Oligomeric procyanidins combined with Parabacteroides distasonis ameliorate high-fat diet-induced atherosclerosis by regulating lipid metabolism,inflammation reaction and bile acid metabolism in ApoE^(-/-)mice

Atherosclerosis(AS)is the main pathological basis of cardiovascular diseases.Hence,the prevention and treatment strategies of AS have attracted great research attention.As a potential probiotic,Pararabacteroides distasonis has a positive regulatory effect on lipid metabolism and bile acids(BAs)profile.Oligomeric procyanidins have been confirmed to be conducive to the prevention and treatment of AS,whose antiatherosclerotic effect may be associated with the promotion of gut probiotics.However,it remains unclear whether and how oligomeric procyanidins and P.distasonis combined(PPC)treatment can effectively alleviate high-fat diet(HFD)-induced AS.In this study,PPC treatment was found to significantly decrease atherosclerotic lesion,as well as alleviate the lipid metabolism disorder,inflammation and oxidative stress injury in ApoE^(-/-)mice.Surprisingly,targeted metabolomics demonstrated that PPC intervention altered the BA profile in mice by regulating the ratio of secondary BAs to primary BAs,and increased fecal BAs excretion.Further,quantitative polymerase chain reaction(qPCR)analysis showed that PPC intervention facilitated reverse cholesterol transport by upregulating Srb1 expression;In addition,PPC intervention promoted BA synthesis from cholesterol in liver by upregulating Cyp7a1 expression via suppression of the farnesoid X receptor(FXR)pathway,thus exhibiting a significant serum cholesterol-lowering effect.In summary,PPC attenuated HFD-induced AS in ApoE^(-/-)mice,which provides new insights into the design of novel and efficient anti-atherosclerotic strategies to prevent AS based on probiotics and prebiotics.

Protective effects of dioscin against Parkinson's disease via regulating bile acid metabolism through remodeling gut microbiome/GLP-1 signaling

It is necessary to explore potent therapeutic agents via regulating gut microbiota and metabolism to combat Parkinson's disease(PD).Dioscin,a bioactive steroidal saponin,shows various activities.However,its effects and mechanisms against PD are limited.In this study,dioscin dramatically alleviated neuroinflammation and oxidative stress,and restored the disorders of mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).16 S rDNA sequencing assay demonstrated that dioscin reversed MPTP-induced gut dysbiosis to decrease Firmicutes-to-Bacteroidetes ratio and the abundances of Enterococcus,Streptococcus,Bacteroides and Lactobacillus genera,which further inhibited bile salt hydrolase(BSH)activity and blocked bile acid(BA)deconjugation.Fecal microbiome transplantation test showed that the anti-PD effect of dioscin was gut microbiota-dependent.In addition,non-targeted fecal metabolomics assays revealed many differential metabolites in adjusting steroid biosynthesis and primary bile acid biosynthesis.Moreover,targeted bile acid metabolomics assay indicated that dioscin increased the levels of ursodeoxycholic acid,tauroursodeoxycholic acid,taurodeoxycholic acid and bmuricholic acid in feces and serum.In addition,ursodeoxycholic acid administration markedly improved the protective effects of dioscin against PD in mice.Mechanistic test indicated that dioscin significantly up-regulated the levels of takeda G protein-coupled receptor 5(TGR5),glucagon-like peptide-1 receptor(GLP-1R),GLP-1,superoxide dismutase(SOD),and down-regulated NADPH oxidases 2(NOX2)and nuclear factor-kappaB(NF-kB)levels.Our data indicated that dioscin ameliorated PD phenotype by restoring gut dysbiosis and regulating bile acid-mediated oxidative stress and neuroinflammation via targeting GLP-1 signal in MPTP-induced PD mice,suggesting that the compound should be considered as a prebiotic agent to treat PD in the future.

