Mycobacterium tuberculosis (Mtb). To combat this global health problem, new antitubercular agents with novel modes of action are needed. We have screened a close family of 17 organometallic half-sandwich Os(II) complexes [(arene)Os(phenyl-azo/imino-pyridine)(Cl/I)]+Y– containing various arenes , and NMe2, F, Cl, or Br phenyl or pyridyl substituents, for activity towards Mtb in comparison with normal human lung cells (MRC5). In general, complexes with a monodentate iodido ligand were more potent than chlorido complexes, and the five most potent iodido complexes (MIC 1.25–2.5 µM) have an electron-donating Me2N or OH substituent on the phenyl ring. As expected, the counter anion Y  had little effect on the activity. The pattern of potency of the complexes towards Mtb is similar to that towards human cells, perhaps because in both cases intracellular thiols are likely to be involved in their activation and their redox mechanism of action. The most active complex against Mtb is the p-cymene Os(II) NMe2-phenyl-azopyridine iodido complex (2), a relatively inert complex that also exhibits potent activity towards cancer cells. The uptake of Os from complex 2 by Mtb is rapid and peaks after 6 h, with temperature-dependence studies suggesting a major role for active transport.The treatment of tuberculosis (TB) poses a major challenge as frontline therapeutic agents become increasingly ineffective with the emergence and spread of drug-resistant strains of Significance to MetallomicsMycobacterium tuberculosis (Mtb), the leading cause of death from a single infectious agent. The patterns of activity of the 17 azo- and imino-pyridine complexes studied here towards Mtb and normal lung cells suggest a common redox mechanism of action involving intracellular thiols.Antimicrobial resistance is a global health problem. New advances are urgently needed in the discovery of new antibiotics with novel mechanisms of action. Half-sandwich organometallic complexes offer a versatile platform for drug design. We show that with an appropriate choice of the arene, an N,N-chelated ligand, and monodentate ligand, half-sandwich organo–osmium(II) complexes can exhibit potent activity towards   Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a major global health challenge. TB is now the leading cause of death from a single infectious agent, killing more people than HIV and malaria combined.1 In 2019, ∼1.4 million people died from this devastating disease and over 10 million new TB cases were reported.1 Drug-sensitive TB is curable; however, the complicated 6–9 months treatment regime introduced into the clinic decades ago has gradually become less effective as drug-resistant TB strains have emerged. Treatment of multi-drug resistant (MDR) TB, characterized by resistance to both isoniazid and rifampicin, and extensively drug-resistant (XDR) TB, characterized by resistance to isoniazid, rifampicin, a fluoroquinolone, and one of the injectable second-line drugs, further complicates therapy and often has poor treatment outcomes.2 The escalation of MDR TB and XDR TB has, in turn, led to totally drug-resistant strains of TB and in these cases there are no effective therapeutic agents for successful treatment.3 Therefore, there is an urgent need to develop new TB drugs that have novel mechanisms of action to reduce the TB burden and tackle this global health crisis.4 Recent studies on the antimicrobial activity of organometallic complexes  show that they are potentially an encouraging source of novel antibiotics, including complexes of Re, Fe, Ru, Os, Ir, and Au. The C-bound ligands include arenes, cyclopentadienyls, CO, CN–, and deprotonated phenyls .5–12An analysis of compounds screened for antimicrobial activity by the Community for Open Antimicrobial Drug Discovery has shown that metal complexes have a significantly higher hit rate against critical bacterial and fungal pathogens than organic compounds.13 Osmium(II)- and Ru(II)-arene complexes, as well as Rh(III)- and Ir(III)-cyclopentadienyl (Cp) complexes have been widely investigated as potential anticancer agents.14–16 More recently, the antimicrobial activity of Ir(III), Rh(III), and Ru(II) complexes has been reported, as both 18-electron (6-coordinate) and 16-electron (5-coordinate) half-sandwich complexes.17 The lipophilicity of the η6/η5-coordinated arene/Cp ring and the complex can be modulated by extension (e.g. phenylation) of the ring, whilst the monodentate and bidentate ligands allow further structural and electronic control of reactivity, including the incorporation of bio-active moieties and fine-tuning of the redox chemistry of the complex. Both the intact complex and the individual ligands can all play a role in the biological mechanism of action. Such mechanisms can therefore be multi-targeting, perhaps an advantage for overcoming resistance. Surprisingly, although compounds of Os(II) have recently found recognition as potential anticancer agents,18–20 their application to the treatment of tuberculosis has not yet been explored. Os(II) arene azopyridine and iminopyridine complexes have been shown to be highly potent towards cancer cells, with some selectivity for cancer cells versus normal human cells.21–23Half-sandwich ‘piano-stool’ complexes containing a π-bonded neutral arene or negatively charged cyclopentadienyl ligand effectively occupying three coordination sites, and three other donors giving pseudooctahedral geometry  are one such class.Mtb and normal lung cells. We also study the time dependence and temperature dependence of intracellular osmium accumulation in Mtb for one of the most potent complexes.In this work, we investigate the activity of a family of structurally related osmium–arene complexes against Mtb were determined for a series of 17 structurally related ‘piano-stool’ osmium(II) p-cymene (para-cymene) or biphenyl arene, azo- and imino-pyridine complexes containing either chloride or iodide as a monodentate ligand  against Previous work has shown that iodido complexes in this series are usually relatively inert, e.g. towards aquation, in comparison with chlorido complexes. It is notable that these small changes in the ligands have a significant effect on the activities, ranging across two orders of magnitude  complexes often exhibit higher activity towards cancer cell lines than their p-cymene analogues.24–26 However, osmium–biphenyl (R1) complexes appear less potent towards Mtb than their p-cymene analogues, e.g. MIC of 31.25 µM for 3 compared with 5 µM for 1. The R1 arene substituent can increase lipophilicity and promote uptake of the complex into cells, which is particularly important for import into Mtb as it has a complicated lipid-rich cell envelope that is highly impenetrable to most antibiotics.27For this family of osmium complexes, biphenyl  suggest that the iodido azopyridine complexes, which are relatively inert towards ligand exchange reactions, are activated inside human carcinoma cells,28 through attack on the azo bond by the thiol of glutathione (GSH), which is abundant (millimolar concentrations) in cells. Initially, this leads to release of the iodide ligand and formation of the more reactive chlorido and hydroxido adducts in cells. Then, thiolato (GS) and sulfenato (GSO) adducts can form, which can react with hydrogen peroxide, generating reactive oxygen species (ROS) inside cells.29 Thiols may also be involved in the intracellular activation of these potent osmium–arene complexes in Mtb cells. Whilst Mtb lacks the glutathione system, there are a number of thiols present at similar levels to those of GSH in mammalian cells: ergothioneine (ERG), mycothiol (MSH), and γ-glutamylcysteine (GGC).31 They probably fulfil similar roles: maintaining the redox balance and counteracting ROS, protecting against oxidative stress (ROS), and are likely to interact with the osmium complexes as GSH does in mammalian cells.32Complexes with an iodido monodentate ligand (X) were between 2 and 12.5× more active against 3 = NMe2 substituent onto the phenyl ring of the azopyridine bidentate ligand (complexes 1 and 2) enhances activity against Mtb, compared with unsubstituted (R3 = H) counterparts, complexes 9 and 10, respectively. This group is known to enhance potency and selectivity in human cell lines,28 and also appears to contribute to the overall antimycobacterial activity. Imino complexes 5 and 6 were less potent towards Mtb than their azo complex analogues 1 and 