-sitosterol, stigmasterol, and Triptoditerpenic acid B, which appeared to be high-potential compounds. Genes with higher degree including VEGFA, PTGS2, JUN, MAPK8, and HSP90AA1 are hub genes of TwHF against DKD, which are involved in inflammation, insulin resistance, and lipid homeostasis. Kaempferol and VEGFA were represented as the uppermost active ingredient and core gene of TwHF in treating DKD, respectively. DAVID results indicated that TwHF may play a role in treating DKD through AGE-RAGE signaling pathway, IL-17 signaling pathway, TNF signaling pathway, insulin resistance, and calcium signaling pathway ( Kaempferol and VEGFA were represented as the uppermost active ingredient and core gene of TwHF in treating DKD, respectively. The key mechanisms of TwHF against DKD might be involved in the reduction of renal inflammation by downregulating VEGFA. Diabetic kidney disease (DKD), a highly prevalent microvascular complication of diabetes, is characterized by thickening of the glomerular basement membrane, mesangial expansion, podocyte, and glomerular injury, leading to glomerular sclerosis and tubulointerstitial fibrosis . It occuGuided by accumulative empirical experience, Chinese herbal medicine  acts as Network pharmacology is an interactive network used to study “compound-proteins/genes-disease” pathways, which own the ability of elucidating complexities for biological systems, drugs, and diseases from a systemic perspective , 17. Thihttp://tcmspw.com/tcmsp.php) was employed to collect the chemical compounds of TwHF, which is a unique systematic pharmacology platform of CHM that displays the associations between drugs, targets, and diseases [https://www.uniprot.org) provides access to a vast amount of sequence and functional information of proteins in a comprehensive, high-quality, and freely accessible way for scientists worldwide [https://pubchem.ncbi.nlm.nih.gov/) for acquiring the compound structures.The Traditional Chinese Medicine Systems Pharmacology Database  and Online Mendelian Inheritance in Man  databases to search for DKD-related targets. GeneCards is a comprehensive, authoritative human gene compendium that enables scientific community to effectively navigate concise genome, transcriptome, proteome, disease, and function data on all predicted and known human genes [The keyword “diabetic kidney disease” was used in the GeneCards  with the organism set as “Homo sapiens” [https://www.cytoscape.org/). The color of the node was displayed in a gradient from red to yellow according to the descending order of the degree value, and a higher degree value node was utilized to present putative crucial targets in the PPI network. Besides, the cytoHubba software was used to select hub genes based on mixed character calculation (MCC) score, and the Molecular Complex Detection (MCODE), a plugin in the Cytoscape software, helped us to screen significant functional modules and get clusters in the PPI network according to K − core = 2.The intersection of the compound targets and DKD disease targets was determined with the semiprogrammed foot software R project (×64 3.6.1) and taken as the candidate targets for the components of TwHF in the treatment of DKD. A protein-protein interaction (PPI) network of overlapped target genes was built based on the STRING database (sapiens” . Afterwahttps://david.ncifcrf.gov) database [P < 0.05 and Q < 0.05 were retained for further analysis about key targets in the PPI network involved in biological processes, molecular functions, cellular components, and functional signaling pathway.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for TwHF in DKD were performed using the DAVID  accumulation, GBM thickening, and albuminuria [β cells [κB and STAT signaling pathways in DM2 mice [Vascular endothelial growth factor A (VEGFA), a predominant proangiogenic factor in the kidney, is secreted by podocytes and provides essential maintenance signals for endothelial cells, podocytes, and mesangial cells in the glomerulus . Chronicuminuria , 36. VEGuminuria . Both en[β cells . c-JUN N[β cells , 38. Not[β cells . Heat sh[β cells , HSP90 iDM2 mice .Overall, these results indicated that the underlying mechanism of TwHF in the treatment of DKD may be involved in the reduction of excessive inflammation and protection against islets dysfunction and lipid deposition.Targets in compound-DKD PPI network were analyzed by MCODE, and 5 central