Intrauterine growth retardation affects liver bile acid metabolism in growing pigs:effects associated with the changes of colonic bile acid derivatives

Background:Intrauterine growth retardation(IUGR)is associated with severely impaired nutrient metabolism and intestinal development of pigs.Our previous study found that IUGR altered intestinal microbiota and metabolites in the colon.However,the consequences of IUGR on bile acid metabolism in pigs remained unclear.The present study aimed to investigate the bile acid metabolism in the liver and the profile of bile acid derivatives in the colon of grow-ing pigs with IUGR using bile acid targeted metabolomics.Furthermore,we determined correlations between colonic microbiota composition and metabolites of IUGR and normal birth weight(NBW)pigs at different growth stages that were 7,21,and 28-day-old,and the average body weight(BW)of 25,50,and 100 kg of the NBW pigs.Results:The results showed that the plasma total bile acid concentration was higher(P<0.05)at the 25 kg BW stage and tended to increase(P=0.08)at 28-day-old in IUGR pigs.The hepatic gene expressions related to bile acid synthe-sis(CYP7A1,CYP27A1,and NTCP)were up-regulated(P<0.05),and the genes related to glucose and lipid metabolism(ATGL,HSL,and PC)were down-regulated(P<0.05)at the 25 kg BW stage in IUGR pigs when compared with the NBW group.Targeted metabolomics analysis showed that 29 bile acids and related compounds were detected in the colon of pigs.The colonic concentrations of dehydrolithocholic acid and apocholic acid were increased(P<0.05),while isodeoxycholic acid and 6,7-diketolithocholic acid were decreased(P<0.05)in IUGR pigs,when compared with the NBW pigs at the 25 kg BW stage.Moreover,Spearman’s correlation analysis revealed that colonic Unclassified_[Mogi-bacteriaceae],Lachnospira,and Slackia abundances were negatively correlated(P<0.05)with dehydrolithocholic acid,as well as the Unclassified_Clostridiaceae abundance with 6,7-diketolithocholic acid at the 25 kg BW stage.Conclusions:These findings suggest that IUGR could affect bile acid and glucolipid metabolism in growing pigs,especially at the 25 kg BW stage,these effects being paralleled by a modification of bile acid derivatives concentra-tions in the colonic content.The plausible links between these modified parameters are discussed.

Detection and analysis of serum bile acid profile in patients with colonic polyps

BACKGROUND Analyzing the variations in serum bile acid(BA)profile can provide a certain biological basis for early warning and prevention of various diseases.There is currently no comprehensive study on the relationship between the serum BA profile and colonic polyps.AIM To study the serum BA profile detection results of patients with colonic polyps,and analyze the correlation between BA and colonic polyps.METHODS From January 1,2022,to June 1,2023,204 patients with colonic polyps who were diagnosed and treated at Zhongda Hospital Southeast University were chosen as the study subjects,and 135 non-polyp people who underwent physical examination were chosen as the control group.Gathering all patients'clinical information,typical biochemical indicators,and BA profile.RESULTS Compared with the control group,the serum levels of taurocholic acid,glycocholic acid,glycochenodeoxycholic acid,and taurochenodeoxycholic acid in the colonic polyp group were significantly higher than those in the control group,while the content of deoxycholic acid(DCA)was lower than that in the control group(P<0.05).When colonic polyps were analyzed as subgroups,it was shown that there was a strong correlation between changes in the BA profile and polyp diameter,location,morphology,pathological kind,etc.CONCLUSION The serum BA profile showed significant changes in patients with colonic polyps,with a significant increase in primary conjugated BA content and a decrease in secondary free bile acid DCA content.There is a certain correlation between primary free BA and pathological parameters of polyps.

Targeted bile acids metabolomics in cholesterol gallbladder polyps and gallstones: From analytical method development towards application to clinical samples

Bile acids(BAs)are synthesized by the liver from cholesterol through several complementary pathways and aberrant cholesterol metabolism plays pivotal roles in the pathogeneses of cholesterol gallbladder polyps(CGP)and cholesterol gallstones(CGS).To date,there is neither systematic study on BAs profile of CGP or CGS,nor the relationship between them.To explore the metabolomics profile of plasma BAs in healthy volunteers,CGP and CGS patients,an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method was developed and validated for simultaneous determination of 42 free and conjugated BAs in human plasma.The developed method was sensitive and reproducible to be applied for the quantification of BAs in the investigation of plasma samples.The results show that,compared to healthy volunteers,CGP and CGS were both characterized by the significant decrease in plasma BAs pool size,furthermore CGP and CGS shared aberrant BAs metabolic characteristics.Chenodeoxycholic acid,glycochenodeoxycholic acid,l-muricholic acid,deoxycholic acid,and 7-ketolithocholic acid were shared potential markers of these two cholesterol gallbladder diseases.Subsequent analysis showed that clinical characteristics including cysteine,ornithine and body mass index might be closely related to metabolisms of certain BA modules.This work provides metabolomic information for the study of gallbladder diseases and analytical methodologies for clinical target analysis and efficacy evaluation related to BAs in medical institutions.