2, respectively, suggesting that the nature of the azo bond is important. It seems likely that redox reactions involving the azo bond may be involved not only in the mechanism of cytotoxicity towards human cancer cells but also towards Mtb.33Introduction of an R4 position was detrimental to both mammalian and mycobacterial potencies (compounds 11–14), with potency inversely correlated with the size of the halide substituent: 11  < 14 (R4 = Cl) < 12  (Table 4 position in R3 = 3-OH complexes (16 and 17) did not appear to affect significantly the potency in either mammalian or mycobacterial cells. The apparent inertness of this site towards substitution reactions suggests that it may be suitable for further investigation (e.g. polymer conjugation as part of a future drug-delivery strategy).Generally, introduction of halide (electron-withdrawing) moieties at the R2 [Os(AzPy-NMe2)I(p-cymene)]PF6 (AzPy: 2-phenylazopyridine) has previously shown promise as a candidate anticancer drug, with selectivity for cancer cells over healthy cells.34 In our current work, this compound also possesses high antitubercular activity, with an MIC potency of 1.25 µM, which is comparable with MIC values of ∼1–9 µM for current clinical TB drugs isoniazid (∼1 µM), ethambutol (∼5 µM), capreomycin (∼3 µM), and streptomycin (∼9 µM). However, complex 2 has a lack of selectivity towards normal human cells compared with the bacteria , or I– (complex 8) had little effect on the antimicrobial activity of the compound (1.25 µM), although this change in halide counter ion has a significant effect on the aqueous solubility of the complexes (7 >> 8 > 2).Complex 1 (12.5 µM), compound 2 (6.25 µM), and compound 4 (6.25 µM). Compared with the determined MIC values  were determined for compound 50  are observed for Mtb and MRC5 normal human lung cell lines , with time-dependent sampling from the culture either with or without shaking. Shaking was found to affect significantly the Os accumulation profile  the rate osmium of influx must decrease by an unknown mechanism (to a rate less than the rate of efflux), or (ii) the rate of osmium efflux must increase (and surpass the rate of influx). Similar accumulation profiles have been observed and modelled for mammalian cancer cells.36 These models demonstrated that, in mammalian cells, the reduction in intracellular Os is a consequence of reduced osmium influx, rather than increased efflux, after reaching a critical intracellular concentration threshold.36For gradient . Mtb celMtb at 277 K with complex 2 in a time-dependent manner in static cultures. These data demonstrate that Os accumulation depends on contributions from passive and active transport mechanisms. Active transport requires energy; therefore, at lower temperatures active transport would be diminished. Osmium accumulation at 277 K suggests passive diffusion of the complex across the lipid envelope , this suggests that its uptake is partially passive and not entirely energy-dependent.The temperature dependence of osmium accumulation was next investigated by incubating ope Fig. . Previou4, a strong oxidant used as a stain for cells, and intriguingly for treatment of synovitis of the knee,37 contains osmium in oxidation state +8, in contrast to the Os(II) complexes used in the present work. The chemical and biological properties of osmium complexes depend not only on the oxidation state of osmium but also on the nature of the coordinated ligands and the geometry of the complex. This is clear from the anti-Mtb activity of complexes 1–17 that ranges from 1.25 to 125 µM. Moreover, these complexes form a close family, with the same half-sandwich structures and only small variations in the ligands. For changes in the N,N-chelated ligand, the activity increases for azo > imino, and for the monodentate ligand I > Cl. The 4–6× increase in activity conferred by the presence of the NMe2 group on the phenyl ring of the N,N-chelated ligand is also notable. This activity pattern is paralleled in activity towards human cancer cells.24–26 Although complex 15 shows selectivity for Mtb versus normal human cells by a factor of 3  were carried out by ion-exchange chromatography as described recently.35Osmium(II) azopyridine and osmium(II) iminopyridine complexes have been previously reported and fully characterized. Changes in the counter anions  was purchased from Lab Tech International. Osmium trichloride was received from Heraeus GmbH. Plasticware for biological experiments was purchased from Greiner Bio-One. Ultrapure nitric acid (72% v/v) was freshly distilled before use. All water used in experiments was Type I Milli-Q (doubly deionized) water. ICP standards for Os were purchased from Inorganic Ventures. All other reagents were purchased from Sigma Aldrich (Merck) and used as received, unless indicated otherwise.p-Benzoquinone  was dissolved in deionized water (50 mL) and perchloric acid  was added. A solution of 5-bromo-2-hydrazinopyridine  in MeOH (10 mL) was added dropwise to the stirring mixture. The mixture turned red-brown and was stirred for 18 h at ambient temperature. The pH was neutralized with the dropwise addition of aqueous sodium hydroxide (6 M). The product was extracted with ethyl acetate (3 × 50 mL) and washed with water (3 × 50 mL). The mixture was concentrated under reduced pressure and placed in a freezer (253 K) overnight. The resulting brown precipitate was collected via vacuum filtration and washed with ice-cold EtOH (2 × 1 mL) and Et2O (2 × 5 mL). Yield: 495.5 mg (67%). 1H NMR : δ 8.73 , 8.19 , 7.93–7.92 , 7.76 , 6.96–6.94 . ESI-MS calculated for C11H9BrN3O+: m/z 278.0. Found: 277.9. CHN analysis: Found: C, 47.28%; H, 2.80%; N, 14.82%. Calculated for C11H8BrN3O: C, 47.51%; H, 2.90%; N, 15.11%.6-p-cym)Cl2]2  was dissolved in EtOH (10 mL), and a solution of 5-Br-AzPy-OH  in EtOH (5 mL) was added dropwise. The mixture was stirred for 18 h at ambient temperature and then filtered through glass microfibre to remove a black precipitate, and NH4PF6  was added. The mixture was concentrated under reduced pressure to ∼3 mL and placed in a freezer (253 K) overnight. A dark crystalline precipitate was collected via vacuum filtration and washed with ice-cold EtOH (2 × 1 mL) and Et2O (2 × 5 mL) and dried overnight in a vacuum desiccator. Yield: 89.6 mg (91%). 1H NMR 2CO): δ 9.66 , 8.75 , 8.56 , 8.15–8.11 , 7.15–7.11 , 6.82–6.81 , 6.53–6.52 , 6.40–6.35 , 2.47 , 2.43 , 0.97 , and 0.91 . ESI-MS calculated for C21H22BrClN3OOs+: m/z 638.0. Found: 637.9. CHN analysis: Found: C, 31.87%; H, 2.70%; N, 5.16%. Calculated for C21H22BrClF6N3OOsP: C, 32.21%; H, 2.83%; N, 5.37%. HPLC purity 98.3%.[Os(η6-p-cymene)I2]2  was dissolved in EtOH (10 mL), and a solution of 5-Br-AzPy-OH  in EtOH (5 mL) was added dropwise. The mixture was stirred for 18 h at ambient temperature and then filtered through glass microfibre to remove a black precipitate, and NH4PF6  was added. The mixture was concentrated under reduced pressure to ∼3 mL and placed in a freezer (253 K) overnight. A dark crystalline precipitate was collected via vacuum filtration and washed with ice-cold EtOH (2 × 1 mL) and Et2O (2 × 5 mL) and dried overnight in a vacuum desiccator. Yield: 76.7 mg (51%). 