T, was isolated from a scallion kimchi sample consisting of fermented chili peppers and vegetables. The isolate was a Gram-positive, rod-shaped, non-motile, catalase-negative and facultatively anaerobic lactic acid bacterium. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain WiKim39T belonged to the genus Lactobacillus, and shared 97.1–98.2 % pair-wise sequence similarities with related type strains, Lactobacillus nodensis, Lactobacillus insicii, Lactobacillus versmoldensis, Lactobacillus tucceti and Lactobacillus furfuricola. The G+C content of the strain based on its genome sequence was 35.3 mol%. The ANI values between WiKim39T and the closest relatives were lower than 80 %. Based on the phenotypic, biochemical, and phylogenetic analyses, strain WiKim39T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus allii sp. nov. is proposed. The type strain is WiKim39T (=KCTC 21077T=JCM 31938T).A novel strain of lactic acid bacteria, WiKim39 Leuconostoc, Weissella and Lactobacillus are involved in the fermentation process of kimchi [Lactobacillus belongs to the large group of LAB producing lactic acid by carbohydrate fermentation. These bacteria are characterized as Gram-positive, non-spore-forming rods, which have a low G+C content, and are catalase-negative, non-motile microorganisms [Lactobacillus are usually found in plants; plant-derived materials such as silage, grains, and foods; and the gastrointestinal tract of humans and animals. Lactobacillus strains are currently used as probiotics, starters, and silage inoculants in food and feed fermentation [Lactobacillus, which is used in the food and feed industries, was isolated from scallion kimchi, and the phenotypic, chemotaxonomic, and molecular characteristics of the novel LAB strain WiKim39T are presented.Fermented foods and beverages not only provide important nutrients but also have great potential for maintaining health and preventing disease, and they play an important role in the human diet worldwide . Kimchi f kimchi . The genrganisms . Membersentation . In the T was isolated from scallion kimchi in Gwangju, Korea, using the dilution plating method with MRS agar medium  at 30 °C for 48 h. Single colonies on the plates were transferred to new plates and incubated on MRS agar at 30 °C under anaerobic conditions. The reference strains used in this study, Lactobacillus nodensis KACC 16346T, Lactobacillus insicii DSM 29801T, Lactobacillus versmoldensis KCTC 3814T, Lactobacillus tucceti KCTC 21005T and Lactobacillus furfuricola KCTC 21034T, were cultured in MRS medium according to the culture collection guidelines. Cells of strain WiKim39T grown on MRS were Gram-stained and visualized under light microscopy and transmission electron microscopy . Bacterial growth at various pH values (3.0–10.0 in 0.5-unit increments) was measured by inoculating pH-adjusted MRS media with HCl or KOH. The optimum growth temperature and tolerance to NaCl of the cells were also measured. For these studies, cells were grown at temperatures ranging from 15 to 60 °C and in 0–10 % (w/v) NaCl for 48 h. Growth was monitored by measuring OD600 using a UV-1600 spectrometer (Shimadzu). Growth under anaerobic conditions was tested on MRS agar (Difco) using GasPak jars (BBL) at 30 °C. Motility was tested in MRS medium with 0.4 % agar. Physiological characteristics  were determined using the API 50CH, API ZYM, API 20E, and API 20 Strep galleries according to the manufacturer’s instructions (bioMérieux). Catalase activity was determined by assessing the production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution. Oxidase activity was measured using an Oxy-swab (bioMérieux) according to the manufacturer’s recommendations. For haemolysis testing, the bacteria were streaked on MRS agar containing 5 % (w/v) sheep's blood and incubated for 48 h at 30 °C. Tellurite tolerance tests were performed by supplementation with 0.04 % K2TeO3 (Sigma-Aldrich). Lactic acid production was quantified using a dl-lactic acid assay kit .Strain WiKim39T were rods with an average length of 0.6×1.8–2.5 µm. The strain was identified as a facultative anaerobe, and it grew at temperatures from 25 to 37 °C and at a pH from 4.5 to 9.0 with optimum growth at 30 °C and pH 6.5–7.0. No growth was observed at ≤20 °C and ≤pH 4.0 or ≥40 °C and ≥7 % (w/v) NaCl l−1. Positive reactions were observed in tests for the production of acid from d-galactose, d-glucose, d-fructose, N-acetylglucosamine, amygdalin, arbutin, aesculin ferric citrate, salicin, d-cellobiose, d-maltose, d-lactose, d-sucrose, and gentiobiose. In addition, enzyme detection with an API zym kit was positive for alkaline phosphatase, esterase, leucine arylamidase, valine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase. No esterase lipase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-mannosidase, or α-fucosidase activities were observed. Biochemical features were negative for nitrate and nitrite reduction; production of indole, H2S, urease, pyrrolidonyl arylamidase, arginine dihydrolase; or hydrolysis of gelatin and hippurate. WiKim39T showed non-haemolysis (γ-haemolysis) and tellurite tolerance. The novel species could be differentiated from all other tested reference type strains using its physiological properties , and phenylalanyl-tRNA synthase alpha subunit (pheS) gene were performed according to the method described by Irisawa et al. [rpoA, and pheS genes of the isolated strain were compared with available sequences from GenBank using the blast program (http://www.ncbi.nlm.nih.gov/blast/) to determine their approximate phylogenetic affiliation. 16S rRNA gene sequence similarities were determined using the EzTaxon-e server (http://www.ezbiocloud.net/) [mega  version 7 [clustal_x [Bacterial DNA extraction was performed using the QIAcube system with a QIAamp DNeasy Blood and Tissue Kit (Qiagen). PCR amplification and sequencing of the 16S rRNA gene, RNA polymerase alpha subunit  . The phylustal_x . PhylogeT were Lactobacillus nodensis DSM 19682T (98.2 %), Lactobacillus insicii TMW1.2011T (98.1 %), Lactobacillus versmoldensis KU-3T (97.5 %), Lactobacillus tucceti CECT 5920T (97.3 %), and Lactobacillus furfuricola JCM 18764T (97.1 %). The 16S rRNA analysis clearly indicated that the isolate represented a novel genomic species in the genus Lactobacillus, as none of the valid described species showed more than 98.7 % 16S rRNA similarity [T and related Lactobacillus species are shown in Based on the pairwise 16S rRNA gene sequence similarities, the closest phylogenetic relatives to strain WiKim39pheS) and RNA polymerase alpha subunit (rpoA) showed that the closest phylogenetic relative strains of strain WiKim39T were type strains of Lactobacillus nodensis, Lactobacillus insicii, and Lactobacillus tucceti, with 84.4–87.6 % pheS gene sequence similarity and 89.5–94.5 % rpoA gene sequence similarity  by Macrogen. The reads were assembled de novo using Hierarchical Genome Assembly Process version 3.0 (HGAP3.0) in SMRT analysis version 2.3.0 [The complete genome of strain WiKim39on 2.3.0 . The comT consists of a circular 2 506 167 bp chromosome and one circular plasmid, totaling 2 534 178 bp. The chromosome contains 2427 predicted protein-coding genes (CDCs), four rRNA operons , 67 tRNAs, and 3 noncoding RNAs. Strain WiKim39T contains 35.3 mol% G+C in its DNA, which is in the reported range of 31.93–57.02 % for Lactobacillus species [Lactobacillus nodensis DSM 19682T (AZDZ00000000). The complete genomes determined in this study have been deposited in the NCBI GenBank database under the accession numbers CP019323 (chromosome) and CP019324 (plasmid).The complete genome of strain WiKim39et al. [Lactobacillus reference strains Lactobacillus nodensis DSM 19682T (AZDZ00000000) and Lactobacillus tucceti DSM 10183T (AZDG01000001), and the strain was therefore concluded to represent a novel species [T represents a novel genomic species that is distinct from its closest relatives.To evaluate the similarity between genome sequences, average nucleotide identity (ANI) values were analyzed between the strain and reported genomes using EzBioCloud as described by Moon et al. . The str species . These DT grown on MRSA plates at 30 °C for 48 h. The fatty acid methyl esters were extracted and analyzed according to the standard protocol of the Sherlock Microbial Identification System . The fatty acid methyl ester mixtures were separated with an automated GC system  and identified using the BHIBLA database of the Microbial Identification Sherlock software package v6.3.The cell-wall peptidoglycan type was determined by methods described previously . CellulaT was A4αl-Lys-d-Asp (A11.31), which is the major type in most species of the closely related reference strains and the L. alimentarius – L. farciminis phylogenetic subgroup within the genus Lactobacillus [T were C16 : 0 and C18 : 1ω9c. The fatty acid composition of WiKim39T was similar to that of L. tucceti KCTC 21005T, with small variations in the proportion, whereas the major fatty acids of the other reference strains were C16 : 0, C18 : 1ω9c, and C19 : 1 cyclo 9, 10  cells are Gram-positive, catalase and oxidase-negative, facultatively anaerobic, and non-motile. Additionally, the cells are non-spore-forming rods, are 0.6×1.8–2.5 µm in size, and occur singly or in pairs. Colonies grown on MRS agar at 30 °C for 48 h are up to 1.0 mm in diameter, off-white, smooth, and round with rough surfaces. They are homofermentative; gas is not produced from glucose. The cells produce d- and l-lactic acid at a ratio of 52 : 48. Growth occurs at 25–37 °C and in the presence of 5 % NaCl but not in the presence of 7 % NaCl. Growth occurs at pH 4.5–9.0 but not at pH 3.5–4.0. Acid is produced from d-galactose, d-glucose, d-fructose, N-acetylglucosamine, amygdalin, arbutin, aesculin ferric citrate, salicin, d-cellobiose, d-maltose, d-lactose, d-sucrose, and gentiobiose. Acid is not produced from glycerol, erythritol, arabinose, ribose, xylose, d- or l-arabinose, adonitol, methyl β-d-xylopyranoside, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl α-d-mannopyranoside, methyl α-d-glucopyranoside, melibiose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, turanose, d-lyxose, d-tagatose, d- or l-fucose, d- or l-arabitol, potassium gluconate, potassium 2-ketogluconate, or potassium 5-ketogluconate. Alkaline phosphatase, estalase, leucine arylamidase, valine arylamidase, acid phosphatase, naphtol phosphohydrolase, α-glucosidase , β-glucosidase (cellulose), and N-acetyl-β-glucosaminidase (chitinase) are produced. Nitrate is not reduced. Indole and H2S gas are not produced. The Voges-Proskauer test is positive. Tellurite tolerance is present. The cell-wall peptidoglycan structure type is A4α l-Lys–d-Asp. The major cellular fatty acids are C16 : 0 and C18 : 1ω9c. The DNA G+C content of the type strain is 35.3 mol%, and the genome size is 2.53 Mb. The type strain WiKim39T (=KCTC 21077T=JCM 31938T) was isolated from scallion kimchi in Gwangju, Korea.