Bile acids and their receptors: Potential therapeutic targets in inflammatory bowel disease

Chronic and recurrent inflammatory disorders of the gastrointestinal tract caused by a complex interplay between genetics and intestinal dysbiosis are called inflammatory bowel disease.As a result of the interaction between the liver and the gut microbiota,bile acids are an atypical class of steroids produced in mammals and traditionally known for their function in food absorption.With the development of genomics and metabolomics,more and more data suggest that the pathophysiological mechanisms of inflammatory bowel disease are regulated by bile acids and their receptors.Bile acids operate as signalling molecules by activating a variety of bile acid receptors that impact intestinal flora,epithelial barrier function,and intestinal immunology.Inflammatory bowel disease can be treated in new ways by using these potential molecules.This paper mainly discusses the increasing function of bile acids and their receptors in inflammatory bowel disease and their prospective therapeutic applications.In addition,we explore bile acid metabolism and the interaction of bile acids and the gut microbiota.

Uridine dynamic administration affects the circadian variation of bile acid metabolism in high-fat-diet-fed mice

High-fat diet(HFD)is demonstrated to disturb the bile acid metabolism.The rhythm of bile acid metabolism can also be affected by uridine,whose metabolism exhibits a daily rhythm.However,the mechanism of dynamic uridine administration affecting bile acid during HFD remains unclear.In this study,C57BL/6J mice were fed HFD(the control group;CON)or HFD with oral administration of uridine in the daytime(DUR)and nighttime(NUR)to investigate the mechanism of the effect of uridine on the bile acid.This study showed that the mRNA expression of uridine transporters and circadian clock genes in the jejunum was affected by zeitgeber time(ZT)(P<0.001).Genes related to the metabolism of pyrimidines in the liver showed a high dependence on daily rhythm(P<0.01),and DUR remarkably up-regulated the expression of ribonucleotide reductase regulatory subunit M2(RRM2)(P<0.05)compared to the CON group.Importantly,the mRNA expression of bile acids nuclear receptors,bile acid synthesis,and transporters in the liver showed significantly rhythmically changed(P<0.05),and the expression of cholesterol 7-alpha-hydroxylase(CYP7A1),fibroblast growth factor receptor 4(FGFR4),Na^(+)/taurocholate co transporting polypeptide(NTCP),and bile salt export pump(BSEP)mRNAs of mice with uridine administration increased significantly(P<0.05).The mRNA expression of the transporters of cholesterol and bile acids in the ileum was also affected by ZT(P<0.01)and significantly dependent on uridine administration(P<0.05).The expression of FXR and SHP was significantly affected by ZT and uridine,respectively.In conclusion,dynamic administration of uridine could regulate the rhythm of gene expression of pyrimidine and bile acid metabolism in the liver and ileum of HFD-fed mice,which contributed to the further study of circadian rhythmic physiological and pathological changes of bile acids.

METABOLISM PATTERN OF FECAL BILE ACIDS IN PATIENTS WITH LARGE BOWEL CANCER

The feca! bite acids were extracted from 21 patients with large bowel (colonic and rectal) cancer and 21 controls, and the bile acid composition and concentration were measured by gas chromatography. The total bile acid concentration and concentration of individual bile acids were not statistically different between colonic and rectal cancer. Bat the concentration and the percentage composition of secondary bile acids (deoxycholic and lithocholic acids) were significantly higher than that in controls. However the percentage composition of primary bile acids (cholic and chenocholic acids) were significantly lower than that in controls. The results suggest that incidence of large bowel cancer is closely related to the metabolism of fecal bile acids, and the etiology of colonic and rectal cancers may be the same.