gene clusters were returned . These cTo further reveal the potential mechanism of the TwHF on DKD, KEGG pathway enrichment analysis was conducted on the genes in the abovementioned five clusters. As shown in Table κB activation, upregulated collagen IV, and glomerulosclerosis in kidney tissues [κB signaling pathway and the release of central proinflammatory cytokine TNF-α, and the interaction of AGE-RAGE and TNF signaling pathway could induce the excessive generation of superoxide in diabetes [The generation and accumulation of AGEs in various tissues are highly facilitated under diabetic conditions due to persistent hyperglycemia and increased insulin resistance. The kidney acts as a crucial target for AGE-mediated impairment as well as a contributor to increasing AGE that results from a decrease in renal function, by the clearance of AGE . Interac tissues . In addidiabetes .κB signaling pathway and MAPK cascade [α generated from renal cells and infiltrating macrophages has shown the ability to modulate several mechanisms involved in the DKD development and progression, including promoting permeability of the glomerular basement membrane and glomerular vasoconstriction, decreasing intraglomerular blood flow [α and its receptor (TNFR) [κB signaling and suppress overexpression of proinflammatory cytokines  in kidney, which may contribute to the improvement in glomerulosclerosis within diabetic rats [γ and TNF-α and upregulated IL-4 and IL-10 [Chronic inflammation has a pivotal role in the development and progression of DKD. Tumor necrosis factor (TNF) functions as a critical cytokine in regulating a wide range of intracellular signal pathways including inflammation and immunity, which has been actively implicated in the renal pathophysiology and activated mainly by NF- cascade . There iood flow . Recent ood flow , and ther (TNFR) . Of notetic rats . Anothernd IL-10 . Furthernd IL-10 . The inhnd IL-10 –54, but 2+ alternation in mesangial cells enhanced the deposition of extracellular matrix proteins and mesangial expansion, thereby leading to glomerular injury [2+-dependent [α pathway in high glucose-treated glomerular endothelial cells and podocytes, thus attenuating oxidative stress and apoptosis [Emerging evidence suggests that calcium signaling pathway appears to be vital in the development of DKD. It has shown that the function of native kidney cells could be controlled by intracellular Ca2+ signaling . High glr injury , and theependent . As for ependent . Additiopoptosis .α expression [α, and IL-17 [Based on the degree value of each gene in compound target-DKD target network, VEGFA was considered as the core gene of TwHF against DKD. The most potent angiogenic factor, VEGFA, is a major contributor to the development of new vessels and is also identified as a proinflammatory cytokine . Data obpression . Lavoz epression . Otherwipression . Therefond IL-17 , and furDKD poses a major public-health burden globally attributed to the dramatically increasing prevalence of diabetes. Despite great efforts in looking for pharmacological strategies to control the disease based on the one-target one-drug paradigm, the incidence of ESRD continues to rise; thus, multiple component-multiple target pattern receives a large amount of attention recently. Tripterygium has been widely used among Chinese practitioners for thousands of years in treating DKD and exhibits remarkable effects in reducing albuminuria excretion. Clinical trials have confirmed that TwHF and its extracts are effective in treating DKD, including decreasing proteinuria and decrease in eGFR . At the Our research is the first report to explain the active ingredients and mechanisms of TwHF against DKD using network pharmacology. A total of 51 active ingredients in TwHF were screened and hit by 88 potential targets related to DKD, and kaempferol and VEGFA were represented as the uppermost active ingredient and core gene of TwHF in treating DKD, respectively. The mechanisms of TwHF against DKD were associated with 5 functional clusters, and the key mechanisms of TwHF against DKD might be involved in the reduction of renal inflammation by downregulating VEGFA. Although the present study provided a methodological exploration for identifying potential active compound and pharmacological mechanism of TwHF on DKD, in vivo and in vitro experimental validation is required in the next step to support our findings.