Penicillium strains with numerous pharmaceutical properties such as antibiotic and immunosuppressive uses. The aim of this work is the survey of the effect of phenylglyoxal and n-ethylmaleimide concentration in culture medium on mycophenolic acid production by Penicillium brevi-compactum ATCC16024 was investigated. Batch submerged fermentation was performed in 250 mL shake flasks at 24 °C and 200 rpm in a rotary shaker for 300 h using a basic culture medium containing different concentrations of phenylglyoxal and n-ethylmaleimide ranged from 0 to 20 mg. L-1. For the basic medium without any amounts of phenylglyoxal and n-ethylmaleimide (control), maximum MPA production, product yield and productivity of process was in order, 1.5042 g. L-1, 20.3 mg. g-1 consumed glucose and 5.37 mg. L-1h-1. Maximum produced MPA of 2.9032 g. L-1, MPA yield of 39.23 mg. g-1 of consumed glucose, productivity of 10.37 mg. L-1 h-1 and total enhancement of 93.11% was obtained when the culture medium was contained 18 mg. L-1 of phenylglyoxal, represented more than 93% raising in compare to control. Maximum MPA concentration, yield and productivity in order was obtained 3.1123 g. L-1, 42.06 mg. g-1 of consumed glucose and 11.11 mg. L-1 h-1, with using 6 mg. L-1 of n-ethylmaleimide. N-ethylmaleimide was caused to 2.138 folds (106.89%) increase in MPA production by P. brevi-compactum ATCC16024.Mycophenolic acid is a secondary extracellular metabolite of  Penicillium and also by some other microbial strains such as Byssochlamys nivea in submerged and solid state fermentation processes (Mycophenolic acid (4E)-6--4-methylhex-4-enoic acid) and its derivatives such as mycophenolate mofetil are new drugs with numerous pharmaceutical properties such as immunosuppressive and decrease the incidence of graft rejection after organ transplantation  on transketolase and phosphoenolpyruvate carboxykinase, two key enzymes in MPA biosynthesis pathway (6H7NO2) created a primary idea to investigate the effects of these compounds on MPA biosynthesis in Penicillium brevi-compactum  acetaldehyde 1-phenylethanedione, C pathway , 14, 15;ompactum . Penicillium brevi-compactum ATCC16024 were evaluated. In this study, the effects of phenylglyoxal and n-ethylmaleimide concentration on MPA production by Material17H20O6) powder with more than 98% purity was prepared from Sigma-Aldrich Co. LLT. All other chemicals and microbial cultures were prepared from Merck and Sigma-Aldrich. Phenylglyoxal hydrate 97% and n-ethylmaleimide crystalline 98% were prepared from Sigma-Aldrich Co. LLT. Analytical standard mycophenolic acid (CMicroorganism and inoculum preparationPenicillium brevi-compactum ATCC16024 was obtained from American Type Culture Collection (ATCC) as an ampoule containing freeze-dried viable cells suspended in cryoprotectant. The stock culture was maintained on the Potato dextrose agar (PDA) slants at 4 °C. PDA containing petri dishes were used for preparation of working cultures. After inoculation, petri dishes were incubated at 24 °C for 3 days. The cell suspension was made by collection of spores grown on petri plates by shaving and extracting the spores with sterile water  was inoculated to 50 mL culture medium contained in 250 mL shake flasks (The number of spores in suspension was counted by Thoma lam and adjusted to 10Medium composition -1): glucose, 80; glycine, 9; enzymatically hydrolyzed casein, 15; methionine, 0.5; KH2PO4, 5; MgSO4.7H2O, 1; and 1 ml. L-1 trace element mixture including (g. L-1): FeSO4·7H2O, 2.2; CuSO4·5H2O, 0.3; ZnSO4·7H2O, 2.4; MnSO4·4H2O, 0.16; and KMoO4, 0.2  and also 36 shake flasks containing basic medium with different n-ethylmaleimide concentrations  were prepared. All shake flasks were autoclaved at 121 °C for 15 min. Amino acids (here glycine and methionine) are heat sensitive compounds and may be decompose in the presence of other medium components at high temperatures. Thus they strolled separately by microfiltration  and then added to the shake flasks contents under sterile conditions. All culture medium pH was adjusted on 6 using 2 N HCl or NaOH solutions . Inoculated shake flasks were incubated at 24 °C with 200 rpm agitation speed in a rotary shaker for 300 h. MPA production was investigated at the end of cultivation time. The effects of phenylglyoxal and n-ethylmaleimide concentration on MPA production were evaluated separately as explained in medium composition section. MPA concentration in each shake flask was measured after 280 h. MPA was extracted from culture medium using a liquid adsorption chromatography column . For this purpose, the first, medium pH was adjusted on 7 and cell biomass separated by centrifugation at 10000 rpm for 10 min. Then, adsorption chromatography was conducted and at the next step, the column was eluted by methanol. MPA white powder was purified from eluted solution by precipitation using chilled water at pH = 2 and drying in oven at 65 °C until constant weight . Analytical methods 18 column at 40 °C was used to determine MPA concentration  and working (2.5–250 µg. mL-1) standards of MPA. All measurements were repeated for three times and the mean values are considered as the experimental results (All contents of each flask were used as sample for MPA concentration measurement. At the end of incubation time after 280 h), MPA concentration was measured for filtered samples through a 0.2 μM filter . High performance liquid chromatography method  with a Cas 50 µL . HPLC gr0 h, MPA Colorimetric method by using spectrophotometer  at 540 nm was used to determine glucose concentration for each shake flask contents at the end of incubation time . Statistical data analysisSPSS 22 software was used for data analysis. Two ways analysis of variance (ANOVA) and Fishers Least Significant Difference (LSD) test were applied to compare the mean values of the produced MPA concentration between all treatments. P≤0.05 was considered as significant difference between each treatment with other experiments. P. brevi-compactum ATCC16024 was appeared as pellets after about 24 h from incubation. These pellets served during the fermentation process until 250 h (the end of the stationary phase) and then broke and disappeared. The major amount of glucose was consumed in the early 180 h of incubation, tropho-phase stage of P. brevi-compactum growth. Then, at the idio-phase stage of growth, glucose consumption rate was decreased and reached to an approximate constant value. Glucose concentration was decreased from 10.51 g. L-1 at the start of the stationary phase to 4.27 g. L-1 at the end of this phase. MPA production as a secondary metabolite was occurred between 180 to 280 h of incubation time. Effect of phenylglyoxal on MPA production -1, at 280 h after incubation. MPA production yield was calculated 20.3 mg. g-1 of consumed glucose and its productivity was determined as 5.37 mg. L-1 h-1. Maximum produced MPA in the basic medium without any amounts of phenylglyoxal, n-ethylmaleimide and 8-hydroxycoinoline-5-sulfonic acid was reached to 1.5042 g. L-1, was obtained when the culture medium was contained 18 mg. L-1 of phenylglyoxal. In this condition, MPA yield and productivity were 39.23 mg. g-1 of consumed glucose and 10.37 mg. L-1 h-1, respectively. These values were higher than MPA production in basic medium without any phenylglyoxal as 93.11% (Table 1.). However, statistical analysis of results didn’t show any considerable differences between treatments with 12 to 20 mg L-1 of phenylglyoxal (p>0.05). Some separate experiments were conducted using basic medium enriched by different concentrations of phenylglyoxal. MPA concentration was determined after 280 hours for each experiment .. MaximuP. brevi-compactum ATCC16024 under the applied conditions. This impact may be related to phenylglyoxal effect on inhibition of certain enzymes involved in the MPA biosynthetic pathway of P. brevi-compactum. Based on some previous reports, phenylglyoxal has an inhibitory effect on transketolase  to phosphoenolpyruvate (PEP)  . OAA is te (PEP) , 21. Theuctivity , 23. TheEffect of n-ethylmaleimide on MPA production Penicillium brevi-compactum was investigated as separate experiments and produced MPA concentration was assayed 280 h after incubation . Adding n-ethylmaleimide more than 6 mg. L-1 not only had not more positive effects on MPA biosynthesis by Penicillium brevi-compactum, but also showed a negative impact. Statistical analysis of results showed considerable difference between the treatments containing of 6 mg. L-1 n-ethylmaleimide and other studied treatments. Effects of different concentrations of n-ethylmaleimide on MPA production by cubation .. MaximuP. brevi-compactum ATCC16024 in the performed fermentation process. N-ethylmaleimide has inhibitory effect on four key enzymes involved in MPA biosynthesis biochemical pathway: squalene synthase, NADH dehydrogenase, pyrroline-5-carboxylate reductase and hydroxymethylglutaryl-CoA synthase. Squalene synthase is the main enzyme responsible for ergosterol (ERG) biosynthesis from farnesyl-diphosphate (F-PP) and s-adenosyl methionine (SAM) as presented in Equation 1that inhibit by n-ethylmaleimide 1F-PP+ + 6NADPH+ 6OThe second inhibited enzyme is NADH dehydrogenase, the biocatalyst undertaking to run the Equation 2. in the section of oxygen transfer chain. N-ethylmaleimide blocks the phosphate porter by reacting with SH groups and prevents respiration by coupled mitochondria and phosphate-mediated swelling. Its inhibitory effect on NADH dehydrogenase was reported by some previous researches  probably2+ 2(P/O) ADP+ 2(P/O) Pi→ 2(P/O) ATP+ 1NAD(p)          (Eq. 2) 1NAD(P)H+ 1OPyrroline-5-carboxylate reductase catalyzes the conversion of glutamine (GLU) to proline (PRO) as presented in Equation 3. Less activity of pyrroline-5-carboxylate reductase affected by n-ethylmaleimide , cause t2O+ 2NADP+ 1ADP+ 1Pi          (Eq. 3)1GLU+ 1ATP+ 2NADPH+ 2H→ 1PRO+ 1HN-ethylmaleimide showed negative effects on the activity of hydroxymethylglutaryl-CoA synthase, the main enzyme to operate the conversion of Acetoacetyl-COA (AACOA) to 3-hydroxy-3-methylglutaryl COA (3H3MGCoA) as presents in Equation 4. Inhibition of this reaction, unlike previous cases, leads to a decrease in MPA biosynthesis. 3H3MGCoA is a precursor for mevalonate synthesis, one of the main parts of MPA molecule. This may be the reason of lower MPA production at higher contents of n-ethylmaleimide.2O+ 1AACCOA→ 1 3H3MGCoA+ 1CoA+ 1CO2           (Eq. 4) 1ACCOA+ 1HPenicillium brevi-compactum ATCC16024 with different content of phenylglyoxal and n-ethylmaleimide concentration was evaluated in batch submerged fermentation. Both two applied agents showed positive impacts on MPA biosynthesis. MPA production was enhanced for 93.11% with adding 18 mg. L-1 of phenylglyoxal to the produced cell culture. While, adding only 6 mg. L-1 of n-ethylmaleimide caused to 106.9% raise in MPA production. MPA production by