Sugarcane leaves-derived polyphenols alleviate metabolic syndrome and modulate gut microbiota of ob/ob mice

Sugarcane leaves-derived polyphenols(SLP)have been demonstrated to have diverse health-promoting benefits,but the mechanism of action has not been fully elucidated.This study aimed to investigate the anti-metabolic disease effects of SLP and the underlying mechanisms in mice.In the current study,we prepared the SLP mainly consisting of three flavonoid glycosides,three phenol derivatives,and two lignans including one new compound,and further demonstrated that SLP reduced body weight gain and fat accumulation,improved glucose and lipid metabolism disorders,ameliorated hepatic steatosis,and regulated short-chain fatty acids(SCFAs)production and secondary bile acids metabolism in ob/ob mice.Notably,SLP largely altered the gut microbiota composition,especially enriching the commensal bacteria Akkermansia muciniphila and Bacteroides acidifaciens.Oral gavage with the above two strains ameliorated metabolic syndrome(MetS),regulated secondary bile acid metabolism,and increased the production of SCFAs in high-fat diet(HFD)-induced obese mice.These results demonstrated that SLP could be used as a prebiotic to attenuate MetS via regulating gut microbiota composition and further activating the secondary bile acids-mediated gut-adipose axis.

Bile acid signaling through farnesoid X and TGR5 receptors in hepatobiliary and intestinal diseases

BACKGROUND： The well-known functions of bile acids（BAs） are the emulsification and absorption of lipophilic xenobiotics. However, the emerging evidences in the past decade showed that BAs act as signaling molecules that not only autoregulate their own metabolism and enterohepatic recirculation, but also as important regulators of integrative metabolism by activating nuclear and membrane-bound G protein-coupled receptors. The present review was to get insight into the role of maintenance of BA homeostasis and BA signaling pathways in development and management of hepatobiliary and intestinal diseases.DATA SOURCES： Detailed and comprehensive search of PubM ed and Scopus databases was carried out for original and review articles.RESULTS： Disturbances in BA homeostasis contribute to the development of several hepatobiliary and intestinal disorders, such as non-alcoholic fatty liver disease, liver cirrhosis, cholesterol gallstone disease, intestinal diseases and both hepatocellular and colorectal carcinoma.CONCLUSION： Further efforts made in order to advance the understanding of sophisticated BA signaling network may be promising in developing novel therapeutic strategies related not only to hepatobiliary and gastrointestinal but also systemic diseases.

Effects of paraprobiotics on bile acid metabolism and liver health in largemouth bass(Micropterus salmoides)fed a cottonseed protein concentrate-based diet

Cottonseed protein concentrate is a sustainable fishmeal alternative in aquafeed.A 10-week experiment was conducted to investigate the effects of a cottonseed protein concentrate-based diet with and without multi-strain yeast fractions(MsYF)on growth,bile acid metabolism,and health in largemouth bass.Four hundred fish(54.0±0.0 g)were casually distributed into 16 tanks(4 replicates/diet).Fish were fed with 4 iso-nitrogen and iso-energetic diets 3 times daily,including a fishmeal diet(FM),a soy protein concentrate-based diet(SPC;replacing 81%fishmeal protein),a cottonseed protein concentrate-based diet(CPC;replacing 81%fishmeal protein),and a CPC diet supplemented with 800 mg/kg MsYF(CPCY).Results showed that the survival of SPC was the lowest,i.e.,48%,with no apparent diet effect among other treatments;we omitted the SPC in additional analyses.Fish fed cottonseed protein concentrate-based diets showed lower growth than FM(P<0.05).Fish fed CPC showed the highest nuclear dense hepatic phenotypes ratio(50%),followed by CPCY(33%)and FM(17%).Further,dietary CPC increased hepatic total cholesterol and triglyceride levels with concurrently increased cholesterol syn-thesis but decreased triglyceride synthesis-associated transcription levels(P<0.05).Furthermore,di-etary CPC increased bile acid synthesis but decreased bile acid transport-associated transcription levels(P<0.05),and then induced an increment of plasma cholic acid and hepatic chenodeoxycholic acid content and the decrement of genus Romboustia(P<0.05).Regarding the effect of MsYF,fish fed CPCY reduced hepatic lipid accumulation and total plasma bile acid content(P<0.05)compared to CPC,suggesting an improvement in liver health.Also,dietary MsYF could reverse the microbiota community structure showing a similar gut microbial composition to FM.In conclusion,81%of fishmeal protein replaced by cottonseed protein concentrate suppressed growth and liver health,while dietary MsYF might mitigate the negative impact of a high cottonseed protein concentrate level diet on liver functions via gut microbiota regulation.