The Massachusetts General Hospital is a large, quaternary care institution with 58 operating rooms, 164 anesthesiologists, 76 certified nurse anesthetists (CRNAs), an anesthesiology residency program that admits 25 residents annually, and 35 surgeons who perform laparoscopic, vaginal, and open hysterectomies. In March of 2018, our institution launched an Enhanced Recovery After Surgery (ERAS) pathway for patients undergoing hysterectomy. To implement the anesthesia bundle of this pathway, an intensive 14-month educational endeavor was created and put into effect. There were no subsequent additional educational interventions.p < 0.001) was achieved during the intervention period. Compliance with the pathway was sustained in the post-intervention period despite no additional actions.We retrospectively reviewed records of 2570 patients who underwent hysterectomy between October 2016 and March 2020 to determine adherence to the anesthesia bundle of the ERAS Hysterectomy pathway. RESULTS: Increased adherence to the four elements of the anesthesia bundle (Implementing the anesthesia bundle of an ERAS pathway in a large anesthesia group with diverse providers successfully occurred using implementation science-based approach of intense interventions, and these results were maintained after the intervention ceased. Enhanced Recovery After Surgery (ERAS) is one of the most significant innovations in perioperative care. As hospiImplementing and maintaining strict adherence to ERAS pathways is challenging, and compliance values of approximately 70% are common. AdditionAs part of a quality improvement project, in March of 2018, an Enhanced Recovery After Surgery pathway for laparoscopic, vaginal, and open hysterectomy patients called “ERAS Hysterectomy” was implemented at the Massachusetts General Hospital. This pathway was designed by surgeons and anesthesiologists and utilized the Consolidated Framework For Implementation Research (CFIR) framework to implement evidence-based clinical care., 17 The To implement ERAS Hysterectomy on March 1, 2018, the entire ERAS pathway was divided into two bundles: surgical and anesthesia. Our team utilized the Consolidated Framework For Implementation Research approach to implement each bundle and ultimately change behaviors.To implement the surgical bundle, published data and best practice position statements were first presented in individualized educational sessions to surgeons, surgical physician assistants, and nurse practitioners. Similar individualized educational sessions were provided to surgical residents when they rotated through gynecology services. Surgical leaders publicly supported the endeavor, and a large-format grand rounds for the gynecology department was held before the pathway launched.Designing and implementing the anesthesia bundle was more complex. For the ERAS program to be successful, all bundles would need to be consensus-driven, derived from evidence-based practices, and complement other bundles. Therefore, after reviewing the most relevant literature with the anesthesia, surgical, and nursing champions, the anesthesia bundle was created.To add to the complexity of implementing the anesthesia bundle, outreach efforts to the 164 anesthesiologists and 76 CRNAs as well as the 130 residents who rotate through different operating theaters would be mandatory.Since duplicating the surgical approach of hosting individualized educational sessions was neither practical nor feasible, the anesthesia bundle was introduced to this group 2 months prior to implementation via email in a communication that described the entire ERAS pathway. The message was inclusive of peer-reviewed literature and data from other hospital-specific ERAS pathways demonstrating improved patient outcomes including decreased length of stay.Next, a large-format grand rounds presentation on ERAS Hysterectomy was given to the anesthesia department. This grand rounds occurred in the month prior to pathway implementation. Evidence supporting the new pathway was presented at this conference.Then, on the night before a patient was scheduled to provide anesthesia for a hysterectomy, each member of the anesthesia care team (attending anesthesiologist and certified nurse anesthetist or anesthesia resident) received an email containing the slide deck that had been presented at the grand rounds and a copy of the pathway with the anesthesia bundle attached. For the next 14 months, nightly emails were sent.Nightly emails ceased 14 months after the pathway was implemented when administrative changes within the department occurred. While the pathway was intended to be posted on a new departmental intranet, it ultimately never was. If a provider requested a copy of the pathway from anesthesia leadership, then it was emailed to that provider. However, it is important to note that after a period of intense intervention, the pathway was not easily accessible to anesthesia providers.To further enhance compliance with the pathway, annual performance reports were emailed