Ligularia cymbulifera is one of the predominant species in the Hengduan Mountains, China, and has led to a decrease in the amount of forage grass in this area. However, little is known about the mechanism behind its predominance. In this study, two novel eremophilane sesquiterpenes, ligulacymirin A and B (1 and 2), together with seven other known terpenoids (3–9), were isolated from the roots of L. cymbulifera. The structures of 1 and 2 were determined by spectroscopic methods and single-crystal X-ray diffraction. Each compound showed phytotoxic activities against Arabidopsis thaliana, and each was detected and identified in rhizosphere soil by UHPLC-MS. Compound 3 was the most potent phytotoxin, showing remarkable inhibition against both seedling growth (EC50 = 30.33 ± 0.94 μg/mL) and seed germination (EC50 = 155.13 ± 0.52 μg/mL), with an average content in rhizosphere soil of 3.44 μg/g. These results indicate that terpenoids in L. cymbulifera roots might be released as phytotoxins in rhizosphere soil to interfere with neighboring plants. Ligularia  Hand. Mazz, belonging to the Asteraceae family, is one of the predominant species in the Hengduan Mountains, China. It is a perennial herb that grows at high density in moist grassland at altitudes from 3000 to 4800 m, being especially abundant in Zhongdian, Yunnan  was found to be an abundant eremophilane sesquiterpene produced by L. cymbulifera, and was thought to be a defensive agent that helps this species to gain an ecological advantage , Chinese Academy of Sciences (CAS). A voucher specimen (KIB H20150913) has been deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, KIB, CAS. Seeds of A. thaliana were of the Columbia wild ecotype.The roots of 3OD or DMSO-d6 on a Bruker AVANCE III 500 MHz spectrometer  with tetramethylsilane as an internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. Electrospray ionization mass spectrometry (ESI-MS) and high-resolution (HR)-ESI-MS were carried out on a Waters Xevo TQ-S mass spectrometer . X-ray diffraction data collection was performed on a Bruker SMART APEX CCD  crystallography system. Normal-pressure column chromatography (CC) was performed on either silica gel  or Sephadex LH-20 . Preparative medium-pressure liquid chromatography (MPLC) was performed on a Buchi Separate system using MCI-gel CHP 20P . Preparative high-performance liquid chromatography (HPLC) was performed on an Agilent 1200 liquid chromatography system  equipped with an XSELECT CSH Prep C18 column  and diode array detector (DAD). Fractions were monitored and analyzed by thin-layer chromatography (TLC) ; spots were visualized under UV254 illumination and/or by heating silica gel plates dipped into 5% H2SO4 in ethanol. All solvents used for extraction and isolation were distilled at their boiling point range prior to use. HPLC-grade acetonitrile and formic acid were from Fisher Scientific . Ultrapure water was prepared by a Milli-Q water purification system .Optical rotation (OR) values were measured with a Jasco P-1020  automatic digital spectropolarimeter. Ultraviolet (UV) spectral data were obtained using a Shimadzu UV-2401PC spectrophotometer . Infrared spectroscopy (IR) was performed using a Bruker Tensor 27 FT-IR spectrometer  with KBr pellets. One-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectra were obtained in CDL. cymbulifera (15.0 kg) were powdered and extracted with 75% MeOH  at 75°C under reflux three times (6 h each). The MeOH extracts were filtered and the solvent was evaporated under a vacuum to afford a crude MeOH extract (1300 g). The crude extract was then suspended in partition between H2O (4 L) and ethyl acetate (EtOAc) (3 × 4 L), and the EtOAc fraction (600 g) was subjected to CC over silica gel  eluting with dichloromethane (CH2Cl2)/acetone (Me2CO)  to afford five fractions, A–E. Fraction A  was further separated by CC on a silica gel (300–400 mesh) using petroleum ether (PE) and Me2CO  to yield five fractions (Fr. A1–A5). Fr. A2 (5.6 g) was subjected to MPLC (MCI gel) using MeOH/H2O (20:80–80:20) and finally purified by Sephadex LH-20 (MeOH) and HPLC to give compounds 4 (167 mg), 5 (158 mg), 6 (210 mg), 7 (480 mg), and 9 (950 mg). Fraction B  was also subjected to MPLC (MCI gel) using MeOH/H2O (40:60–80:20) to yield five main fractions (Fr. B1–B5). Fr. B1  was purified by Sephadex LH-20 chromatography (MeOH) and then recrystallized to yield compound 8 (220 mg) and compound 3 (960 mg). Fr. B2  was separated by CC on a silica gel (300–400 mesh) using PE/Me2CO (20:1), and then further purified by preparative HPLC  using 30% aqueous acetonitrile (v/v) at a flow rate of 10 mL/min to afford compound 1 (retention time (tR) of 23 min, 680 mg) and compound 2 .Air-dried roots of 1)Ligulacymirin A ; Mp: 175–176°C; c 0.17, MeOH) : 203.6 (3.62) nm  spectroscopic data, see Table m/z 343 [M+Na]+; positive HRESIMS m/z 359.1627   Figure ; UV  Figure .2)Ligulacymirin B ; Mp: 180–182°C; c 0.1, MeOH) : 204.5 (3.62)  spectroscopic data, see Table m/z 343 [M+Na]+; positive HRESIMS m/z 359.1623   Figure ; UV (MeO) Figure ; IR  Figure .1 and 2 were obtained in MeOH at room temperature. Crystallographic data were collected at 100 K on a Bruker APEX DUO diffractometer with APEX II CCD, using CuKα radiation. All calculations were performed using the SHELXS-97 program and refined by full-matrix least-squares refinements based on F2 with SHELXL-97. The absolute configurations of 1 and 2 were analyzed using Hooft methods. Crystallographic data for the reported structures have been deposited with the Cambridge Crystallographic Data Center as supplementary publication deposition number CCDC 1475236 for compound 1 and CCDC 1475237 for compound 2. Copies of these data can be obtained free of charge from the Cambridge Crystallographic Data Center via http://www.ccdc.cam.ac.uk/data_request/cif.Colorless crystals of 1: C19H28O4, M = 320.41, orthorhombic, size 0.95 × 0.70 × 0.50 mm3, a = 7.9425 (2) Å, b = 10.8992 (2) Å, c = 19.3930 (4) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1678.79 (6) Å3, T = 100(2) K, space group P212121, Z = 4, μ (CuKα) = 0.701 mm−1, 9417 reflections measured, 2878 independent reflections (Rint = 0.0301). The final R1 values were 0.0340 (I > 2σ (I)). The final wR (F2) values were 0.0849 (I > 2σ (I)). The final R1 values were 0.0340 . The final wR (F2) values were 0.0850 . The goodness of fit on F2 was 1.139. Flack parameter = 0.12 (16). The Hooft parameter is 0.11(4) for 1168 Bijvoet pairs.Crystal data for 2: C19H28O4, M = 320.41, Monoclinic, size 0.970 × 0.380 × 0.260 mm3, a = 7.8489 (6) Å, b = 10.8350(8) Å, c = 10.2850 (8) Å, α = 90°, β = 104.762 (2)°, γ = 90°, V = 845.79(11) Å3, T = 100(2) K, space group P21, Z = 2, μ (CuKα) = 0.696 mm−1, 8987 reflections measured, 2924 independent reflections (Rint = 0.0362). The final R1 values were 0.0300 (I > 2σ (I)). The final wR (F2) values were 0.0807 (I > 2σ (I)). The final R1 values were 0.0300 . The final wR (F2) values were 0.0807 . The goodness of fit on F2 was 1.089. Flack parameter = 0.06 (5).Crystal data for A. thaliana seeds were surface-sterilized with ethanol  for 2 min and sodium hypochlorite  for 2 min, and then rinsed three times with sterile distilled water. The surface-sterilized seeds were cold-stratified for 3 days at 4°C before use. The seeds were then sown on MS agar plates that contained 0.4% gellan gum  and 1% sucrose. These glass Petri dishes (9 cm) were placed vertically for growth, and the conditions of the growth chamber were 23/18°C, a 12/12-h light/dark cycle, photosynthetic photon flux density of 150 μM·m−2·s−1, and relative humidity of 65%.1–5 and 7–9 were prepared using methanol, and that of compound 6 was prepared using dimethylsulfoxide (DMSO). After the sterilized MS medium had been cooled to 50°C, tested compounds were added to the medium to obtain the final serial concentrations. To assess the toxic effects of methanol or DMSO, MS medium containing 0.8% MeOH or DMSO (v/v) was used as a control. Three replicates were set for each treatment; in each replicate, 20 surface-sterilized seeds were sown equidistantly on MS medium. Subsequently, Petri dishes were sealed with Parafilm to retard moisture loss and then placed in the growth chamber. The germination rate of seeds was determined after 7 days, when over 95% of the control seeds had germinated, using emergence of the radicle (≥ 1 mm) as the index of germination. The seed germination inhibition (IG) was evaluated using the following equation: IG% = (1−NT/N) × 100. NT is the number of germinated seeds for each treatment and N is the number of seeds used in the bioassay.The phytotoxic effects of the nine compounds were evaluated as described by Zheng et al., with minor modifications  was calculated using the following equation: IR% = (1−T/C) × 100. T is the average root length (cm) of treated seeds and C is the average root length (cm) of the control. Seeds that produced a radicle but no coleoptile were scored as zero. To detect root death, roots of A. thaliana were stained with 5 μg/mL fluorescein diacetate  for 5 min, and then rinsed three times with MS liquid medium. After staining and rinsing, the roots were observed under a confocal laser scanning microscope . FDA fluorescence decreased as the dye leaked from dead cells.To test the effects of compounds L. cymbulifera was collected in Zhongdian, Yunnan Province, China, in August 2016. The plants (ca. 60–80 cm in height) were randomly collected and carefully uprooted, and the rhizosphere soil was shaken off the roots. The soil was picked and crushed, and residues were then removed with a sieve (30 mesh). Three replicated sieved soil samples (100 g each) were stored at 75% MeOH (300 mL) for 3 days at room temperature, and then extracted ultrasonically at 75°C for 60 min. The extracts were filtered and concentrated in a vacuum and was then dissolved in MeOH (10 mL). The solution was centrifuged at 12,000 rpm for 10 min, the supernatant was passed through a 0.45 μm nylon membrane filter and then analyzed by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS).The rhizosphere soil of The LC analysis was carried out using an Agilent 1290 Infinity Series UHPLC system comprising a quaternary pump , an autosampler , a column compartment , and a DAD. Samples were separated on a Phenomenex Kinetex C18 column  at room temperature. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B) and the elution gradient was set as follows: 32% B (0 min), 38% B (12 min), 85% B (15 min), 95% B (18 min). The mobile phase flow rate was 350 μL/min and the injected volume was set at 2 μL of standard and 5 μL of MeOH extracts of rhizosphere soil.n experiments, a quadrupole time-of-flight high-resolution mass spectrometer  was connected to the UHPLC instrument via an ESI interface. The data were acquired using Mass Hunter workstation software. Detection was performed in positive ESI mode and the full scan mass range was set from m/z 100 to m/z 700. The MS parameters were optimized as follows: the fragment voltage was set at 135 V; the capillary was set at 3500 V; the skimmer was set at 65 V; and nitrogen was used as the drying  and nebulizing (25 psi) gas.For the LC-ESI-MS1–9 in the rhizosphere soil samples of L. cymbulifera was undertaken by UHPLC-MS. Identification of potential phytotoxins was performed by comparing the retention times and MS/MS data with those of standards. Quantification of 3 in the rhizosphere soil was also undertaken using the same UHPLC method, with the isolated authentic sample as an external standard. Samples were also prepared in the same way as rhizosphere soil sample described above. For quantification, a calibration curve for 3 was prepared. Triplicate injections were carried out at four concentrations , and standard curves were constructed by the linear regression method. The equation and correlation coefficient obtained from the linearity study for 3 were as follows: y = 2.492x+21.1673 (r2 = 0.9997).Identification of compounds 50) were calculated using SPSS.Each treatment was conducted with three replicates in a completely randomized design. The data on the inhibition of seed germination and root elongation are expressed as mean ± standard deviation (SD). The values of effective