1H NMR : δ 9.10–9.09 , 8.12–8.08 , 8.02–8.01 , 6.43–6.39 , 7.15–7.11 , 6.20–6.19 , 6.07–6.06 , 6.03–6.02 , 5.97–5.96 , 2.73 , 2.39 , 0.97 , and 0.89 . ESI-MS calculated for C21H22BrIN3OOs+: m/z 730.0. Found: 729.8. CHN analysis: Found: C, 28.62%; H, 2.63%; N, 4.73%. Calculated for C21H22BrF6IN3OOsP: C, 28.85%; H, 2.54%; N, 4.81%. HPLC purity 99.7%. and the inoculum was standardized to 1 × 106 colony-forming units (CFUs)/mL before addition to a prepared 96-well flat bottom microtitre plate with 2-fold serial dilutions of each compound in media. Control experiments in which either no drug, or rifampicin as an antibiotic control were also included on each microtitre plate. The plates were incubated for 7 days at 310 K before the addition of 25 µL resazurin [one tablet of resazurin (VWR) dissolved in 30 mL of sterile phosphate buffered saline (PBS). Following a further incubation for 24 h at 310 K, the plates were assessed for colour development. The MIC values were determined as the lowest concentration of drug that prevented the turnover of resazurin from blue  to resorufin, pink . The results represent three independent biological repeats.The MIC of all compounds was determined using the resazurin microtitre assay, as described previously.The minimum bactericidal activity (MBC) was determined by setting up a microtitre plate as performed for the MIC determination. Instead of adding resazurin, each well from the microtitre plate was plated on to solid 7H10 medium and the CFUs were determined after incubation at 37°C. The lowest concentration at which no CFUs were counted was taken as the MBC. The MBCs were carried out with three independent experimental repeats.Mtb H37Rv was grown to an OD600 of 0.5 and then either compound 2  or water was added. The cultures were incubated at either 277 or 310 K over 24 h or 7 days. At each time point, Mtb culture (1.5 mL) was removed, centrifuged , the supernatant removed, and the pellet washed three times with Tris-buffered-saline containing 0.05% Tween 80 (TBST). The pellet was resuspended in 72% v/v nitric acid (200 µL), heated to 353 K for 16 h, cooled to room temperature, and diluted with Milli-Q water containing 100 mg L–1 ascorbic acid and 10 mM thiourea (3.8 mL). The results represent three independent experimental repeats.l-glutamine) and grown as adherent monolayers at 310 K in a 5% CO2 humidified atmosphere. Cells were passaged using trypsin/EDTA after reaching ∼90% confluence. Trypsin activity was quenched by addition of excess medium, and the single-cell suspension passaged twice weekly. MRC5 fibroblasts were discarded after five passages.MRC5  cell lines were obtained from Public Health England . They were thawed upon receipt and the supernatant removed after centrifugation . Cells were re-suspended in DMEM culture medium  were seeded in a 96-well plate using 0.15 mL of culture medium. After 48 h pre-incubation at 310 K, the supernatant was removed and cells exposed to six concentrations (0–100 µM) of osmium complex, for which exact concentrations were determined using ICP-OES . DMSO was used to aid solubility of Os complexes, but never exceeded 0.5% v/v in the final culture medium. Cells were exposed to Os complexes for 24 h, and then washed with PBS and allowed 72 h recovery time in drug-free medium. Cell viability was determined using the sulforhodamine B (SRB) assay. Cells were fixed using 50 µL of 50% TCA, incubated at 277 K for 1 h, and then stained using SRB dye (0.4% dye in 1% acetic acid). After 30 min, excess dye was removed by acetic acid washing, and then the stain liberated using Tris base . Absorbance was measured using a Thermo Scientific Multiskan FC microplate reader fitted with a 492-nm filter. Data were processed using Microsoft Excel and are reported as percentage survival relative to the untreated control. Sigmoidal dose/response curves were fitted using Origin 2016 to determine IC50. Data were acquired as duplicate of triplicate experiments, with a positive control of cisplatin (CDDP).Briefly, 5 × 10–1 ascorbic acid and 10 mM thiourea to stabilize Os in nitric acid solution. Samples were analysed using an Agilent Technologies 7900 Series ICP-MS (inductively coupled plasma-mass spectrometry) in He gas mode. Calibration standards (0–1000 ppb) for 189Os and 31P were freshly prepared in 3.6% v/v nitric acid containing 100 mg L–1 ascorbic acid and 10 mM thiourea, and all samples analysed using an internal standard of 166Er. Osmium accumulation was normalized to phosphorus content (µg Os/mg P) and standard deviations are reported.Cell pellets were obtained and subjected to digestion using 200 µL of 72% v/v nitric acid. After 24 h incubation at 353 K, samples were diluted using 3800 µL Milli-Q water containing 100 mg Lmfab007_Supplemental_FileClick here for additional data file.


Candidatus Micrarchaeum harzensis A_DKE”, remains largely enigmatic, as they highly express numerous genes encoding hypothetical proteins. Due to a lacking genetic system, it is difficult to elucidate the biological function of the corresponding proteins and heterologous expression is required. In order to prove the viability of this approach, A_DKE’s isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified to electrophoretic homogeneity for biochemical characterization. MhIDH showed optimal activity around pH 8 and appeared to be specific for NADP+ yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed MK-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and catk-values of 38.48 ± 1.62 and 43.99 ± 1.46 s−1 resulting in catk/MK-values of 725 ± 107.62 and 22.69 ± 2.15 mM−1 s−1 for DL-isocitrate and NADP+, respectively. MhIDH’s exceptionally low affinity for NADP+, potentially limiting its reaction rate, can likely be attributed to the presence of a proline residue in the NADP+ binding pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure.Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota, such as “ Microorganisms can survive and thrive under extreme environmental conditions ,2,3. BacThermoplasmatales [Although neutralophilic forms have been identified, recently , Micrarcsmatales ,13,14,15Candidatus Micrarchaeum harzensis A_DKE” in co-culture with its putative host “Ca. Scheffleriplasma hospitalis B_DKE” [To our best knowledge, the only acidophilic Micrarchaeon currently cultivated under laboratory conditions is “s B_DKE” . The culs B_DKE” ,16. Alths B_DKE” ,17, detas B_DKE” , still rs B_DKE” . Of notes B_DKE” ,14, thesE. coli. As a target protein, its isocitrate dehydrogenase (IDH) was chosen, which is a key enzyme of the tricarboxylic acid cycle catalyzing the oxidative decarboxylation of isocitrate to α-ketoglutarate and CO2 [+ binding pocket compared to IDH model enzymes.The goal of this study was to investigate the viability of biochemical characterization of A_DKE proteins via recombinant expression in  and CO2 . Here, wMhIDH were obtained from the BRENDA database ([www.brenda-enzymes.org) (accessed on 6 November 2021).Genomic (accession number: CP060530) and transcriptomic data (accession numbers: SRX8933312-SRX8933315) of A_DKE were accessed via the National Center for Biotechnology Information NCBI  . Conserved sequence motifs and protein domains were detected using the Pfam database ([http://pfam.xfam.org/) (accessed on 6 November 2021).The theoretical molecular weight and isoelectric point of atabase , Aeropyrum pernix K1 , Archaeoglobus fulgidus DSM 4304 , Haloferax volcanii DS2 , and Sulfolobus tokodaii strain 7  was carried out using the Clustal Omega algorithm [www.espript.ibcp.fr) (accessed on 6 November 2021).a BLASTp  search oa BLASTp  via NCBIlgorithm ,24,25 aslgorithm , www.espMhIDH structure was achieved via the CLC Main Workbench 20.0.1 using the crystal structure of EcIDH in complex with Ca2+, isocitric acid and NADP+  (accessed on 7 November 2021)). All protein structures were visualized using PyMOL 2.3.3 .Homology modeling of a putative d NADP+  and assembled via isothermal in vitro ligation [6x Hisicd2 was transformed into E. coli Rosetta pRARE .The s B_DKE”  via oligligation . The resMhIDH6x His over time, E. coli Rosetta pRARE pBAD202_ 6x Hisicd2 was cultivated in shaking flasks containing 50 mL of Terrific Broth medium (1.2% (w/v) tryptone, 2.4% (w/v) yeast extract, 0.5% (w/v) glycerol, 17 mM of KH2PO4, 72 mM of K2HPO4) supplemented with 50 µg mL−1 of kanamycin and 30 µg mL−1 of chloramphenicol at 37 °C and 180 rpm. Upon reaching an OD600 of 0.6–0.8, expression of 6x Hisicd2 was induced by addition of 1 mM of L-(+)-arabinose. From this point forth, the culture was incubated at 30 °C and 180 rpm and samples (1 mL) were taken at different time points after induction , and subjected to OD600-measurement using a GENESYSTM 20 spectrophotometer  and preparation for SDS-PAGE analysis. Samples were centrifuged for 2 min at 16,000× g and cell pellets were resuspended in 75 µL of 2× SDS loading dye (240 mM of TRIS/HCl (pH 6.8), 20% (v/v) glycerol, 2% (w/v) SDS, 100 mM of DTT, 0.02% (w/v) Orange G) per OD600 of 0.2, boiled for 10 min at 95 °C and centrifuged for 5 min at 16,000× g. After determination of the optimal induction time, over-expression was carried out in a total volume of 1 L as described above. Cells were harvested for 15 min at 16,000× g and 4 °C, 4 h after induction, and stored at −20 °C until used.In order to monitor production of ® Pressure Cell Press  at 137.8 MPa.The cell pellet of an expression culture was resuspended in IMAC buffer (50 mM of HEPES/NaOH (pH 7.4), 500 mM of NaCl) followed by the addition of a spatula tip of Deoxyribonuclease I . Cell extracts were prepared using mechanical disruption in an FA-078 FRENCHg and 4 °C. Membranes were separated from the plasma