to anesthesia providers showing individual compliance with different elements of the pathway. The first and only report that occurred during this period was emailed to providers 15 months after the pathway was implemented. Technical constraints limited our ability to email reports more frequently. Fortunately, these constraints have been addressed and are now resolved.To assess the impact of our approach to implement and sustain ERAS Hysterectomy, ethical approval was obtained through the Institutional Review Board (IRB) at the Massachusetts General Hospital IRB: 2017P000443). Requirement for written informed consent was waived by the IRB. Next, 2570 consecutive charts between October of 2016 and March of 2020 were retrospectively reviewed as part of this cohort study. The objective of this study was to evaluate compliance of pre-determined ERAS metrics during intervention and post intervention periods. The outcome measures are seen in Fig. 7P000443.We recognize that patient comorbidities may impact patient care, and we controlled for the following demographic data: patient age, heart rate, systolic blood pressure, BMI category, and American Society of Anesthesiology (ASA) category. We also recognize that clinical factors such as hysterectomy type and subspecialty surgical division may impact our analysis. Therefore, we controlled for these factors. Finally, we controlled for compliance with four ERAS measures  when performing our analysis. Of note, heart rate and systolic blood pressure serve as very rudimentary proxies for intra-operative pain and fluid status.We used standard descriptive statistics to characterize the sample. Multivariable regression analyses were used to evaluate changes in ERAS measures during the intervention and post-intervention periods. For binary outcomes logistic regression was used and for continuous outcomes generalized linear models were used. A significance level of 0.05 was used to establish statistical significance and regression results are reported as odds ratio or rate ratio depending on the nature of outcome measure. All statistical analyses were performed using SAS version 9.4.A total of 1059 surgeries were performed during the baseline period, 852 surgeries during the intervention period, and 659 during the post-intervention period. See Table Compared to the baseline period, compliance with four of five ERAS metrics measured showed statistically significant improvement during the intervention period Table . CompliaImportantly, the improvements during the intervention period were sustained during the post-intervention period  a “decrease in long-acting narcotic administration” and 2) a “decrease in short-acting narcotic administration.” This guidance allowed clinicians to use their best judgment when caring for their patients. However, it did not identify a target quantity of narcotic to administer and could have led to confusion.A third limitation to our study is that our post-intervention period is 10 months. As anesthesia residency is 3 years, it is possible that we will not capture post-intervention activities in their entirety.Finally, while our intention had been to ultimately post the ERAS Hysterectomy pathway on a departmental intranet, technical constraints prevented this from happening. Providers relied on their familiarity with the pathway or on previously sent emails that contained the pathway to guide their care. This oversight likely decreased compliance in the post-intervention period.ERAS pathways can be implemented and sustained in large anesthesia practices. Most of the effort to successfully implement the pathway occurs in the planning/early implementation period. Obtaining buy-in from surgeons, anesthesiologists, and nurses is key. Therefore, we recommend assembling a multidisciplinary team to examine the latest evidence before creating the pathway, and we advise hosting small format meetings with impacted stakeholders prior to launching a pathway.Once the pathway is designed, senior leadership support is necessary. This support can occur via large-format grand rounds. After this public display of support, emailing the entire pathway and evidence supporting the pathway to all providers who will be involved in the care of this type of surgical patient is useful. This approach, when supplemented with individual emails to providers on the night before the procedures, is effective. Also, providing individual feedback on compliance with the pathway is impactful. After a certain amount of time, in our case 14 months, sending continual reminders may not be necessary. However, we do recommend posting the pathway in a central repository as a reference for providers.As our ERAS pathways mature, we will incorporate additional reporting structures for practitioners in addition to integrating complication data into our reports. We also plan to refine various elements of the pathway as evidence-based practices improve. This will keep our providers engaged and make sure we provide the highest-quality care to our patients.