concentration producing 50% inhibition (EC1 (680 mg), 2 (920 mg), 3 (960 mg), 4 (167 mg), 5 (158 mg), 6 (210 mg), 7 (480 mg) 8 (220 g), and 9 (950 mg) were isolated from the MeOH extracts of the air-dried roots of L. cymbulifera  was obtained as optically active colorless cubic crystals from MeOH. Its molecular formula C19H28O4 was determined on the basis of the positive HRESIMS at m/z359.1627 , corresponding to six degrees of unsaturation. The IR spectrum indicated the presence of hydroxyl (3386 cm−1) and lactone (1778 cm−1) groups. The 13C NMR , 129.6 (C-7), and a carboxyl at δC 180.3 (C-18) were occupied with two degrees of unsaturation. Thus, the remaining four degrees of unsaturation indicated that 1 is a compound with four rings. Upon careful comparison of the NMR data of 1 with furanoeremophilan-10β-ol (3) , 0.79 , and 1.30 , which are characteristic of eremophilane sesquiterpenes, were observed. These data suggested that compound 1 is an eremophilane sesquiterpene derivative with the skeleton with 19 carbons.ymirin A  was obtaymirin A  was obta1H-1H COSY spectrum, the cross peaks between δH 5.70  and δH 3.77 suggested one free hydroxyl group link to C-12  and 1.30  were located at C-19 and C-13, respectively  was located at C-10 due to the significant HMBC correlations from OH-10 to C-1. In addition, carboxyl at δC 180.3 (C-18) indicated a lactone moiety, which linked to C-11 through an ester bridge to occupy the last degrees of unsaturation. Thus, the planar structure of 1 was thus identified as shown in Figure 1 was definitively determined to be 4S, 5R, 10S, 11R, 12S, 17S, and it was named ligulacymirin A. It is noteworthy that 1 is a novel eremophilane derivative with the skeleton with 19 carbons featuring an unusual 6/6/6/5 ring system.In the 2) showed the same molecular formula, C19H28O4, as ligulacymirin A. The 1D and 2D NMR spectra of 2 closely resemble those of 1, clearly revealing that the planar structure of 2 was the same as that of 1       -3,6-dihydroxy-1-menthene (9) , given that the absence of these two moieties caused a noticeable reduction in activity . Similarly, upon comparing the inhibitory activity between compounds 6 and 7, it is possible that 8-OH weakens this activity. Furthermore, compound 3 showed stronger activity than compounds 6 and 8, revealing that the furanoeremophilane-type sesquiterpenes are more active than the eremophilanolide-type ones, and simple eremophilane-type ones show the weakest activities. In addition, compounds 1 and 2 showed almost the same inhibitory activities against seed germination , indicating that their stereochemistry should not influence their activity. Together, these results suggest that the basic eremophilane structures, as well as the co-existence of the tri-substituted furan ring and the OH-10, appeared to be important for phytotoxicity.The above results indicate that compounds  Figures , 4, 5, t1–9 were released from the roots of L. cymbulifera into the surrounding rhizosphere soil, rhizosphere soil samples were collected, extracted with MeOH, and then analyzed by UHPLC-MS under the conditions described above. Compounds 1–9 in the rhizosphere soil samples were readily identified by comparing their retention times and MS/MS data with the isolated authentic sample standards  and B (2) were isolated from L. cymbulifera roots for the first time and their structures were found to differ from those of other known eremophilane sesquiterpenes  including two novel eremophilane sesquiterpene derivatives (1 and 2) were isolated from the roots of L. cymbulifera. Compounds 3, 5, and 6 exhibited significant phytotoxic activities, while 3 was the most phytotoxic chemical in L. cymbulifera. The average content of compound 3 in rhizosphere soil was 3.44 μg/g. These results indicate that terpenoids in L. cymbulifera roots might be released into the surrounding rhizosphere soil as phytotoxins. These phytotoxic terpenoids would synergistically interfere with the germination and root elongation of neighboring plants to help L. cymbulifera gain an advantage in the particular habitat. Additional studies will be performed to obtain a better understanding of the mechanisms of action associated with these phytotoxic terpenoids in the roots of L. cymbulifera.In conclusion, nine terpenoids , Technological Leading Talent Project of Yunnan (2015HA020), and National Natural Science Foundation of China (31401313).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Butyricicoccus desmolans and Clostridium cadaveris have previously been reported for steroid-17,20-desmolase and 20β-hydroxysteroid dehydrogenase (HSDH) activities that are responsible for androstane formation from cortisol; however, the genes encoding these enzymes have yet to be reported. In this work, we identified and located a gene encoding 20β-HSDH in both B. desmolans and C. cadaveris. The 20β-HSDH of B. desmolans was heterologously overexpressed and purified from Escherichia coli. The enzyme was determined to be a homotetramer with subunit molecular mass of 33.8 ± 3.7 kDa. The r20β-HSDH displayed pH optimum in the reductive direction at pH 9.0 and in the oxidative direction at pH 7.0–7.5 with (20β-dihydro)cortisol and NAD(H) as substrates. Cortisol is the preferred substrate with mK, 0.80 ± 0.06 μM; maxV, 30.36 ± 1.97 μmol·min−1; catK, 607 ± 39 μmol·μM−1·min−1; catK/mK, 760 ± 7.67. Phylogenetic analysis of the 20β-HSDH from B. desmolans suggested that the 20β-HSDH is found in several Bifidobacterium spp., one of which was shown to express 20β-HSDH activity. Notably, we also identified a novel steroid-17,20-desmolase-elaborating bacterium, Propionimicrobium lymphophilum, a normal inhabitant of the urinary tract.Members of the gastrointestinal microbiota are known to convert glucocorticoids to androstanes, which are subsequently converted to potent androgens by other members of the gut microbiota or host tissues.  The desAB genes are annotated as “transketolase” N-terminal and C-terminal subunits  . Krafft irements . Howeverirements  identifisubunits . The twosubunits . A corti20α-HSDH . In thes20α-HSDH .Clostridium cadaveris and Butyricicoccus desmolans (formerly Eubacterium desmolans)  and C. cadaveris  became available, prompting us to determine whether these anaerobic bacteria encode the desAB genes in addition to an NAD(P)H-dependent dehydrogenase encoding 20β-HSDH.Steroid-17,20-desmolase activity has also been reported in smolans) , 11. Unlactivity . FollowiB. desmolans ATCC 43058 and Bifidobacterium scardovii ATCC BAA-773 were purchased from ATCC. C. scindens ATCC 35704 was maintained as −80°C glycerol stocks in our laboratory. Propionimicrobium lymphophilum ACS-093-V-SCH5 was obtained from the Culture Collection, University of Götesborg, Sweden. Escherichia coli DH5α (turbo) competent cells were from New England Biolabs  and E. coli BL21-CodonPlus(DE3) RIPL was purchased from Stratagene . The pET-51b(+) vector was obtained from Novagen . Restriction enzymes were purchased from New England Biolabs; QIAprep Spin Miniprep kit was obtained from Qiagen . Isopropyl β-D-1-thiogalactopyranoside was purchased from Gold Biotechnology . Strep-Tactin resin was purchased from IBA GmbH . Steroids were purchased from Steraloids . Amicon Ultra-15 centrifugal filter units with 30 and 50 kDa molecular-mass cutoffs were obtained from Millipore . All other reagents were of the highest possible purity and were purchased from Fisher Scientific .B. desmolans ATCC 43058, B. scardovii ATCC BAA-773, and C. scindens ATCC 35704 were cultivated in supplemented brain heart infusion (BHI) broth, containing 5 g l−1 of yeast extract. P. lymphophilum ACS-093-V-SCH5 was grown in Gifu anaerobic medium. Steroids were dissolved in methanol and added to the sterilized medium at a concentration of 50 μM, unless otherwise indicated; the concentration of methanol (v/v) per culture was kept at 0.5%. The medium was then seeded with 0.1 ml of a pure bacterial culture and incubated at 37°C for 2 days. After incubation, the products were extracted by vortexing the culture media with 2 vol of ethyl acetate for 1–2 min and then the organic phase was recovered. The organic phase was evaporated under nitrogen gas. The residue was dissolved in 50 μl methanol and analyzed using HPLC  equipped with a C-18 analytical column . The mobile phase consisted of acetonitrile/water with 0.01% formic acid and the flow rate was maintained at 0.2 ml·min−1. A DAD detector was used at a wavelength of 254 nm. Peak retention times and peak areas of samples were compared with standard steroid molecules.B. desmolans ATCC 43058 was cultivated in BHI broth in the presence or absence of 50 μM cortisol until mid-log phase. Cultures were centrifuged at 16,000 g for 5 min and the pellet suspended in RNALater™ (Ambion) overnight at 4°C followed by centrifugation and storage at −70°C until further processing. Total RNA was isolated using the Ribopure bacteria kit (Ambion) according to the manufacturer’s instructions, including the DNase step. One microgram of total RNA was converted to cDNA using the Advantage RT-for-PCR kit (Clontech) with random hexamer primers. Intergenic PCR was performed using TITANIUM Taq PCR kit (Clontech) with oligonucleotides synthesized by Integrated DNA Technologies, Inc. . All primers used in this study are listed in supplemental Table S1. The mRNA start-site was determined by 5′-RACE PCR using the SMARTer RACE PCR kit (Clontech) according to the manufacturer’s instructions with some modifications. RACE PCR products were purified from 1.5% agarose gels by GENECLEAN spin kit according to the manufacturer’s instructions (Bio 101) and cloned into a TA vector. The plasmid DNA was sequenced to identify the mRNA start sequence .B. desmolans ATCC 43058 genomic DNA was extracted using the Fast DNA isolation kit from Mo-Bio  according to the manufacturer’s protocol. Oligonucleotide primers used for amplifying the 20β-HSDH gene are reported in supplemental Table S1. The coding sequence for 20β-HSDH was amplified using the Phusion high-fidelity polymerase (Stratagene) and cloned into a pET-51b vector using BamHI and HindIII restriction sites. Recombinant plasmid was transformed into chemically competent E. coli DH5α cells via the heat stock method, plated, and grown for 16 h at 37°C on lysogeny broth (LB) agar plates supplemented with ampicillin (100 μg/ml). A single colony from each transformation was inoculated into LB medium (5 ml) containing ampicillin (100 μg/ml) and grown to saturation. The cells were subsequently centrifuged  and plasmids were extracted from the resulting cell pellet using the QIAprep Spin Miniprep kit (Qiagen). The foreign DNA inserts in the recombinant plasmids were sequenced to confirm the correctness of the gene sequence .E. coli DH5α cells were transformed into E. coli BL-21 CodonPlus (DE3) RIPL chemically competent cells by the heat shock method and grown overnight at 37°C on LB agar plates supplemented with ampicillin (100 μg/ml) and chloramphenicol (50 μg/ml). After 16 h, five isolated colonies were used to inoculate 10 ml of fresh LB medium supplemented with antibiotics and grown at 37°C for 6 h with vigorous aeration. The precultures were then added to fresh LB medium (1 liter), supplemented with the same antibiotics at the same concentrations, and grown with vigorous aeration at 37°C. At an OD600 of 0.3, isopropyl β-D-1-thiogalactopyranoside was added to each culture at a final concentration of 0.1 mM and the temperature was decreased to 16°C. Following 16 h of culturing, cells were pelleted by centrifugation  and resuspended in 30 ml of binding buffer . The cell suspension was subjected to four passages through an EmulsiFlex C-3 cell homogenizer  and the cell lysate was clarified by centrifugation at 20,000 g for 30 min at 4°C.For protein expression, the correct recombinant plasmids extracted from the 3 M−1·cm−1) (The recombinant 20β-HSDH (r20β-HSDH) was then purified using Strep-Tactin® resin (IBA GmbH) as per the manufacturer’s protocol. The recombinant protein was eluted using an elution buffer composed of 20 mM Tris-HCl, 150 mM NaCl, 20% glycerol, 10 mM 2-mercaptoethanol (pH 7.9), and 2.5 mM desthiobiotin. The protein purity was assessed by SDS-PAGE and protein bands were visualized by staining with Coomassie brilliant blue G-250. The protein concentrations were calculated based on the molecular mass (41.3 kDa) and computed extinction coefficient (31.6 × 10−1·cm−1) .Gel filtration chromatography was carried out on Superdex 200 analytical column  attached to Akta FPLC  at 4°C. The eluted protein from the Strep-tag column was concentrated and loaded on the column with a buffer composed of 50 mM Tris-Cl, 300 mM NaCl, 10% glycerol, 10 mM 2- mercaptoethanol, and 0.1% Triton X-100 (pH 7.5). The native molecular mass of the protein was calculated by peak retention volume of protein to retention volume of standard proteins. Eluted fractions were visualized by staining with Coomassie brilliant blue G-250. The activity of the enzyme in the eluted fraction was calculated as described below in the standard enzyme assay section.