fraction via ultracentrifugation for 60 min at 138,000× g and 4 °C. The membrane pellet was resuspended in solubilization buffer (20 mM HEPES/NaOH (pH 8.0), 150 mM of NaCl, 2% (v/v) Triton X-100) and the plasma fraction was passed through a 0.2-µm syringe filter  to remove remaining insoluble particles. Samples of the raw lysate, as well as the membrane and plasma fraction were used for SDS-PAGE.The raw lysate was fractioned by successive steps of centrifugation. Intact cells and cell debris were pelleted for 15 min at 6000× 2+-IMAC) for protein purification was conducted using a HisTrap® HP 5-mL column  coupled with a BioLogic DuoFlow™ Chromatography System . The column was equilibrated with IMAC buffer, prior to loading with plasma fraction. Non-specifically bound proteins were removed by washing with IMAC buffer containing 80 mM of imidazole. Elution of the target protein was achieved with IMAC buffer containing 500 mM of imidazole. The eluted fraction was concentrated using a 3-kDa MWCO centrifugal filter . Samples of the column flow-through, wash, and eluate were used for SDS-PAGE.Nickel immobilized metal ion affinity chromatography  coupled to the aforementioned chromatography system. The column was equilibrated and run isocratically with IDH buffer (50 mM of HEPES/NaOH (pH 7.4), 150 mM of NaCl, 1 mM of DTT, 0.5 mM of MgCl2). The eluted fractions were collected, concentrated, and analyzed via SDS-PAGE. For long-term storage at −20 °C, 50% (v/v) glycerol was added.Size exclusion chromatography (SEC) of the concentrated protein solution was conducted using a HiLoadProtein quantification of samples collected for analysis via SDS-PAGE was carried out colorimetrically according to . AlternaTM Prestained Protein Marker  or PageRulerTM Prestained Protein Ladder  was used. After separation, the gels were subjected to either colloidal staining using Quick Coomassie Stain  or transfer of the separated proteins to a nitrocellulose membrane  via a semi-dry blot. The latter was carried out with a Trans-Blot® Turbo™ device  at 1.3 A for 10 min using a continuous blotting buffer system  ethanol, 5% (v/v) methanol, pH 8.8).Samples containing 20 µg of total protein (5 µg in case of purified protein) were mixed with 2× SDS loading dye and separated via denaturing SDS-PAGE in hand cast 12% TRIS/Glycine gels according to . As refeDensitometric estimation of protein purity from Coomassie-stained acrylamide gels was carried out using the Image Studio Lite 5.2 software .v/v) Tween® 20) containing 3% (w/v) skim milk powder. After a few brief rinses with TBST, the blot was incubated with a mouse anti-His-tag primary antibody , diluted 1:1000 in TBS (10 mM TRIS/HCl (pH 7.5), 150 mM NaCl) containing 3% (w/v) BSA for 1 h, followed by washing with TBST (4 × 5 min) and incubation with a goat anti-mouse alkaline phosphatase secondary antibody  diluted 1:30,000 in TBST containing 3% (w/v) skim milk powder for 45 min. After washing with TBST (4 × 5 min) and several brief rinses with dH2O, protein bands were visualized colori-metrically using the AP conjugate substrate kit  in accordance with the manufacturer’s instructions.For immuno-staining the membrane was blocked for at least 1 h at room temperature with TBST (20 mM of TRIS/HCl (pH 7.5), 500 mM of NaCl, 0.05% , 1 mM of DL-Na3-isocitrate, 5 mM of MgCl2, 2 mM of Na2NADP and 0.6–2.5 µg of enzyme in a total volume of 200 µL. Each reaction was started individually by addition of either NADP+ or enzyme using a TeInjectTM Dispenser  followed by measurement of A340 each 200 ms for 15–30 s using an Infinite® M 200 PRO plate reader . Investigation of cofactor-specificity was conducted by measuring specific activity with 20 mM of NADP+ or NAD+ in the presence of Mg2+ and cation-dependency was determined by measuring specific activity in presence of 5 mM of MgCl2, MnCl2, CaCl2, ZnCl2, NiCl2, CuCl2, CoCl2, and Na2EDTA, respectively, with 2 mM of NADP+.3CO2Na/CH3CO2H (pH 5.0–6.0), 0.1 M of Na2HPO4/NaH2PO4 (pH 5.5–7.5), and 0.1 M of TRIS/HCl (pH 7.0–9.0).The pH optimum was determined by measuring specific activity in buffers with varying pH values. A corresponding polynomial fitting curve of fifth order was calculated using Origin Pro 2020. In order to span a range from pH 5 to 9.0, three different buffer systems were applied as described in : 0.1 M o+ (0–10 mM) and DL-isocitrate (0–500 µM), respectively. MK and maxV were calculated from a non-linear fit based on the Michaelis–Menten model [Enzyme kinetics were determined by measuring the initial reaction rate at increasing concentrations of NADPen model ,37 usingicd2, Micr_00902) annotated to be encoding a putative NADP-dependent IDH, which is actively expressed, according to available transcriptomic data [A_DKE possesses only one gene  or NADP+ (EC 1.1.1.42) [+ [2+ and Mn2+ [MhIDH, its dependency on different cofactors was tested.IDHs catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate and CO.1.1.42) ,38. Type1.42) [+ . Still, 1.42) [+ ,41,42. Fand Mn2+ . In orde−1 mg−1 MhIDH6x His activity was about 55-fold higher using NADP+ as cofactor relative to NAD+ with only 0.74 ± 0.09 µmol min−1 mg−1  in the active site , MhIDH6x His ranks among the three IDHs with the lowest catalytic efficiency (cat/KMk = 22.69 ± 2.15 mM−1 s−1).Regarding NADPIDHs see . A KM of+ could be the bottleneck limiting the overall reaction rate of MhIDH6x His and possibly the metabolic rate of the whole organism, given that IDH is a key enzyme of the tricarboxylic acid cycle, which is the central metabolic pathway in A_DKE [All in all, low affinity to NADPin A_DKE . This poin A_DKE .MhIDH6x His could be an artefact resulting from the His-tag located at its N-terminus, there is strong evidence suggesting that this might be a natural characteristic of the enzyme itself, when considering potential ligand binding mechanics.While the low cofactor affinity of MhIDH, we conducted a multiple sequence alignment with other experimentally verified IDHs and modeled a putative structure , the NADP+ binding loop (residues 336–352), as well as helix α12 (residues 390–397) [+ via hydrogen bonds or salt bridges  . In partbridges , the only exception being Tyr391 (see MhIDH (Pro382). While this appears to be a common feature among isopropylmalate dehydrogenases rather than IDHs , MhIDH tter see . Since Tee + see a,d this ee EcIDH ,45,51. Mee EcIDH , which cE. coli and biochemically characterized. Compared to other known IDHs, the NADP+ and divalent cation-dependent protein from A_DKE shows optimal activity at near-neutral pH and seems to be highly inefficient because of the architecture of its NADP+ binding pocket. Since MhIDH plays a role in A_DKE’s main pathway for generation of reducing equivalents, its inefficiency is in line with the slow growth rates of the Micrarchaeon and is hypothesized to be partly overcome via glutamate dehydrogenase. Lastly, this study proves the viability of recombinant production of functional A_DKE proteins in E. coli, which opens numerous possibilities for the biochemical characterization of proteins of unknown function in A_DKE.Although several approaches lead to new findings about Micrarchaeota in the last decade, the survival strategies of these ultra-small, acidophilic organisms are still not fully understood. In this study, we presented the first biochemical description of a recombinant micrarchaeal enzyme. The enzyme was successfully produced in