The food frequency questionnaire (FFQ) is the most frequently used method to assess dietary intake in epidemiological studies evaluating diet-disease association. The objective of this study was to validate a FFQ for use among Lebanese adults by evaluating various facets of validity and reproducibility.The quantitative 164-items FFQ was validated against the average of six 24-h dietary recalls (DRs) in a sample of 238 Lebanese adults. Reproducibility of the FFQ was assessed by administering it twice within 1 month’ time interval.Positive statistically significant Pearson correlations were observed in most macro and micronutrients between the FFQ and the six 24-h DRs, ranging from 0.16 to 0.65, with two thirds of the correlation coefficients exceeding 0.3. Energy, gender, and age-adjusted statistically significant Pearson correlation coefficients ranged from 0.14 to 0.64, with two thirds of the coefficients exceeding 0.2. Intakes from the FFQ were mostly higher than those of the 24-h DRs. Mean percent difference between nutrient intakes from both dietary methods decreased remarkably after using energy-adjusted mean intakes. Values were acceptable to good for all macronutrients and several micronutrients. Cross-classification analysis revealed that around 64.3 to 83.9% of participants were classified into the same and adjacent quartile whereas grossly misclassified proportions ranged from 3.7 to 12.2%. Weighted kappa values ranged from 0.02 to 0.36 with most of them exceeding 0.2. In indirect validity analysis, key nutrient mean intakes estimated from the six 24-h DRs were significantly positively associated with tertiles of food groups derived from the FFQ. Bland Altman plots showed that the majority of data points fell within the limits of agreement (LOA) for all nutrients. As for reproducibility analysis, ICC values were all statistically significant ranging from 0.645 to 0.959 and Bland Altman plots confirmed these results.Based on various aspects of validity and reproducibility, and an extensive range of statistical tests, the present FFQ developed for a Lebanese community is an acceptable tool for dietary assessment and is useful for evaluating diet-disease associations in future studies. Several epidemiological studies investigate the effect of diet on health and non-communicable diseases such as obesity, diabetes, cancers, as well as neurological, endocrine, and immunological disorders . Such stThus, the aim of the current study was to validate a FFQ for use among Lebanese adults, by investigating various facets of validity and reproducibility. The objectives of the present study were to i) determine the relative validity of the developed FFQ in measuring energy and nutrient intakes as compared to six non-consecutive 24-h DRs ii) compare the means of nutrient intakes across tertiles of food groups obtained by the FFQ iii) evaluate the reproducibility of the FFQ.A sample of 500 participants was drawn from the university database covering both students and employees, using a stratified random cluster sampling, with a status and sex distribution proportionate to that of the university population per campus. To be included in the study, the participants had to be Lebanese, aged between 18 and 64 years, not having medical conditions or taking medications that affect food intake. Selected participants received a letter by email explaining the procedure of the study. The agreement for participation was requested by phone, 5 days after sending the letter. Out of the 500 participants, 305 agreed to participate in the study. After providing written consent, socio-demographic, anthropometric, and dietary data were collected. Based on the “non-individualized method” , also caThe quantitative 164-items FFQ was developed by a panel of nutritionists who drafted a pre-final version of the questionnaire that was tested on a representative sample of the target population, composed of 50 students from the university. The respondents completed the FFQ during in-depth interviews where they were asked about its comprehensibility and acceptability . The finThe FFQ was validated against the average of six non-consecutive 24-h DRs. Participants filled 24-h DRs of 3 days (two weekdays and a day of the weekend) repeated twice within one-month interval to obtain a total of six non-consecutive 24-h DRs. The average of six 24-h DRs was used: in order to estimate the usual intake from 24-h DRs and investigate its association with biochemical variables and indicators of health status, it is recommended to collect multiple days. In general, 4 to 5 days are collected as an appropriate compromise between scientific rigor and practicability for assessing energy and macronutrients intake . This