The blind men 'see' the elephant-the many faces of fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a group of diseases with excess fat in liver in the absence of a poorly defined limit of alcohol consumption. Most common variety, a universal public health problem, is associated with insulin resistance caused by a host of genetic and epigenetic defects modulated by life style and environmental factors. In fact the term NAFLD is loose to incorporate so many etiologies except alcoholism and few other etiologies, presenting as fat in liver. However as a sign fatty liver is very important in predicting the risk of diabetes, cardiovascular disease, stroke, cirrhosis and cancer. Abnormal fat accumulation can result from several defects in nuclear receptors associated with lipid sensing, synthesis and oxidation like LXR, FXR, SREBP, ChREBP and PPAR; defects in the lipid influx-efflux channels, insulin signaling, proteins involved in fatty acid catabolism, defects in adipose tissue development and function, inappropriate nutrition and finally defects in neural regulatory mechanisms. The progress of the disease is determined by the basic defects which results in fat accumulation, an individual’s immunological response to the accumulated fat and its derivatives and the oxidant stress response. Congregation of unrelated genetic defects under same diagnosis ‘NAFLD’ can result in inefficient patient management. Further studies are required to understand the molecular basis of fatty liver to enable a personalized management of diseases presenting as fatty liver in the absence of alcohol abuse.

Bile acid metabolism and signaling in liver disease and therapy

Bile acids play a critical role in the regulation of glucose,lipid,and energy metabolism through activation of the nuclear bile acid receptor farnesoid X receptor(FXR)and membrane G protein-coupled bile acid receptor-1(Gpbar-1,aka TGR5).Agonist activation of FXR and TGR5 improves insulin and glucose sensitivity and stimulates energy metabolism to prevent diabetes,obesity,and non-alcoholic fatty liver disease(NAFLD).Bile acids have both pro-and anti-inflammatory actions through FXR and TGR5 in the intestine and liver.In the intestine,bile acids activate FXR and TGR5 to stimulate fibroblast growth factor 15 and glucagon-like peptide-1 secretion.FXR and TGR5 agonists may have therapeutic potential for treating liver-related metabolic diseases,such as diabetes and NAFLD.

Influence of fever on biliary elements of guinea pigs

IM To study the influence of fever on biliary elements and gallstone formation in guinea pigs. METHODS Sixty guinea pigs were divided into lithogenic (group B) and normal diet (group A) groups, and each group was further divided into control and fever subgroups. The animals in the fever groups were injected subcutaneously with boiled defat milk 1ml/kg, once a week for 4 times to run fever. After 45 days, all the animals were killed. Among them the guinea pigs in the fever groups were injected subcutaneously with turpentine 1ml/kg 36 hours before they were killed, and kept in fever condition. Their gallbladder lumens were examined and bile samples were analyzed. RESULTS The gallstone incidence of the fever group fed with lithogenic diet was the highest, accounting for 40% (6/15). All the animals in fever group had significantly higher concentration of total bile protein and bilirubin than that of the control groups. CONCLUSION The effect of fever on biliary elements plays an important role in the gallstone formation in guinea pigs.

High rumen degradable starch diet induced blood bile acids profile changes and hepatic inflammatory response in dairy goats