ε = 6,220 M−1·cm−1). Linearity with respect to time and enzyme concentration was determined. The standard reaction mixture contained 50 mM phosphate buffer (pH 7.5), 50 μM of cortisol, 150 μM NADH, 0.05 μM enzyme, and buffer to a final volume of 0.5 ml. The reaction was started by the addition of the enzyme. The initial velocity of enzyme-catalyzed reactions was plotted against the substrate concentrations, and the kinetic parameters were estimated by fitting the data to the Michaelis-Menten equation by the nonlinear regression method using the enzyme kinetics module in GraphPad Prism .The 20β-HSDH activity was measured aerobically by monitoring the conversion of cortisol into 20β-dihydrocortisol using a continuous spectrophotometric method, monitored by measuring the oxidation of NADH at 340 nM , 50 mM sodium phosphate (pH 6.5–7.5), 50 mM Tris-Cl (pH 8–9), and 50 mM glycine-NaOH (pH 10–11). In order to determine the optimal pH in the reductive direction, purified recombinant 20β-HSDH enzyme at a final concentration of 0.05 μM was added to 50 μM cortisol and 150 μM NADH in 500 μl buffer. The oxidation of NADH was measured continuously at 340 nM for 5 min by spectrophotometry and the initial activities were calculated. For the oxidative reaction, 0.05 μM of enzyme was added to 50 μM 20β-dihydrocortisol and 150 μM NADB. desmolans ATCC 43058. Seventy sequences with an identity cutoff of 30% with the 20β-HSDH were selected and aligned using MUSCLE v3.8.31 (Candidate protein sequences for phylogeny reconstruction were retrieved from NCBI after BLAST using as query the short-chain dehydrogenase/reductase (SDR) family oxidoreductase WP_051643274.1) from  v3.8.31 . The res v3.8.31  using th v3.8.31  was used43274.1 fC. scindens ATCC 35704 and B. desmolans ATCC 43058  from C. scindens ATCC 35704 was used to query the nonredundant protein database (BLASTP). DesA is annotated in the thiamine-pyrophosphate-dependent enzymes superfamily domain, particularly N-terminal subunit transketolase (EC 2.2.1.1). The highest hits  were against amino acid sequences encoded by C. cadaveris (WP_027640052) and B. desmolans (WP_031476417) with 82% identity/89% similarity and 81% identity/88% similarity, respectively . A deduced amino acid sequence (WP_016455356.1) from the taxon, P. lymphophilum ACS-093-V-SCH5, shared 75% identity/86% similarity with DesA from C. scindens ATCC 35704.The desA revealed what appeared to be conserved desB gene in addition to an open reading frame named WP_051643274.1, whose gene product consisted of 282 amino acids containing the canonical Gly-X-X-X-Gly-X-Gly N-terminal pyridine nucleotide-binding motif typical of SDR family enzymes  (desC from C. scindens ATCC 35704 encodes a 20α-HSDH with annotation in the L-threonine dehydrogenase family (EC 1.1.1.103) and B. desmolans and C. cadaveris are reported to express 20β-HSDH  , 18. TheEC 1.1.1.3 and B. desEAB genes are coexpressed as a polycistronic operon, we isolated total RNA from mid-log phase B. desmolans and designed oligonucleotides  spanning the intergenic regions between desE-desA and desA-desB. Amplification from the cDNA demonstrates that the 20β-HSDH (desE) is coexpressed with the putative steroid-17,20-desmolase (desAB) . The transcription initiation site was determined by SMARTER-RACE PCR to be an adenine residue at position −144. The putative ribosome-binding site (AGGAGGA) was identified starting at position −9. Conserved upstream elements (−149 to −182 and −183 to −196) were identified in alignments between the region between lysR and desA in C. scindens ATCC 35704 and lysR and desE from C. cadavaris AGR2141 and B. desmolans ATCC 43058 .To determine whether the To determine whether WP_051643274.1 encodes 20β-HSDH, the gene was cloned into pET51b(+) expression vector. The N-terminal streptavidin-tagged recombinant protein was purified by Strep-Tactin® affinity chromatography. The recombinant 20β-HSDH protein was resolved as a single band on SDS-PAGE with subunit molecular mass of 33.8 ± 3.7 kDa   . The 20βP < 0.05).The pH optima of the 20β-HSDH enzyme was determined in the oxidative and reductive directions by spectrophotometry. Steroid reduction was observed within the pH range 4.0–10, retaining 19.9 ± 3.1% of maximal activity at pH 4.0 and 29.5 ± 3.7% of maximal activity at pH 10.0 , the oxidative direction represented only 3.9% of the maximal activity in the reductive direction (20.8 ± 1.2 μmol·min−1). These results indicate that under in vitro conditions, the enzyme catalyzes reductive reactions far more readily than oxidative reactions.Similarly steroid oxidation, as measured by the conversion of NADand 9.25 . Strikin+ or NADP+ was added as cofactor, as expected  is the preferred pyridine nucleotide. Nevertheless, the purified enzyme (50 nM) catalyzed the net reduction of cortisol with either NADH or NADPH as the cosubstrate after 12 h incubation; however, formation of 20β-dihydrocortisol was not detected if NADexpected . Km and urk plot . The calThe substrate-specificity of recombinant 20β-HSDH is shown in desE, the sequences formed three major clusters (Mycobacteriumspp. (cluster 3), followed by a cluster inhabited by anaerobic gut bacteria inhabiting the gastrointestinal tracts of ruminants and monogastric fermenters (cluster 2). Several of these genes are found in succinate-producing gram-negative ruminants . Several others belong to members of Clostridium cluster XVIa, including species of Dorea and Blautia producta, and Clostridium cluster IV, including Clostridium leptum. Notably, a conserved active-site tetrad, NSYK, was identified in the majority of sequences within each cluster, with the exception of B. desmolans and C. cadaveris, whose active-site residues, according to the multiple-sequence alignment, are DSYK , and most closely related to the 20β-HSDH from B. desmolans. Examination of their genomic location revealed that the two putative 20β-HSDH encoding genes share 91% amino acid sequence similarity, suggesting the likelihood of gene duplication  convert 7-oxo-bile acids generated by other gut bacteria (2) regulate the flux of bile acids into the 7α-dehydroxylation pathway by reversible oxidation-reduction of the 7α-hydroxy group  –31. The P. lymphophilum, which may be members of the normal urinary microbiota . Mycobacteria encode numerous genes involved in the catabolism of host fatty acids, cholesterol, and oxysterols for the generation of energy and mycolic acid (M. tuberculosis, which is suggested to be involved in steroid metabolism (M. tuberculosis SDR-family proteins. It is possible that the proteins identified in our phylogeny are utilized by mycobacteria to metabolize host sterols or perhaps environmental sterols from plants and animal material; however, this remains the subject of future investigation.Finally, our phylogenic analysis of lic acid , 37, butlic acid . A 3α,20tabolism . Rv2002 tabolism  or in ou


Complement C3a is an important protein in innate and adaptive immunity, but its specific roles in vivo remain uncertain because C3a degrades rapidly to form the C3a-desArg protein, which does not bind to the C3a receptor and is indistinguishable from C3a using antibodies. Here we develop the most potent, stable and highly selective small molecule modulators of C3a receptor, using a heterocyclic hinge to switch between agonist and antagonist ligand conformations. This enables characterization of C3 areceptor-selective pro- vs. anti-inflammatory actions in human mast cells and macrophages, and in rats. A C3a receptor-selective agonist induces acute rat paw inflammation by first degranulating mast cells before activating macrophages and neutrophils. An orally administered C3a receptor-selective antagonist inhibits mast cell degranulation, thereby blocking recruitment and activation of macrophages and neutrophils, expression of inflammatory mediators and inflammation in a rat paw edema model. These novel tools reveal the mechanism of C3a-induced inflammation and provide new insights to complement-based medicines. Complement C3a is an important protein in innate and adaptive immunity, but its roles in vivo are unclear. Here the authors develop novel chemical agonists and antagonists for the C3a receptor, and show that they modulate mast cell degranulation and inflammation in a rat paw edema model Most PPIs are currently thought to be ‘undruggable’ using conventional small drug-like molecules3. A significant challenge in chemical biology is to rationally downsize a protein to an equipotent small molecule that could be cheaper to manufacture, easier to structurally and functionally modify, non-immunogenic, and likely more stable and more orally active4. Complement C3a is a ~ 9 kDa helix bundle inflammatory protein that binds to a ~ 100 kDa G protein-coupled receptor called C3aR expressed on the cell surface. C3a is thought to be important in mediating inflammatory responses to infection and injury6. C3a concentrations are reportedly elevated during inflammatory diseases7, recombinant C3a reportedly induces hypertension and delayed neutrophilia in rats over 24 h8, while sustained activation of C3aR vs knockouts support a role in allergies9, asthma10, arthritis11, sepsis12, lupus13, diabetes14, ischemia-reperfusion injury15, obesity and metabolic dysfunction16. However, the actions of C3a in vivo remain uncertain because C3a is synthesized at the cell surface and very rapidly degraded by extracellular carboxypeptidases, which cleave off the C-terminal residue Arg to form C3a des-Arg6 that does not bind to C3aR and has a completely different pharmacological profile. Moreover, commercially available antibodies used in many in vivo studies do not discriminate between C3a and C3a-desArg15. Thus, most claims for detection of C3a in vivo or for properties of exogenous C3a administered in vivo may be compromised. Synthetic agonists that act through C3aR, but do not degrade rapidly like C3a, could aid the characterization of C3a biology in vivo and may be valuable immunostimulants or antimicrobial agents17, while metabolically stable and orally active