Moschus berezovskii Flerov) in Sichuan and Shaanxi Provinces, China, for coccidian oocysts and 65% were infected with Eimeria spp. Previously, only four Eimeria species were known from Moschus spp. Here we describe six new Eimeria species. Eimeria aquae n. sp., in 38% deer, has ovoidal oocysts, 32.0 × 23.0 μm, micropyle (M) and scattered polar granules (PGs) of various sizes are present, sometimes oocyst residuum (OR) is present; ovoidal sporocysts, 14.1 × 7.5 μm, with Stieda body (SB) and sporocyst residuum (SR). Eimeria dolichocystis n. sp., in 11% deer; cylindroidal oocysts, 36.6 × 18.9, with a M, 1 PG and OR; ovoidal sporocysts, 13.9 × 7.7, with SB and SR. Eimeria fengxianensis n. sp., in 7% deer; ovoidal oocysts, 36.3 × 25.2, a M and PGs present but OR absent; ovoidal sporocysts, 13.9 × 7.3, with SB and SR. Eimeria helini n. sp. in 24% deer; subspheroidal oocysts, 27.0 × 24.1, OR and PGs often present, but M absent; ovoidal sporocysts, 13.5 × 7.7, with SB and SR. Eimeria kaii n. sp. in 26% deer; ovoidal oocysts, 33.2 × 20.7, M and PGs present, but OR absent; ovoidal sporocysts, 14.4 × 7.5, with SB and SR. Eimeria oocylindrica n. sp., in 17% deer; cylindroidal oocysts, 36.0 × 21.4, M and 1-2 PGs present but OR absent; ovoidal sporocysts, 13.8 × 7.7, with SB and SR. Eimeria dujiangyanensis n. nom. is proposed to replace E. moschus Sha, Zhang, Cai, Wang & Liu, 1994, a junior homonym of E. moschus Matschoulsky, 1947.We examined 674 fresh fecal samples from forest musk deer ( Moschus is the only genus in the family Moschidae, with seven extant species, which are all called musk deer  vs. 32 × 23 [27–33 × 21–26]), they always have a much thicker oocyst wall (1.6 vs. 1.2), slightly larger L/W ratio (1.4 vs. 1.3), and they lack any remnant of either PG and/or OR, which some of the oocysts of E. aquae often display as 1, 2, or more spheroidal bodies , Sichuan Province, China.Other hosts: Unknown, none to date.Description of sporulated oocyst: Oocyst shape: spheroidal to subspheroidal; L × W (n = 9): 27.0 × 24.1 (20–30 × 16–27), L/W ratio: 1.1 (1–1.3); oocyst wall smooth and colorless to pale yellow, ~1.1 thick, bi-layered; oocyst wall that may thin slightly at one end; M, MC: both absent; PG: always one present (1.9–2.6 wide); OR: present (n = 2): 0.3–3.2 wide.Description of sporocyst and sporozoites: Sporocyst shape: elongate ovoidal; L × W (n = 9): 13.5 × 7.7 (9–16 × 6–10); L/W ratio: 1.8 (1.4–2.0); SB present; SSB, PSB: both absent; SR: present as small granules disbursed between SZ; SZ: sausage-like, usually with one large RB at more rounded end of SZ and sometimes a second, smaller RB is seen in the more pointed end; N: when visible, in middle of SZ between RBs.Prevalence: Oocysts of this morphotype were found in 159/674 (24%) M. berezovskii, being found in 53/185 (29%) juveniles, in 60/263 (23%) adult males, and in 46/226 (20%) adult females.Sporulation: Exogenous. Oocysts were shed unsporulated and they completely sporulated after 2 weeks in 2.5% (w/v) potassium dichromate solution (K2Cr2O7).Prepatent and patent periods: Unknown.Site of infection: Unknown, oocysts were recovered from feces after the animals defecated.Pathogeny: Unknown.Materials deposited: Photosyntypes [syntypes  of sporuEtymology: The species name is given in honor of Helin Sheng, a retired professor at East China Normal University, who has been devoted to musk deer conservation and research for more than 30 years.Remarks: The spheroidal to subspheroidal shape of the sporulated oocysts is a distinctive feature in E. helini that allows it to be distinguished from all other eimerians now known to infect Moschus species.urn:lsid:zoobank.org:act:46620F9C-97ED-4B63-A16C-7E1B376EE6DCType host: Artiodactyla: Moschidae: Moschus berezovskii Flerov, 1929, Chinese forest musk deer.Type locality: Baoji , Shaanxi Province, China.Other localities: Hanzhong , Shaanxi Province, China. Aba , Sichuan Province, China. Guangyuan , Sichuan Province, China.Other hosts: Unknown, none to date.Description of sporulated oocyst: Oocyst shape: ovoidal; L × W (n = 94): 33.2 × 20.7 (32–37 × 19–24), L/W ratio: 1.5 (1.5–1.7); oocyst wall smooth and colourless to pale yellow, ~1.2 thick, bi-layered; oocyst wall thickest on sides and thinnest at end opposite the M; MC: absent; M: present (n = 81): ~1.9 (0.9–4.9) wide; PG: present only as a few scattered granules or 1–2 spheroidal bodies, 0.6–2.8 wide; OR: absent.Description of sporocyst and sporozoites: Sporocyst shape: elongate-ovoidal; L × W (n = 92): 14.4 × 7.5 (12–16 × 6–8); L/W ratio: 1.9 (1.6–2.2); SB: present; SSB, PSB: both absent; SR: present as small granules disbursed between SZ; SZ: usually with one large RB at more rounded end of SZ, and a second smaller RB in the thinner end of the SZ; N: when visible, in middle of SZ between RBs.Prevalence: Oocysts of this morphotype were found in 174/674 (26%) M. berezovskii, being found in 79/185 (43%) juveniles, in 42/263 (16%) adult males, and in 53/226 (23%) adult females.Sporulation: Exogenous. Oocysts were shed unsporulated and became completely sporulated after 2 weeks in 2.5% (w/v) potassium dichromate solution (K2Cr2O7).Prepatent and patent periods: Unknown.Site of infection: Unknown, oocysts were recovered from feces after the animals defecated.Pathogeny: Unknown.Materials deposited: Photosyntypes [syntypes  of sporuEtymology: This species was named in honor of Kai Li, a professor of the Key Laboratory of Non-Invasive Research Technology for Endangered Species, School of Nature Conservation, Beijing Forestry University, Beijing, China, in recognition of his numerous contributions to our understanding of wildlife conservation and research.Remarks: The L/W ratio of sporulated oocysts of this species is similar as the L/W ratio of E. jinfengshanenisis. However, many features truly distinguish them from each other including oocyst and sporocyst size ranges, the number and shape of their RBs in the SZ, and the presence of a SB in the sporocysts of E. kaii that the sporocysts of or absent of E. jinfengshanenisis lack , Sichuan Province, China.Other hosts: Unknown, none to date.Description of sporulated oocyst: Oocyst shape: cylindroidal, ellipsoid, to elongate ovoidal; L × W (n = 27): 36.0 × 21.4 (33–41 × 19–26), L/W ratio: 1.7 (1.6–1.8); oocyst wall smooth and colorless to pale yellow, ~1.3 thick, bi-layered; oocyst wall thickest on sides and thinnest at end opposite M; MC: absent; M: present (n = 23), ~2.6 (0.8–6.7) wide; 1 or 2 obvious PG present (1.6–2.0 wide), OR absent.Description of sporocyst and sporozoites: Sporocyst shape: elongate ovoidal; L × W (n = 25): 13.8 × 7.7 (11–16 × 6–9.5); L/W ratio: 1.8 (1.5–2.2); SB present; SSB, PSB: both absent; SR: present as small granules disbursed between SZ; SZ sausage-shaped with large RB at more rounded end and a smaller RB at the more pointed end; N, when visible, in middle of SZ between RBs.Prevalence: Oocysts of this morphotype were found in 113/674 (17%) M. berezovskii, being found in 49/185 (26%) juveniles, in 26/263 (10%) adult males, and in 38/226 (17%) adult females.Sporulation: Exogenous. Oocysts were shed unsporulated and became completely sporulated after 2 weeks in 2.5% (w/v) potassium dichromate solution (K2Cr2O7).Prepatent and patent periods: Unknown.Site of infection: Unknown, oocysts were recovered from feces after the animals defecated.Pathogeny: Unknown.Materials deposited: Photosyntypes [syntypes  of sporuEtymology: The species name is derived from oocyst shape based on oocyst and cylindr- .Remarks: The sporulated oocysts of this species are similar in shape to those of E. jinfengshanenisis described by Sha et al. [E. oocylindrica oocyst is most similar to those of E. dolichocystis and E. fengxianensis. They differ from those of E. dolichocystis by having a much smaller L/W ratio (1.7 vs. 2.0), and they lack an OR which is present in oocysts of E. dolichocystis ; L/W ratio: 1.6 (sic) (1.4–1.8) with a thin, 2–layered oocyst wall, ~0.5–0.8 thick and an inconspicuous M and lacking OR and PG. Their sporocysts were ellipsoidal, 11.1 × 5.9 (10–11 × 5–7); L/W ratio: 1.9 (1.7–2.0), without SB, SSB, or PSB, but a SR of small granules between SZ was present  eimerians in the Artiodactyla are reported without a SB .The sporulated oocysts of this species are among the smallest of all eimerians now known from ures see , 2. One Type host: Artiodactyla: Moschidae: Moschus berezovskii Flerov, 1929, Chinese forest musk deer.Remarks: As far as we know, Matschoulsky [Eimeria parasites in musk deer, but the paper was in an obscure journal and most scholars who have summarized the coccidia known in Artiodactyla and/or in Moschus, as indicated by Pellérdy [E. moschus and E. sajanica. Later authors [E. moschus, and seemed to presume that E. sajanica was found in Saiga tatarica “found in Asian territory of the Soviet Union.” In 1947, Matschoulsky [L/W ratio: 1.3; OR, PG: both absent. Sporocysts were ellipsoidal, 10–10.5 × 5–7 (means not given) and SB, SSB, PSB were all absent while SR was present as scattered small granules and SZ had one small RB at its more rounded end (line drawing). Matschoulsky [M. moschiferus, which is found in Siberia. The description of this species is marginal by even the most lenient current standard, but Matschoulsky [choulsky  publishePellérdy , Levine Pellérdy , 12, LevPellérdy , Sha et Pellérdy , were unPellérdy . FortunaPellérdy  said, “W authors , 12, 17, authors  who refechoulsky  said thechoulsky  mentionechoulsky  did