The objective of this study was to reveal the effect of rumen degradable starch(RDS)on bile acid metabolism and liver transcription in dairy goats using metabolomics and transcriptomics.Eighteen Guanzhong dairy goats of a similar weight and production level(body weight=45.8±1.54 kg,milk yield=1.75±0.08 kg,and second parity)were randomly assigned to 3 treatment groups where they were fed a low RDS(LRDS,RDS=20.52%DM)diet,medium RDS(MRDS,RDS=22.15%DM)diet,or high RDS(HRDS,RDS=24.88%DM)diet,respectively.The goats were fed with the experimental diets for 5weeks.On the last day of the experiment,all goats were anesthetized,and peripheral blood and liver tissue samples were collected.The peripheral blood samples were used in metabolomic analysis and white blood cell(WBC)count,whereas the liver tissue samples were used in transcriptomic analysis.Based on the metabolomics results,the relative abundances of primary bile acids in the peripheral blood were significantly reduced in the group that was fed the HRDS diet(P<0.05).The WBC count was significantly increased in the HRDS group compared with that in the LRDS and MRDS groups(P<0.01),indicating that there was inflammation in the HRDS group.Transcriptomic analysis showed that 4 genes related to bile acid secretion(genes:MDR1,RXRa,AE2,SULT2A1)were significantly downregulated in the HRDS group.In addition,genes related to the immune response were upregulated in the HRDS group,suggesting the HRDS diet induced a hepatic inflammatory response mediated by lipopolysaccharides(LPS)(gene:LBP),activated the Toll-like receptor 4 binding(genes:S100A8,S100A9)and the NF-kappa B signaling pathway(genes:LOC106503980,LOC108638497,CD40,LOC102180880,LOC102170970,LOC102175177,LBP,LOC102168903,LOC102185461,LY96 and CXCL8),triggered inflammation and complement responses(genes:C1QB,C1QC,and CFD).The HRDS diet induced a hepatic inflammatory response may be mediated by activating the Toll-like receptor 4 binding and NF-kappa B signaling pathway after free LPS entered the liver.The changes of bile acids profile in blood and the downregulation of 4 key genes(MDR1,RXRa,AE2,SULT2A1)involved in bile secretion in liver are probably related to liver inflammation.

Bile acid receptors and signaling crosstalk in the liver, gut and brain

Bile acids are physiological detergents derived from cholesterol that aid in digestion and nutrient ab-sorption,and they play roles in glucose,lipid,and energy metabolism and in gut microbiome and metabolic homeostasis.Bile acids mediate crosstalk between the liver and gut through bactericidal modulation of the gut microbiome,while gut microbes influence the composition of the circulating bile acid pool.Recent research indicates bile acids may also be important mediators of neurological disease by acting as peripheral signaling molecules that activate bile acid receptors in the blood-brain barrier and in the brain itself.This review highlights the role of bile acids in maintaining liver and gut microbe homeostasis,as well as their function as mediators of cellular signaling in the liver-gut-brain axis.

Bile acids control inflammation and metabolic disorder through inhibition of NLRP3 inflammasome

With the support by the National Natural Science Foundation of China,the research team led by Prof.Wang Di(王迪)at the Immuno metabolism Lab,Institute of Immunology,Zhejiang University School of Medicine,uncovered the mystery of Bile Acids control inflammation and metabolic disorder,which was published in Immunity(2016,45:802—816)).

Pyrrolizidine alkaloids:An update on their metabolism and hepatotoxicity mechanism

Pyrrolizidine alkaloids(PAs)are among the most hepatotoxic natural compounds that are widely distributed throughout the world.Most PAs are metabolically activated to trigger toxicity.Exposure to herbal medicine containing PAs and food supplements contaminated by PAs is considered to be one of the two main causes of hepatic sinusoidal obstruction syndrome(HSOS),which is a rare hepatic vascular disease with a high mortality rate.PAs-induced HSOS cases have been reported worldwide.However,there is no clinically effective therapy for PAs-induced HSOS,which is partially because the toxic mechanism is not fully understood.This review focuses on updating the information on the metabolism and the molecular mechanisms of PAs hepatotoxicity,including oxidative stress,apoptosis,and dysfunction of bile acid metabolism,and their interactions.

Up to date on cholesterol 7 alpha-hydroxylase (CYP7A1) in bile acid synthesis

Cholesterol 7 alpha-hydroxylase(CYP7A1,EC1.14)is the first and rate-limiting enzyme in the classic bile acid synthesis pathway.Much progress has been made in understanding the transcriptional regulation of CYP7A1 gene expression and the underlying molecular mechanisms of bile acid feedback regulation of CYP7A1 and bile acid synthesis in the last three decades.Discovery of bile acid-activated receptors and their roles in the regulation of lipid,glucose and energy metabolism have been translated to the development of bile acid-based drug therapies for the treatment of liver-related metabolic diseases such as alcoholic and non-alcoholic fatty liver diseases,liver cirrhosis,diabetes,obesity and hepatocellular carcinoma.This review will provide an update on the advances in our understanding of the molecular biology and mechanistic insights of the regulation of CYP7A1 in bile acid synthesis in the last 40 years.