antagonists may be valuable new anti-inflammatory agents with therapeutic potential18. No drug-like small molecule agonists or antagonists of C3aR have been found yet with high potency, selectivity, metabolic stability and oral bioavailability for interrogating C3a-mediated functions in vivo4.Protein-protein interactions (PPIs) mediate most physiological processes and involve large interacting protein surfaces that are proving to be extremely difficult to mimic, or interfere with, using small molecules in vivo4. These compounds were built from the C-terminal arginine of C3a and displayed the same capacity in vitro as human C3a to induce calcium release and the expression of inflammatory cytokines in human macrophages4. While C3a is degraded within minutes in plasma, these small molecule proxies for C3a are stable in plasma and may be useful with some modifications as pharmacological tools to probe C3a properties in vivo. We have also probed how the relative hydrogen-bonding potential of different heterocycles contributes to binding affinity19 and used theoretical calculations to predict barriers to rotation, and hence the probable population of different conformers which was then related to functional activity20. Here we have significantly extended that work by incorporating different heterocycles to switch from agonist to antagonist conformations, culminating in the most potent small molecule activators and inhibitors known for the C3a receptor in vitro and in vivo. We characterize their solution structures, their activities on human mast cells and macrophages, their target specificity, and their effects on C3a-dependent innate immune responses in a rat model of acute inflammation. This is an important advance in (i) identifying the actions of the C3a protein, which degrades rapidly in biological fluids, (ii) rationally developing small molecule agonists and antagonists of C3aR for use in vivo in physiology and disease, and (iii) identifying the temporal sequence of cellular immune responses to activation of C3aR in rodents. This novel approach to downsizing a protein to conformationally restricted small molecules may lead to complement-based medicines and encourage similar approaches to modulate other protein–protein interactions.Recently, we described an approach to rationally downsizing the 77 residue human complement C3a protein to small molecule agonists (MW < 500) equivalent in size to just the last 3–4 amino acid residues of the C-terminus of C3a21. Here we report , which is a partial agonist at sub-µM concentrations in inducing histamine release from human LAD2 mast cells  were measured from molecular models. Conversely, agonists 1, 2 and 3 showed no amide-NH….H or CH3-heterocycle cross-peaks . We attribute this conformational switching to an attractive S…O interaction between the thiazole/thiophene sulfur and the amide carbonyl oxygen in 4–6, fixing the carbonyl oxygen in such a conformation that evidently facilitates antagonist activity. This kind of attractive S…O interaction has been shown in crystal structures of other thiazoles and thiophenes22. On the other hand, in 1–3 there is no attractive heterocycle–N…O–amide carbonyl interaction, instead the opposite amide orientation is preferred and this conformation clearly favors agonist activity. These highly original findings demonstrate that key functional activities  and found no binding , which do not express the C3a receptor23 , consistent with their specificity and on-target action on C3aR   Fig. . The dec3, designated BR103) and antagonist  in vivo, the in vitro stability of these compounds were examined in rat plasma and in rat liver microsomes. These assays provide a measure of stability towards degradation by proteolytic and cytochrome P450 enzymes, respectively. Compounds 3 and 6 remained intact after exposure to plasma, whereas C3a degraded within minutes in plasma , a property previously attributed to cleavage by circulating carboxypeptidases26. Furthermore, compounds 3 and 6 were much more stable than C3a in liver microsomes, known to be rich in cytochrome P450 enzymes, as reflected by their lower intrinsic clearance rates . These results suggested that compounds 3 and 6 could potentially be more stable and more bioavailable in vivo than C3a or its peptide analogues, and thus represent important and novel small molecule leads for modulating C3aR biology in vivo.In preparation for evaluating the most potent agonist  to characterize for the first time a C3aR-induced innate immune response in vivo in rats. An acute inflammatory response and edema can be induced in rodents by an intraplantar injection of substances such, as λ-carrageenan into, the rear paws leading to leukocytosis and a hyperemic response, which produces localized swelling27. Similarly, we aimed here to investigate if selective activation of C3aR in vivo in rats would induce an acute inflammatory edema and to characterize the innate immune responses over a 24 h time course , induced an acute inflammatory edema, similar to that shown by λ-carrageenan and other irritants, with localized paw swelling, mast cell activation, histamine and tryptase release, myeloperoxidase (MPO) activation, leukocyte recruitment and expression of various proinflammatory cytokines in a time-dependent manner . The BR103-induced paw swelling peaked at 0.5 h and returned to baseline after 24 h . Histological evaluation at the various time points after BR103 administration suggested that resident mast cell activation and degranulation  was associated with paw inflammation, which preceded the infiltration and activation of other leukocytes such as inflammatory macrophages and neutrophils. The BR103-induced peak paw swelling at 0.5 h was concurrent with an increased number of activated mast cells, together with increased histamine secretion and decreased tryptase-positive cells in paw tissue compared to vehicle-treated animals . This increase in mast cell activation and degranulation at 0.5 h was a temporal response, since activated mast cells subsided at 6 h and 24 h time points after C3aR activation .A single intraplantar injection of the C3aR agonist, BR103 . Further, BR103 also increased infiltration and activation of other granulocytes and leukocytes such as neutrophils compared to vehicle-treated animals . However, activation of neutrophils (measured as increased activity of MPO) and accumulation of other granulocytes and leukocytes were not observed until 6 h after BR103 administration , suggesting a temporal involvement following recruitment to the site of inflammation.Interestingly, BR103 also induced a temporal increase in infiltration of inflammatory ED15 h Fig.  without als Fig. . Furtheals Fig. . Howeveion Fig. , sugges6, we further characterized changes in inflammatory gene expression in rat paw tissue at 0.5, 6 and 24 h after BR103 administration . BR103 induced the upregulation of many proinflammatory genes, such as Il1β, Il6, Tnf, Ccl3, and Mcp1, compared to vehicle-treated animals . Most of these C3aR-induced inflammatory markers are early response genes, peaking at 0.5 h and slowly subsiding over 6 h and 24 h time points after BR103 administration .Moreover, since both immune and non-immune cells have been implicated in C3aR-induced innate immune responsesion Fig. . BR103 als Fig. . Most oion Fig. .6. Verifying that our novel small molecule C3aR antagonists have in vivo functionality would furnish useful therapeutic tools for regulating acute and chronic inflammatory disease conditions. BR111 (compound 6) was assessed in vivo in the above rat model of acute inflammatory edema induced by C3aR agonist, BR103. When given as an oral pretreatment, BR111 strongly inhibited acute rat paw edema, induced 2 h later by i.pl. injection of BR103 . Orally administered BR111 inhibited BR103-induced paw swelling at 30 min in a dose-dependent manner, suggesting specificity and competition for C3aR in vivo . Furthermore, this pre-treatment with BR111 attenuated BR103-induced acute inflammatory responses such as mast cell activation and degranulation  and secretion of histamine , in these rat paws at 0.5 h after agonist administration. BR111 also attenuated neutrophil activation at 6 h after BR103 administration . BR111 also prevented the increased infiltration of inflammatory ED1 macrophages at 0.5 h in paws compared to BR103-treated animals . These effects were observed together with the suppression of BR103-induced gene expression of proinflammatory cytokines and chemokines by oral BR111 treatment .Dysregulated C3aR expression and activation have been shown to be involved in various chronic inflammatory disease conditions103 Fig. . Orallyivo Fig. . Furtheion Fig.  and secine Fig. , in theion Fig. . BR111 als Fig. . These ent Fig. .Fig. 6A6), is a potent, selective, orally active antagonist of C3aR with beneficial anti-inflammatory properties in vivo. In summary, the data are consistent with C3aR being activated in vivo by BR103 (compound 3) and antagonized by BR111 and have helped to characterize for the first time the direct responses of selective C3aR activation and inhibition in vivo on acute innate immune responses.These results strongly suggest that the novel C3aR ligand, BR111 . Cromolyn is believed to prevent mast cell degranulation by stabilizing the cell membrane and it is currently used to treat various mast cell related diseases such as asthma, conjunctivitis, allergic rhinitis, and mastocytosis31. When rats were pretreated with cromolyn  there was a significant reduction in BR103-induced paw swelling at 0.5 h, almost back to baseline levels . Cromolyn treatment in vivo also attenuated BR103-induced increased activation  and degranulation  of mast cells together with reduced histamine expression  at 0.5 h in rat paws. This was followed by a reduced infiltration of inflammatory ED1+ macrophages at 0.5 h in rat paws compared to BR103-treated animals  and a reduced expression of early response inflammatory genes, including Il1β, Il6, Tnf, Ccl3, and Mcp1 at 0.5 h . Taken together, these findings indicate that the direct activation of C3aR in vivo by BR103 (compound 3) results in acute inflammatory edema and innate immune responses that are specifically initiated via a mast cell-dependent mechanism. Thus, C3aR on resident mast cells clearly has a key initiating and amplifying role in acute events such as degranulation, histamine and tryptase secretion, leading to paw inflammation, cytokine expression and other associated innate immune responses in vivo. Other key innate immune cells such as infiltrating macrophages and neutrophils are subsequently recruited to the site of inflammation and can drive a sustained immune response, but they do not seem to be major contributors to the early acute innate immune response induced in vivo in rats through C3aR activation.Finally, we investigated whether the C3aR agonist-induced acute inflammatory edema and immune response in vivo was specifically mediated by a mast cell-dependent mechanism or whether resident or infiltrating macrophages or neutrophils were contributors. Resident mast cells are generally localized at peripheral sites, including the skin, airways, gut, and other tissues. Rapid activation of mast cells has been shown to initiate inflammation through the release of both pre-formed and synthesized mediators, which induce increased vasopermeability, vasodilation, accumulation of inflammatory mediators, and chemotaxis of other immune cellsels Fig. . Cromolion Fig.  and degion Fig.  of mastion Fig.  at 0.5 als Fig.  and a r5 h Fig. . Taken 6. Collectively, they “complement” immune cells and antibodies to identify, tag, destroy, and eliminate pathogens and infected or damaged cells, and repair wounds6. If the inflammatory stimulus is not removed, complement activation becomes prolonged, or misdirected to healthy cells, causing or exacerbating chronic inflammatory or auto-immune diseases. Multiple pathways of complement activation produce C3a, which is thought to stimulate important immune and metabolic responses. Its human receptor, hC3aR is considered to be a key mediator of inflammatory disease6, but current small molecule ligands for hC3aR are not sufficiently drug-like to be effective probes for in vivo studies. Development of potent, selective, metabolically stable and bioavailable ligands for C3aR could be valuable new leads to a new class of therapies for treating complement-mediated diseases.Human C3a (hC3a) is one of ~ 30 complement proteins produced in blood or on cell surfaces through activation by infection or injury3, BR103) or antagonists . This chemical discovery has been then exploited to unravel new cell and in vivo biology, by examining the effects of potent and selective small molecule agonists that mimic C3a in activating mast cells, macrophages and neutrophils, and of potent small molecule antagonists that block the actions of both C3a and BR103 on these innate immune cells. These small molecule modulators were selective for C3aR, showing functional responses on human LAD2 mast cells, HMDM cells, and HEK293 cells that expressed C3aR but not on C3aR−/− HEK293 cells. They were also selective for C3aR over C5aR, and were metabolically stable chemical tools for use in animal models.Here we describe different heterocycles adjacent to a carboxamide that can be used to control ligand conformation, as established by 2D NMR spectroscopic techniques. We have demonstrated how this conformational change, triggered by a simple change of heteroatom, profoundly impacts on ligand function, resulting in potent agonists , and macrophage and neutrophil infiltration. All of these measures of acute inflammatory responses were inhibited by oral pretreatment of a potent C3aR antagonist delivered systemically to rats. An important mechanistic finding was that the mast cell stabilizer cromolyn, which blocks degranulation of mast cells but does not inhibit neutrophil or macrophage functions, was also able to inhibit agonist-induced paw inflammation in rats. C3a and its small molecule agonist mimics are able to activate a range of innate immune cells such as mast cells, macrophages and neutrophils. However, these results support the conclusion that it is the mast cell that initiates the acute innate immune response and that other immune cells that infiltrate damaged or infected tissues subsequently drive inflammation.32. They are special in containing as many as 500,000 secretory granules32, filled with numerous pre-formed and pre-activated immunomodulators32 that are released upon degranulation, but are also known to secrete inflammatory mediators in the absence of degranulation32. Although mast cells have been regarded as sentinels for infection and drivers of allergic and anaphylactic responses through IgE33, they are also emerging as potentially important contributors to chronic inflammatory diseases such as inflammatory bowel disease, irritable bowel syndrome, multiple sclerosis, arthritis, wound healing, graft rejection, angiogenesis, cancers, cardiovascular diseases, diabetes, obesity, and others28. Recent single-cell transcriptomic studies have identified new progenitor populations with mast-cell potential that are distinct from the neutrophil-monocyte lineage and that segregate early in hematopoietic development34. Transcriptomic analysis in isolated constitutive connective-tissue mast cells from skin, tongue, oesophagus, trachea, and the peritoneal cavity have identified distinct gene clusters for mast cells independent of other lymphoid and myeloid-cell populations35. These studies are revealing previously unappreciated mast-cell turnover rates and pre-activated secretory granule turnover in the periphery in the absence of tissue inflammation35. Human C3a is known to degranulate mast cells in vitro to release histamine and tryptase21, but evidence for the action of C3a in vivo on mast cells remains clouded. Our data for C3aR activation in rat paws by an agonist and blockade by an antagonist suggests that C3a is proinflammatory in vivo through its action on resident mast cells in the skin, and that mast cell degranulation is a key initiator of acute inflammation in the paw and a stimulator of recruitment of other immune cells  that are subsequently activated. Further studies using our novel ligands may help identify specific roles of mast cells in other distinct anatomical locations in health and disease, and in more chronic inflammatory diseases where mast cell turnover could be important in sustaining an inflammatory stimulus.Mast cells have been almost the forgotten innate immune cells until recently. They were identified over 100 years ago, are conserved across many species, yet their roles in immunity, defence and disease are still not completely defined36, but it is now emerging that many cell types, including cells of myeloid, lymphoid, and non-myeloid, non-lymphoid origin can generate C3a from transported C3 presented at the cell surface where C3 convertases are formed36. For example, activation of T cells induces the local expression of C3, factor B and factor D that results in the extracellular assembly of C3 convertase and cleavage to C3a that acts on the cell surface C3aR37. Rapid cleavage of the C-terminal Arg of C3a to C3a-desArg by carboxypeptidases may be an efficient defence mechanism to dampen an inflammatory response and restrict the action of C3a to the cell surface. Availability of the first truly potent, stable, selective, and bioavailable C3a agonists and antagonists reported here could be valuable new molecular tools for interrogating the sequence of cellular immune responses to C3aR activation in vivo in different pathological settings.Historically, C3a has been thought to be formed in the liver through proteolytic cleavage of serum complement C3This report is the culmination of attempts to downsize the 77-residue complement C3a protein to potent and selective small molecule modulators that are stable enough to be used in vivo as chemical tools to probe C3a-dependent biology. Unlike many protein–protein interactions, the bioactive effector region of C3a is localized to a small region around its C-terminal arginine. However, the C-terminal tetrapeptide segment of C3a alone has no detectable C3aR activity and it has been challenging to obtain a small molecule agonist with equal potency and functional selectivity as the C3a protein. Further, the rational development of a potent and C3aR-selective antagonist suitable for use in vivo was also a challenging goal. Here we have shown how to use different heterocycles to produce potent and selective C3aR ligands, exploiting a novel conformational switch by positioning the heterocycle adjacent to a carboxamide to change agonists into antagonists. This resulted in potent and selective agonists and antagonists that are very effective inflammatory modulators and valuable chemical biology tools when administered to rats. This approach to potent small molecule protein mimics exemplified here for one protein, might also be more generally applicable and transferable to other protein–protein interactions, where site-directed mutagenesis has identified hot spots responsible for protein function. It remains to be determined whether this approach also leads to small molecules with similarly potent and selective functional activities and selectivity for other protein targets.2O + 0.1% TFA, and solvent A was H2O + 0.1% TFA, at a flow rate of 40 mL min−1. Compounds were detected by UV spectroscopy and pure fractions were lyophilized. Analytical rpHPLC was used to assess compound purity . Standard conditions  were used for all compounds at a flow rate of 1 mL min−1. All final compounds were ≥ 95% pure by analytical HPLC.Preparative-scale reversed-phase HPLC (rpHPLC) separations were performed on a Phenomenex Luna C18 10 μm, 250 × 30.0 mm column. Standard conditions were used for elution of all compounds: 100% A to 100% B linear gradient over 20 min, followed by a further 10 min at 100% B, where solvent B was 90% MeCN, 10% H−1 using sodium formate clusters as an internal calibrant. The data were processed using Bruker Daltonics DataAnalysis 3.4 software. Mass accuracy was consistently better than ± 1 ppm.Electrospray ionization mass spectral measurements were obtained on a Micromass LCT spectrometer. High-resolution mass spectra (HRMS) measurements were obtained on a Bruker microTOF mass spectrometer equipped with a Dionex LC system (Chromeleon) in positive ion mode by direct infusion in MeCN at 100 μL h1H and 13C NMR spectra were recorded on Bruker Avance III HD 600 equipped with a cryoprobe or Varian 400 spectrometers at 298 K using deuterated solvents and were referenced to the residual solvent signal DMSO-d61H 2.50 ppm, 13C 39.51 ppm.  CDCl3 solutions were referenced to internal tetramethylsilane (TMS). The exact concentration of the compound for assay was confirmed by the quantitative NMR integration ‘PULCON’ experiment. Settings for all PULCON experiments: relaxation delay d1 = 30 s, 16 scans, 2 dummy scans, 90° pulse and temperature at 298 K. ROESY spectra were acquired using the standard Bruker pulse sequence roesyph with a spinlock pulse of 350 ms and relaxation delay 2 s.16 is Ac-cyclo--Phe-[Orn-Pro-dCha-Trp-Arg], synthesized by Boc chemistry on a 0.20 mmol scale using HBTU/DIPEA activation and in situ neutralization on Boc-l-Arg(Tos)-4-hydroxymethyl-phenylacetamidomethyl resin (PAM resin), cleavage and deprotection with HF and p-cresol (1 ml), cyclization with BOP and DIPEA in DMF38, or it was synthesized in solution as described39.All reagents were purchased from Sigma-Aldrich or Chem-Impex International Inc. C3a-desArg (cat. No. C3A16-N-25) was purchased from Jomar Life Research, Australia. All compounds were synthesized via solid- or solution-phase chemistry approaches. The C5a receptor antagonist 1–6 in Supplementary Figs. tR) measured under standard HPLC conditions, for all compounds used in this study are catalogued below.Supplementary Figs. Compound 1.1H NMR : δ 8.28 , 8.23 , 7.50 , 7.37–7.27 , 6.03 , 4.42–4.39 , 3.11–3.07 , 1.90–1.83 , 1.81–1.74 , 1.51–1.45 . 13C NMR : δ 173.5, 173.0, 160.4, 156.6, 149.5, 141.8, 128.7, 128.67, 128.66, 127.3, 127.2, 124.9, 53.6, 51.6, 40.3, 28.0, 25.3. HRMS calculated for C23H26N5O3S+ 452.1751, found 452.1750. HPLC tR 14.6 min.Compound 2.1H NMR : δ 8.14 , 7.54 , 7.39–7.33 , 7.33–7.25 , 5.94 , 4.39 , 3.14–3.06 , 2.68 , 1.86 , 1.76 , 1.53–1.43 . 13C NMR : δ 173.1, 168.4, 161.8, 156.6, 142.3, 141.8, 140.8, 128.7, 128.6, 127.2, 53.5, 51.3, 40.3, 28.2, 25.3, 12.2. HRMS calculated for C24H28N5O3S+ 466.1907, found 466.1907. HPLC tR 15.4 min.Compound 3.(BR103)1H NMR : δ 7.91 , 7.65 , 7.37–7.32 , 7.30–7.24 , 5.69 , 4.38 , 3.16–3.05 , 2.41 , 1.83 , 1.72 , 1.55–1.46 . 13C NMR : δ 173.4, 161.7, 156.8, 146.7, 140.3, 132.4, 128.7, 128.6, 127.1, 51.1, 49.4, 40.3, 28.5, 25.3, 10.6. HRMS calculated for C24H29N6O3+ 449.2296, found 449.2295. HPLC tR 11.5 min.Compound 4.1H NMR : δ 8.84 , 8.43 , 7.61 , 7.29–7.37 , 6.00 , 4.32–4.35 , 3.08–3.15 , 1.82–1.88 , 1.67–1.73 , 1.49–1.59 . 