provurn:lsid:zoobank.org:act:8E81F0A4-9E6A-4AB1-ABC6-FBD13CC54963Synonym: Eimeria moschus Sha, Zhang, Cai, Wang and Liu 1994.Type host: Artiodactyla: Moschidae: Moschus berezovskii Flerov, 1929, Chinese forest musk deer.Remarks: Sha et al. [Eimeria moschus) for an eimerian they described from M. berezovskii in China. Their oocysts had ovoidal sporulated oocysts that measured, 35.2 × 26.9 (32.5–37.5 × 25–30); L/W ratio: 1.3; M: present, ~6–7 wide; OR, PG: both absent. Their sporocysts were spindle-shaped, pointed at both ends, 17.5 × 8.2 (16–20 × 7.5–10); L/W ratio: 2.1 (1.9–2.3); SB, SSB, PSB: all absent; SR: present as scattered small granules between SZ; SZ: with one RB at its more rounded end (line drawing).a et al.  did not a et al.  paper usEtymology: We propose a new name for the species to avoid synonymy. The new species name is derived from the name of the location within Sichuan Province where Sha et al. (1994) found this species and -ensis .Type host: Artiodactyla: Moschidae: Moschus moschiferus L., 1758, Siberian musk deer.Remarks: Matschoulsky [Moschus, as indicated by Pellérdy [E. moschus and E. sajanica. Later authors, as noted above, referred to information given by Svanbaev [E. sajanica was found in Saiga tatarica “in Asian territory of the Soviet Union.” However, if we trust that Matschoulsky [M. moschiferus, the Siberian musk deer. Matschoulsky [L/W ratio: 1.1; OR, PG: both absent. Sporocysts were ellipsoidal, 5–10 × 3–5 (means not given); SB, SSB, PSB: all absent; SR: present as scattered small granules between SZ; SZ: with one small RB at more rounded end (line drawing). The description of this species is marginal by even the most lenient current standard, but Matschoulsky [choulsky  publishePellérdy , Levine Pellérdy , 12, LevPellérdy , Sha et Pellérdy , either Pellérdy . Our traPellérdy  is: “We Svanbaev , who menSvanbaev , but prechoulsky  said thechoulsky  said thechoulsky  providedEimeria are now known to parasitize two of the seven extant Moschus species; the remaining five Moschus species have never been examined for coccidian oocysts. These 10 coccidians include the six new species described here from M. berezovskii , two species described by Sha et al. [M. berezovskii , and the first two species that were  described by Matschoulsky [M. moschiferus . This is the first comprehensive report of the coccidian species known from all forest musk deer (Moschus spp.).In summary, 10 species of a et al. , also frchoulsky  from M. Eimeria species in the forest musk deer we sampled, especially in juveniles (85%), which is similar to infection rates found by other workers in China [Eimeria between female and male adults was not significantly different in our research, which had been observed in Cai et al. [Eimeria oocysts between males (46%) and females (45%) in 2016, and did not find any difference in genders (p > 0.05). In all fecal samples, E. aquae was the most prevalent species , while the other five eimerians we discovered have lower prevalence in these endangered deer: E. kaii 26% (174/674), E. helini 24% (159/674), E. oocylindrica 17% (113/674), E. dolichocystis 11% (73/674), and E. fengxianensis 7% (45/674). Of all positive samples, 60% were infected with multiple Eimeria species. These results suggest to us that Eimeria spp. in these musk deer may be the main risk factor in limiting their health and contribute as the leading negative effect on juvenile musk deer survival. We suggest that integrated strategies are essential to prevent and/or at least control coccidial infection in these populations.Our study demonstrated a high prevalence (65%) of six in China , 3. The i et al. , when thEimeria species so we do not know if each can infect other wild or domestic Artiodactyla; we know nothing about the host’s immune response to each of these eimerians or if previous infection produces lasting or only transient immunity to that species or even to other species; we do know that many of the 10 known eimerians have sporulated oocysts that share many similarities, but there have not yet been any genes sequenced of any of these oocysts and, thus, no phylogenetic analyses have been possible to determine whether or not these oocysts, or certain combinations thereof, represent 10 or perhaps fewer real species. Clearly, we are only at the discovery stage and there is a lot more work to be done before we can gain any real understanding of the biology and consequences of Eimeria species infections in our endangered and protected musk deer.Unfortunately, this is the sum total of our knowledge about the biology of all species in the Eimeriidae, now known to infect musk deer. We know nothing about any of their endogenous stages  or the cells and locations of these stages in infected cells or even within the gastrointestinal tract (or elsewhere?); we know nothing about the pathology each of these eimerians may cause in musk deer and whether some are more pathogenic than others; we know nothing about how long these eimerians can persist within their hosts (prepatent and patent periods); there have not yet been any cross-transmission studies with any of the 10 known Eimeria leads to severe enteric disease in captive musk deer, while little is known about these species, even for their classification. To the best of our knowledge, our work is the first comprehensive report on the coccidian species known from all musk deer. We summarized 10 species of Eimeria known to parasitize musk deer, including six new species: E. helini, E. huangi, E. oocylindrica, E. shengi, E. dolichocystis, E. kaii, Our research also demonstrated a high prevalence of infection in forest musk deer, usually with multiple Eimeria species. Thus, we argue that coccidia in the wildlife, especially for endangered animals in captivity, should be identified more deeply.In captivity, the confined areas and high stress would accelerate host susceptibility to parasites. Additionally, coccidiosis caused by


All the synthesized compounds were not toxic to the growth of normal cells, HEK-293. They appear to have a higher binding affinity for the target protein, tubulin, PDB ID = 5LYJ (beta chain), relative to the reference compounds, CA4  and doxorubicin  except for 4E, 4M, 4N and 4O. The high binding affinity for beta-tubulin did not translate into enhanced cytotoxicity but the compounds  that have a higher cell permeability (as predicted in-silico) demonstrated an optimum cytotoxicity against the tested cell lines in an almost uniform manner for all tested cell lines.The in-silico study provided insight into the role that cell permeability plays in enhancing the cytotoxicity of this class of compounds and as potential antiproliferative agents.Novel 1,1-diaryl vinyl-sulfones analogues of combretastatin CA-4 were synthesized via Suzuki–Miyaura coupling method and screened for in-vitro antiproliferative activity against four human cancer cell lines: MDA-MB 231(breast cancer), HeLa , A549 (lung cancer), and IMR-32 (neuroblast cancer), along with a normal cell line HEK-293 (human embryonic kidney cell) by employing 3--2,5-diphenyltetrazolium bromide (MTT) assay. The compounds synthesised had better cytotoxicity against the A549 and IMR-32 cell lines compared to HeLa and MDA-MB-231 cell lines. The synthesized compounds also showed significant activity on MDA-MB-231 cancer cell line with IC Its occurrence annually and globally is in millions, especially in developed countries, resulting to well over 5 million mortality rates annually2. This has necessitated the drive to find a lasting solution from both synthetic compounds5 and several other therapeutic medicinal agents inspired from plants or aquatic organisms11 to arrest the situation  Kuntze (Combretaceae) , one of the best known and most potent combretastatin, has been reported as a promising cytotoxic agent, which strongly prevents tubulin polymerization by binding to the colchicine site. Combretastatin CA-4, a simple stilbene [cis 1 -2-(30-hydroxyl-40-methoxy phenyl)-ethane] has been observed to vie for binding sites on tubulin in competition with colchicines22. CA-4 has therefore been considered as an appealing lead molecule for the improvement of antitumor drugs23. A number of CA-4 analogues such as CA-4P, AVE8062 (ombrabulin), ABT-751 (E7010), EPC2407 (Crolibulin), MPC-6827, OXi4503, T138067, BNC-105P etc. are in different phases of clinical trials24.Combretastatin is a naturally occurring well known tubulin polymerization