meThe reproducibility of the FFQ was tested on a smaller sample from the same target population. A number of 52 participants completed the questionnaire twice within 1 month’ time interval . The perNutritional data deriving from the FFQ and the 24-h DRs of 6 days were assessed using the Nutrilog software . Nutrient composition of the Lebanese traditional dishes was derived from the American University of Beirut (AUB) food composition table . For the2 and ≥ 30 kg/m2 respectively [Data were collected regarding age, gender, and crowding index. The latter is defined as the total number of co-residents per household, divided by the total number of rooms, excluding the kitchen and the bathrooms. Anthropometric measurements were taken for the description of the population. Weight and height were measured using a scale and stadiometer . Body mass index (BMI) was calculated and the participants were classified as overweight or obese if the BMI value ranged between 25 and 29.9 kg/mectively .Frequencies and percentages were used for categorical variables and means (standard deviation SD) were used for quantitative data. Validity of the FFQ as compared to the 24-h DRs was assessed for all nutrients using the Pearson correlation coefficient, with adjustment for energy intake, age, and gender. The analysis was repeated for men and women separately because they have several physiological and behavioral differences that affect distinctively their response to health problems . Mean peGeneral characteristics of the sample are presented in Table Pearson correlations between FFQ and six 24-h DRs are presented in Table Pearson correlations between FFQ and six 24-h DRs for men and women are presented in Table S2 in the additional file The Mean (SD) of energy intake and all nutrient intakes as estimated by the FFQ and the average of six 24-h DRs are presented in Table Table In cross-classification analysis, around 64.3% (% polyunsaturated fatty acids) to 83.9%  were classified into the same and adjacent category. Grossly misclassified proportions ranged from 3.7% (iron) to 12.2% (% polyunsaturated fatty acids). Weighted kappa values ranged from 0.02 (% polyunsaturated fatty acids) to 0.36 (energy intake). Results are shown in Table The indirect validity of the FFQ is shown in table S5 in additional file Bland Altman analysis was conducted by plotting the mean of nutrient intakes between the FFQ and 24-h DRs against the difference between the two methods . Bland AParticipants in the reproducibility and the validity phase had a similar gender distribution , with median age ~ 25.1 years  mean (SD) = 31.72 (12.08) for reproducibility group vs 22 years  mean (SD) = 27.83 (11.95) for validity group. The two groups also had similar BMI means (mean = 24.5 (5.0) for reproducibility group vs 23.6 (3.7) for validity group). Concerning reproducibility analysis, ICC ranged from 0.645 (vitamin D) to 0.959 (riboflavin). All correlations were statistically significant as shown in Table Based on various aspects of validity and an extensive range of statistical tests, we demonstrated that the present FFQ developed for a Lebanese community is a useful tool for dietary assessment, when compared to six 24-h DRs. We obtained an acceptable agreement between nutrient intakes of both dietary instruments, given that most participants were correctly classified into the same and adjacent quartiles, with a low level of misclassification. Weighted kappa statistics also showed acceptable results. These findings were further confirmed in the Bland Altman plots and the indirect validity analysis relating nutrient intakes from the 24-h DRs to food groups from the FFQ, indicating a satisfactory agreement between the two methods.There is no perfect reference method in validation studies. Objective methods such as biochemical indicators are relatively invasive and expensive especially when they aim to test many nutrients. Moreover, biochemical indicators do not exist for some nutrients . They are also influenced by dietary factors including day-to-day variation and physiological factors such as nutrient absorption and metabolism, diurnal and menstrual cycles . Diet reIn validation studies, it is important to cover many aspects of validity. An in-depth literature review carried out in 2015 showed that the mostly used statistical tests in FFQ validation studies were combinations of two to three tests, which may not be sufficient to provide a comprehensive perception of various facets of validity . MoreoveRegarding correlation analyses, a desirable Pearson correlation coefficient generally ranges from 0.5 to 0.7 , with coResults showed that the FFQ tended to overestimate nutrient intakes as compared to 24-h DRs. This finding is