13C NMR : δ 176.9, 173.2, 160.0, 156.6, 143.9, 141.7, 134.6, 128.71, 128.67, 127.2, 53.8, 52.0, 40.3, 27.7, 25.4. HRMS: [MH]+ calc. for C23H26N5O3S+ 452.1751, found 452.1751. HPLC tR 14.2 min.Compound 5.1H NMR : δ 8.41 , 7.52 , 7.39–7.26 , 5.94 , 4.29 , 3.13–3.04 , 2.52 , 1.81 , 1.67 , 1.57–1.44 . 13C NMR : δ 173.1, 172.6, 161.4, 156.6, 154.9, 141.7, 128.7, 127.2, 125.5, 53.7, 52.2, 40.3, 27.5, 25.4, 17.0. HRMS calculated for C24H28N5O3S+ 466.1907, found 466.1907. HPLC tR 14.3 min.Compound 6. (BR111) 1H NMR : δ 8.60 , 7.73 , 7.58 , 7.37–7.22 , 6.79 , 5.83 , 4.32 , 3.16–3.04 , 1.85 , 1.70 , 1.61–1.46 . 13C NMR : δ 173.4, 161.2, 156.7, 153.0, 143.2, 137.8, 128.6, 128.5, 126.9, 51.9, 51.2, 40.3, 27.7, 25.4. HRMS calculated for C24H27N4O3S+ 451.1798, found 451.1798. HPLC tR 15.2 min.®-34 Serum Free Media  supplemented with StemPro®-34 nutrient, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM GlutaMAX, and 100 ng/mL human stem cell factor (SCF). Cells were hemi-depleted weekly and were not allowed to grow beyond a density of 0.5 × 106/mL. This cell line was authenticated by expression of tryptase and histamine granules. For the histamine assay, 1 × 104 cells per well were seeded in StemPro®-34 SFM in 96-well plates for 30 min before any treatments. For agonist studies, the cells were treated with compounds prepared in media for 30 min. For antagonist studies, the cells were pre-treated with compounds prepared in SFM for 30 min before addition of C3aR agonists for another 30 min. Cells were centrifuged  and cell-free supernatants were collected for measuring histamine. Histamine content was measured using the histamine EIA kit according to the manufacturer’s instructions . The readings were recorded on a FLUOstar Optima .LAD2 human mast cells  were cultured in StemPro+ monocytes were positively selected using CD14 MicroBeads 41. CD14+ monocytes were differentiated to HMDMs using 100 ng/mL of recombinant human macrophage colony-stimulating factor  for 7 days in Iscove’s modified Dulbecco’s media supplemented with 10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 µg/mL) and L-glutamine (2 mM). HMDMs were supplemented after 5 days with fresh medium containing 10 ng/ml macrophage colony-stimulating factor and collected by gentle scraping on day 7.HMDMs were isolated using Ficoll-paque density centrifugation  from buffy coat (of anonymous human donors) provided by the Australian Red Cross Blood Service, Brisbane. CD144 cells per well and allowed to adhere overnight. Prior to assay, cells were incubated with dye-loading buffer  buffer, 4 μM Fluo-3 AM, 25 μl Pluronic acid F-127 and 1% fetal bovine serum) for 1 h at 37 °C. Cells were then washed once with assay buffer . Intracellular calcium release was monitored using a fluorescence imaging plate reader . The percentage responses were plotted against logarithmic concentrations of each test compound42.HMDMs were seeded to a 96-well clear-bottom black-wall assay plate at a density of 5 × 10125I]-C3a or 25 pM [125I]-C5a  on HMDMs (1.2 × 106 cells/ml). HMDMs were treated with [125I]-C3a or [125I]-C5a ± various concentrations of unlabelled C3a/C5a or experimental ligands were diluted with assay buffer  BSA, pH 7.4) for 60 min at room temperature. Unbound [125I]-C3a or [125I]-C5a was removed by filtration through glass microfibre filter GF/B  and washed three times with cold buffer (50 mM Tris-HCl pH 7.4). Bound [125I]-C3a or [125I]-C5a were assessed by scintillation counting on Microbeta counter. Specific binding was defined as the difference between total binding and nonspecific binding as determined in the presence of 1 μM unlabelled C3a or C5a4.Receptor binding was performed by ligand competition with labelled 80 pM [16-C3aR cells were non-enzymatically lifted using Versene Solution and seeded at 30,000 cells per well in 2% BSA in phosphate buffered saline (PBS) in a round-bottom 96-well plate. Cells were simultaneously treated with Eu-DTPA-C3a (2 nM) and various concentrations of C3a or C3a-desArg for 60 min at room temperature with shaking. Cells were then washed thrice with PBS supplemented with 0.2% BSA, 20 µM EDTA and 0.01% Tween-20 by repeated centrifugation. Cells were then resuspended with 20 µL of DELFIA enhancement solution (PerkinElmer) and then transferred to a white 384-well ProxiPlate (PerkinElmer). Time-resolved fluorescence was measured using a PHERAstar plate reader (BMG Labtech) at 337 nm excitation followed by 400 µs delay before 620 nm emission.HEK293 Gα2a  and secondary HRP-linked anti-mouse IgG . GAPDH was detected using affinity isolated rabbit anti-GAPDH  and secondary HRP-linked anti-rabbit IgG . An example of a full blot is shown in Supplementary Fig. Human Embryonic Kidney (HEK) 293 cells  and HMDMs were lysed on ice. Equal amounts of cell lysates were separated, transferred using iBlot Dry Blotting System (Invitrogen). Human C3aR was detected with mouse monoclonal IgG2O . The samples were centrifuged at 13,000 g for 3 min before supernatants were isolated and analyzed for test compound degradation via LC-MS. For liver microsomal stability studies, commercially available rat liver microsomes from Life Technologies, GIBCO were used. In brief, microsomes stored at −80 °C were slowly defrosted on ice. Test compounds  were added to microsomes (5 μL) and NADPH . Test solutions were heated in a circulating water bath at 37 °C for 5 min before additional cofactor NADPH  was added. Aliquots (20 μL) were taken at time points . Aliquots were added to MeCN/H2O  and centrifuged at 13,000 g for 3 min. Supernatants were analyzed for test compound degradation by LC-MSMS. Concentrations were plotted as a function of time on a semi-logarithmic graph. Rat plasma and microsomal activity was confirmed by rapid degradation of SLIGRL-NH2 (t1/2 < 5 min) in a parallel experiment.For plasma stability studies, C3a and experimental compounds  were added to neat rat plasma (190 μL) and heated to 37 °C in a circulating water bath. Aliquots (20 µL) were collected at time points  and added directly to MeCN/H43. A total of 109 animals were used in the experiments described here. Numbers were determined using power analysis  Male Wistar rats  were bred at the Australia Animal Resource Centre . Animals were housed in the appropriate temperature/pressure environment in a 12 h light/dark cycle, according to the standards of the accredited holding facility, with food and water provided ad libitum. At least 48 h habituation in the UQBR facility was provided prior to any experimental intervention. After experimentation, animals were humanely killed by CO2 inhalation as stipulated by approved ethical agreements.All experiments were approved by the Molecular Biosciences Animal Ethics Committee of The University of Queensland, and adhere to The Australian Code of Practice for Use Of Animals for Scientific Purposes (2013) and The Australian Government Guidelines to Promote the Wellbeing of Animals used For Scientific Purposes (2013). Studies involving animals are reported in accordance with the ARRIVE guidelines3, 350 μg in 100 μL isotonic saline) into the plantar surface of both hind paw pads using a 30G needle. The inhibitor cromolyn (20 mg/kg in 5% DMSO) was injected subcutaneously 30 min before the agonist. The antagonist BR111  was administered orally as a single dose 2 h before the agonist. Paw thickness and width were measured using digital calipers (WPI) at 0, 0.5, 2, 6 and 24 h after agonist BR103 administration. Hind paw size is expressed as % change in area from baseline after 0.5 h and then normalized against maximum swelling induced by agonist alone. Researchers were not blinded to treatments given.Male Wistar rats (8–9 weeks) were injected with BR103 . Researchers who performed histological analysis and scoring were blinded to the sample identity.Rat paw tissue was collected and fixed in 4% paraformaldehyde (pH 7.4) for 2 h in the cold room (4 °C). The rat paw tissue was then transferred into falcon tubes containing 30% sucrose solution in PBS and left overnight in the cold room. The next day, rat paw tissue was embedded with the Optimal Cutting Temperature compound  and stored in the −80 °C freezer before being used for making cryo-sections for histological analysis. Frozen sections of 5 microns were prepared using a cryostat-microtome  and stained using standard protocols on the same day. In normal histological staining, such as with alcian blue and safranin O and hematoxylin and eosin (H&E), samples were briefly washed with distilled water before staining. In alcian blue and safranin O, samples were stained with 0.1% alcian blue  and 0.5% safranin O  before being mounted in mounting medium. In H&E, samples were stained with hematoxylin  and 0.1% eosin  before being mounted in mounting medium. In immunohistochemistry, samples were briefly rinsed with PBS to remove OCT and incubated with blocking medium  for 1 h at room temperature. Samples were incubated with primary antibody medium  overnight at 4 °C. The next day, samples were washed with PBS and were incubated with the secondary antibody medium  for 1 h at room temperature. The samples were briefly rinsed, dried and counterstained with DAPI . Primary antibodies for histamine , tryptase , ED1 , ED2  were purchased from commercial sources. Secondary detection antibody was purchased from . ED1 & ED2 positive cells were quantified using FIJI/ImageJ 1.42q software, U.S. National Institutes of Health, Bethesda, MD, USA2O2) in a clear 96-well plate. The plate was read immediately at 460 nm every min for 20 min using a plate reader . O-dianisidine HCl was purchased from Sigma-Aldrich (USA).Rat paw tissues were collected and immediately frozen with liquid nitrogen. The frozen rat paw tissue samples were weighed and crushed into smaller pieces with a mortar and pestle in liquid nitrogen. The crushed tissues were added to MPO buffer  to make up a concentration of 200 mg/mL and homogenized using Zirconium oxide beads  and the Bullet Blender® homogeniser . The homogenized tissues were then diluted to 100 mg/mL with the MPO buffer containing 1% hexadecyl trimethylamonium bromide  and underwent three cycles of freeze, thaw, and sonication. The samples were centrifuged at 13,000 g for 10 min at 4 °C and the clear supernatant for each samples were collected. To measure the MPO release, 10 μL of the sample supernatant was added with 290 μL of substrate solution  and Oligo(dT)12-18 primer  according to manufacturer’s instructions. Real-time PCR was measured on a ABI PRISM 7900HT (Applied Biosystems), each target gene was normalized to housekeeping 18S rRNA and fold change was calculated relative to control sample (Sham). All samples were done in duplicates. Primer sequences  prior to homogenizing according to manufacturer’s instructions. Homogenized tissue samples were added with 200 μL of chloroform and mixed thoroughly. The samples were centrifuged at 11,000 g for 15 min. The top clear solution for each sample was collected and isolated using the ISOLATE II RNA Mini Kit  according to the manufacturer’s instructions. RNA was converted to cDNA using SuperScriptt-test for paired comparison or a two-way repeated measures ANOVA as appropriate. All values of independent parameters are shown as mean ± SEM of at least three independent experiments, unless otherwise stated. Significance was set at *P < 0.05, **P < 0.01 and ***P < 0.001.All experimental results are expressed as means ± standard error. The data were plotted and analyzed using GraphPad Prism version 5.0c for Mac OS X (GraphPad Software). Statistically significant differences were assessed either using student’s The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary Information