inhibitor. Tubulin polymerizes to form microtubules which furnish eukaryotic cells structure, thus playing a pivotal role in cancer development which therefore makes it an attractive target for the development of anticancer agents. A vast variety of drugs are able to bind to tubulin and modify its assembly properties, e.g. colchicine, vincristine, combretastatin, etc. The interference with microtubule dynamics can have the effect of stopping a cell-cycle and eventual apoptosis. Thus, a tubulin polymerization inhibitor is a potential anticancer agent26. Intrinsically, these isolates are stilbene like, having biphenyl rings separated via a C=C bond. Ring-A possess 3-OCH3 substituents at the 3,4,5-positions while in ring B, 1-OH group is at the C3 position and 1-OH group at the C4 position  of combretastatinsVery many interesting analogues of CA-4 have been designed. In some analogues, the variation was carried out on the alkene functional group alone Fig. d, while Worthy of note is that, in virtually every case, 3,4,5-trimethoxy aromatic ring (ring A), was kept constant, since it is considered very necessary for cytotoxicity of the compound as seen in Fig. 2R) group at the 2-carbon (C2) position, owing to its great medicinal properties. Sulphur based functional moieties  have been found in several natural products to have potent pharmacological activities  to investigate its likely interaction as well as pharmacokinetic property predictions. The compounds were synthesized and tested in-vitro against four human cancer cell lines: MDA-MB 231(breast cancer), HeLa , A549 (lung cancer), and IMR-32 (neuroblast cancer), along with a normal cell line HEK-293 (human embryonic kidney cell) by employing 3--2,5-diphenyltetrazolium bromide (MTT) assay to access their cytotoxicity and antiproliferative properties, and expressed as IC50 (µM) values.In this study, steps have been taken to replace the ethylene bridge in CA-4 with a sulfonyl  were synthesized from the readily available starting material of 3,4,5-trimethoxy benzaldehyde, prepared by Corey Fuchs olefination in the presence of carbon tetrabromide and triphenylphosphine in dichloromethane to afford the 5--1,2,3-trimethoxybenzene (1A) which in turn reacts with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in acetonitrie to give 5-ethynyl-1,2,3-trimethoxybenzene (2A). Further treatment of (2A) with diacetoiodobenzene (DIB), sodium-p-toluenesulfinate and potassium iodide in acetonitrile yields ((E)-5-(1-iodo-2-tosylvinyl)-1,2,3-trimethoxybenzene) (3A). ((E)-5-(1-iodo-2-tosylvinyl)-1,2,3-trimethoxybenzene) (3A) undergoes Suzuki Miyaura coupling with different substituted arylboronic acids in tetrahydrofuran (THF), in the sight of a base, potassium phosphate with palladium acetate as catalyst to give different combretastatin sulfonyl compounds (4a–v), as represented in Fig. The desired targeted analogues  δ 6.69 , 6.55 , 3.82 , 3.78 ; 13CNMR  153.45, 139.75, 136.80, 125.30, 108.70, 88.00, 60.91, 56.15.Light brown liquid, 3.786 g, 80% yield, mp: 51–53 °C; 1H NMR  δ 6.90 , 3.82 , 3.77 , 2.83 ; 13CNMR  152.88, 139.75, 116.30, 111.66, 84.00, 60.91, 56.15, 40.05.Yellow oil, 2.560 g 92% yield; mp: 68–70 °C; 1H NMR  δ 7.44 , 7.39 , 7.17 , 6.38 , 3.87 , 3.78 , 2.38 ; 13CNMR  152.36, 144.40, 142.08, 139.07, 137.27, 134.48, 129.41, 128.01, 113.76, 105.37, 61.01, 56.09, 21.59; MS (ESI): m/z 475 [M + 1]+; HRMS (ESI): calcd for C18H20IO5S [M + 1]+ 475.00706, found 475.00934. IR (KBr) νmax: 3039, 2928, 2835, 1582, 1270, 1130, 994, 802 cm−1.Brown solid, 240 mg, 92% yield; mp: 169–171 °C; 1H NMR  δ 7.44 , 7.39 , 7.35 , 7.28 , 7.13 , 7.05 (1H s), 6.23 , 3.90 , 3.71 , 2.36 ; 13CNMR  154.56, 152.61, 143.53, 138.64, 138.43, 130.58, 130.46, 129.67, 129.05, 128.65, 128.21, 127.85, 107.36, 60.96, 55.99, 21.48. MS (ESI): m/z 425 [M + 1]+ ; HRMS (ESI) calcd for C24H25O5S [M + 1]+ 425.14172, found 425.14311; IR (KBr) νmax: 3035, 2934, 2837, 1583, 1309, 1140, 1083,760 cm−1.White solid; 52 mg, 85% yield; mp: 139–141 °C; Synthesised Sulfonyl Analogues of CA-4 (4b–v). provides detailed report and characterization of compounds 4c–v, pls see the entire list as Supplementary documents Session 1.0). relative to the reference compound doxorubicin having IC50 values 0.23 and 2.06 µM respectively for IMR32 and A549 cell lines 40. There were a few exceptions to the generally observed trend, such as compounds 4A and 4B which did not follow the observed general trend among the test compounds  relative to the reference compounds, CA4 − 7.1 kcal/mol) and doxorubicin − 7.2 kcal/mol) except for 4E, 4M, 4N and 4O  as predicted, are those with the halogen substituents, 4G (R3 = F), 4M (R3 = Cl), 4N (R2 & R3 = Cl), 4R (R5 = F). It is worthy to note that the halogen groups confer extra lipophilicity which may facilitate cell-permeability. Compound 4T (R4 = F) also has cell-line inhibition profile similar to those of 4M, 4N, 4R and 4G but its predicted cell permeability is quite low compared to the four compounds—it however, has a halogen group like the others that follow the general trend.The docking poses of the compounds revealed that the compounds occupy the colchicine binding site Fig. . Only tw41, pyridine-bridge analogues by Zheng et al.43 and biaryl aryl stilbenes by Kumar et al.44, it was observed that the pyridine-bridge analogues, appear to perform better than the sulfonyl bridge analogues in this study, based on a comparison of IC50 values on a relative scale—the pyridine-bridge analogues are active at a generally lower threshold of IC50 values compared to the compounds being reported here. However, the sulfonyl bridge analogues studied in this work appear to have better cytotoxicity compared to the cyclopropyl amide analogues and biaryl aryl stilbenes against the tested cell lines (HeLa and A549) comparing the reported IC50 values for the compounds synthesized and tested by Huan et al.41, and Kumar et al.44, with those reported for the compounds in this study—the cyclopropyl amides and biaryl aryl stilbenes appear to be active generally at a higher threshold of IC50 values relative to the compounds being reported here which means that the compounds being reported here are more active than the cyclopropyl amides and biaryl aryl stilbenes reported by Huan et al.41 and Zheng et al.42 respectively. It therefore, implies that the introduction of the sulphonyl group as a bridge between the stilbeneoid aromatic rings may moderately improve the antiproliferative activity of the combretastatin analogues relative to many other synthetic analogues.Comparing the sulfonyl bridge analogues prepared in this study with other analogues prepared by other groups of researchers, such as the cyclopropyl amide analogues by Huan et al.B provided remarkable SAR information about these compound CA-4 congeners. The introduction of substituents on ring B were exemplified by preparing analogues possessing electron donating groups (4B–4E), electron withdrawing groups (4G–4J and 4I–4Q) as well as both electron donating and withdrawing groups . However, compound possessing biphenyl group (4U) on ring B shows poor cytotoxicity compared to the simple phenyl group (4A) thereby increase in the number of phenyl moieties, activity was declined. Compound 4B displayed prominent potent antiproliferative activity against lung cancer (A549), human neuroblastoma (IMR32), and cervical cancer (HeLa). So, the presence of 4-methoxy phenyl on ring B is essential for cytotoxicity. In contrast, order of activity of electron donating substituents on ring B was 4-methoxy (4B) > 3,5-dimethoxy (4E) > 3,4-dimethoxy (4D) > 4,6-dimethoxy (4C). It was observed that compounds with electron withdrawing substituents on the B-ring such as halogens, like fluoro, difluoro, chloro, dichloro, and trifluoro methyl on ring B which also enhance the lipophilicity of the compounds must have improved the cell permeability making the compounds more bioavailable40 within the cell to carry out its predicted biological function of inhibiting tubulin polymerization and this includes the compound with the nitrile group, noting that it has also been established that the nitrile group is sufficiently lipophilic38 when it replaces an halogen attached to an aromatic ring and can enhance cell permeability as postulated in this study.In addition, it was envisioned that variation of the substituents on ring-, MDA-MB 231 (breast cancer), HeLa , A549 (lung cancer), and IMR-32 (neuroblast cancer) showed