consistent with most of the FFQ validation studies , 38, 39.Regarding the mean percent difference, it was calculated for both crude and energy-adjusted nutrient intakes. The difference remarkably decreased with the energy-adjusted values. It showed acceptable to good results for macronutrients, vitamins such as vitamin D, thiamin, and niacin, and minerals like phosphorus, potassium, sodium, and iron. Given that the FFQ overestimates energy intake, it seemed more plausible to compare intakes when they are energy-adjusted. This allowed evaluating the nutrient composition of the diet as assessed by both dietary instruments, rather than only crude intakes. In future epidemiological studies, especially those evaluating diet-disease associations, it is crucial to consider adjusting for energy intake among other confounding factors; diet-disease associations should not be the sole result of differences in total energy intake between cases and non-cases .Cross-classification of nutrient intakes into quartiles and weighted kappa calculation showed promising results as per the agreement between the dietary instruments. Regarding the quartile categorization, misclassification was less than 10% among most nutrients, while a relatively high proportion of participants were classified into the same or adjacent quartile. Results were similar to previous FFQ validation studies , 37, 40.Bland Altman plots showed a good level of agreement between the two methods. While the positive mean in most plots indicated that the FFQ overestimated intakes, plots show that the majority of data points fell within the LOA around the mean intake.Indirect validity assesses the relationship between the food consumption categories derived from the FFQ, and the nutrient intakes extracted from the 24-h DRs . This tyTest-retest reliability displays not only the degree of correlation but also the agreement between measurements. In contrast to Pearson correlation coefficient, paired t-test, and Bland Altman plots, ICC is an advisable measure of reliability that assesses both degree of correlation and agreement between two measures . In the This is the first FFQ validation study conducted in Lebanon to assess most aspects of validity, for a complete range of macro- and micronutrients. In fact, only a few number of studies worldwide used an extensive number of statistical tests for FFQ validation. Another strength of this study is the number of 24-h DRs (6 days) collected as a reference method for the FFQ validation, which was not common in previous studies. In addition, the sample size which is relatively higher than other validation studies, appears sufficient in the context of deriving useful information on questionnaire validity, when combined with 24-h DRs of 6 days .We acknowledge the present validation study has some limitations. First, the length of the FFQ could have increased the burden on participants, hence impairing the cooperation of the respondents and raising the risk of biased responses and the overestimation of intakes. Therefore, in order to account for this limitation, the FFQ was interviewer-administered which assured a more accurate completion of answers , 24. DesIn the present study, we performed an extensive range of statistical tests and evaluated various aspects of validity and reproducibility. We demonstrated that the FFQ developed for a Lebanese adult community is an acceptable tool for dietary assessment, namely in the context of nutrients distribution and ranking individuals according to their dietary intake. Hence, it is valuable for use in future epidemiological studies evaluating diet-disease associations. This study also showed that caution must be taken in the quantitative assessment of the diet by accounting for energy intake, in addition to gender and other confounding variables.Additional file 1: Table S1. Food items included in the FFQ; Table S2. Validity of the FFQ: Pearson correlations between first FFQ and mean of six 24-h DRs, stratified by gender (n = 238); Table S3. Mean ± SD comparison of nutrient intake estimated by the FFQ and the average of six 24-h dietary recalls, stratified by gender (n = 238); Table S4. Mean ± SD comparison of energy-adjusted nutrient intake estimated by the FFQ and the average of six 24-h dietary recalls, stratified by gender (n = 238); Table S5. Indirect validity: mean daily nutrient intake as assessed by 24-h DRs of 6 days according to tertiles of food group consumption (FFQ)Additional file 2: Figure S1. Bland-Altman plots of difference between nutrients as predicted by the first FFQ and the mean of six 24-h recalls (n = 238); Figure S2. Bland-Altman plots of difference between nutrients as predicted by the first and second FFQs (n = 52)