moderate to good activity and non were cytotoxic towards normal healthy cell lines (HEK 293) except compound 4V which showed some level of cytotoxicity. The observed results from both in-silico and in-vitro study shows that the compounds are a source of potential lead compounds that can be optimized further especially by improving their cell permeability. Based on the observation of improved cell-lines inhibition profile of test compounds by halogen substitution, the compounds may be optimized further by incorporating more halogen substituents at different positions (and perhaps on the other aromatic ring) and investigate if there would be an improvement in the anticancer profile of the optimized compounds.A series of 20 compounds of the analogues of CA-4 using Suzuki Miyaura coupling method were successfully synthesized and fully characterized using different spectroscopic techniques. The compounds possessing electron donating substituents gave higher percentage yields than those possessing electron withdrawing substituent. This is due to their positive electronic induction effect. All the synthesized compounds evaluated for their anticancer activity against four cancer cell lines® Digital Melting Point Apparatus from Cole-Palmer Limited and are unuttered. The infrared spectra were documented on Thermo Nicolet Nexus 670 Spectrometer with 4 cm−1 resolution using KBr beam splitter and wave numbers of maximal absorption peaks were presented n cm−1. NMR spectra were recorded on Bruker 300, 400, 500 and 600 MHz NMR Spectrometers, 13C-NMR spectra were documented at 75, 100, 125, 150 MHz. The proton resonances are annotated as chemical shifts δ parts per million (ppm) relative to tetramethylsilane (δ 0.0) using the residual solvent signal as an internal standard or tetramethylsilane itself: chloroform-d , multiplicity , coupling constant J, in hertz (Hz), and the number of protons for a given resonance indicated by nH. The chemical shifts of 13C NMR are reported in ppm, relative to the central line of the triplet at ẟ 77.0 ppm for CDCl3. ESI spectra were recorded on Micro mass Quattro LC using ESI+ software with a capillary voltage of 3.98 kV and an ESI mode positive ion trap detector. High-resolution mass spectra (HRMS) were recorded on a QSTAR XL hybrid MS–MS mass spectrometer.Melting points were obtained in open capillary tubes with a 1101D Mel-Temp254 precoated silica gel flexible plates. Column chromatography was conducted using silica gel (60–120 mesh and 100–200 mesh) packed in glass columns. Technical grade solvents which include: n-hexane, methanol, ethyl acetate, acetone, chloroform, dichloromethane, and ethanol were distilled before use. All chemicals used were supplied by Sigma-Aldrich Company, USA. These include: Phenylacetylene, Iodosobenzene diacetate, p-Toluenesulfinic acid sodium salt, Boronic Acids, 1,2,3-trimethoxy benzaldehyde, Sodium thiosulphate, Potassium iodide, etc.All reactions were carried out in flame-dried round-bottom flasks, fitted with rubber septa under nitrogen gas. Removal of solvent was done using a Heidolph Hei-VAP Value Digital G3: 560-01302-00 rotary evaporator attached to a vacuum pump (∼ 3 mmHg). Analytical thin-layer chromatography (TLC) was performed using aluminum foil UVFA panel of four cancer cell lines used for testing the in-vitro cytotoxicity include: MDA-MB-231 derived from human breast adenocarcinoma cells (ATCC No HTB-26), HeLa derived from human cervical cancer cells (ATCC No. CCL-2), A549 derived from human alveolar adenocarcinoma epithelial cells (ATCC No. CCL-185) and IMR-32 derived from human neuroblastoma cell line (ATCC No CCL-127), with a normal cell line HEK-293 (human embryonic kidney cell). These cancer cell lines were obtained from the American Type Culture Collection (ATCC), Manassas, VA, USA, and stored before use.2SO4 and evaporated to dryness. The crude was purified on SiO2 (60–120 mesh) column using Hexanes/EtOAc (95:5) as eluents to afford the analytically pure 1A  and carbon tetrabromide  in anhydrous dichloromethane was added triphenylphosphine  at 0 °C under inert atmosphere. After completion of reaction (monitored by TLC), reaction mixture was quenched with water and extracted with dichloromethane. The organic layer was dried over anhydrous Na3CN was added DBU  drop wise over a period of 10 min at ambient temperature (25–30 °C). The reaction mixture was allowed to stir at ambient temperature for 16 h. After completion of reaction (monitored by TLC), reaction mixture was cooled at 15 °C and quenched by drop wise addition of 5 N aqueous HCl (10 mL) over a period of 15 min then continued stirring for 5 min. The reaction mixture was extracted with EtOAc/hexane ; organic layers were washed with water (10 mL). The organic layers were dried over anhydrous Na2SO4, solvent was evaporated under reduced pressure, and resulting residues were dried in high vacuum to afford the analytically pure 2A  in anhydrous CH6.552 g, 3.10 mmol2  was added to a suspension of the alkyne 3A , sodium arenesulfinate , and KI  in CH3CN, and the reaction mixture was vigorously stirred at room temperature for 1 h. Upon completion of the reaction, the reaction mixture was quenched by the addition of a saturated aqueous solution of Na2S2O3 (5 mL) and basified with a saturated aqueous solution of NaHCO3 (5 mL). Further stirring was followed by extraction with EtOAc (3 × 15 mL). The combined organic extract was washed with H2O (15 mL) and brine (15 mL), dried (Na2SO4), filtered, and concentrated. The residue was purified by column chromatography to afford 3A 2CO3  in water and dimethylformalmide (1:2), with palladium acetate (Pd(OAc) 2) catalyst  was added and reaction was stirred at room temperature under nitrogen for 3 h. The residue was purified by chromatography (EtOAc/hexanes) to afford the corresponding coupled product 4A-V -5-(1-iodo-2-tosylvinyl)-1,2,3-trimethoxybenzene 4A , different aryl boronic acids  and Na51,52. The pale yellow coloured tetrazolium salt (MTT) reduced to a dark blue water-insoluble formazan by metabolically active cells and the product was measured quantitatively after solubility in DMSO. The absorbance of the soluble formazan is directly proportional to the number of viable cells. A panel of four cancer cell lines were used for testing the in-vitro cytotoxicity. These cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing non-essential amino acids and 10% FBS. All the cells were maintained under humidified conditions of 5% CO2 atmosphere at 37 °C in a CO2 incubator . The 96-well micro titre plates were incubated for 24 h prior to addition of the experimental compounds. Cells were treated with vehicle alone (DMSO) or compounds (drugs (1 µg/mL) were dissolved in 100 µL DMSO previously) at different concentrations  of test compounds for 48 h. The assay was completed with the addition of MTT  and incubated for 60 min at 37 °C. The supernatant was aspirated and plates were air dried and the MTT-formazan crystals were dissolved in 100 µL of DMSO.The cytotoxicity of synthesized compounds 4A–V were determined in triplicates by 3--2,5-diphenyltetrazolium bromide (MTT) assay® M200Pro, Tecan, Switzerland). The percent cell viability of each treated well of 96 well plate was calculated based on test wells relative to control wells. Doxorubicin was used as positive controls for comparison purpose and 1% DMSO as a vehicle control. The cytotoxicity of test compounds was expressed in terms of IC50 value, which is defined as a concentration of compound that produced 50% reduction absorbance relative to control, and the absorbance at 570 nm wavelength was recorded51.The optical density (O.D) was measured at 570 nm using TECAN multimode reader (Infinite2+ and Mg2+) and saved in a pdb format. This extracted chain B was processed further using the AutoDock program (MGL Tools 1.5.6). Gasteiger charges were added and the search space area was set by centering the grid box at the colchicine site, capturing all the residues within 4 Å of the native combretastatin within the protein. The grid box parameters  in Angstrom units are  for size and  for centre and the processed protein chain was saved as a pdbqt file. The native doxorubicin ligand was redocked with the protein and the rmsd of the native ligand and redocked native ligand was estimated (0.7029 Å) in order to validate the docking protocol. The 3D structures of the synthesized compounds were prepared using chem3D and their energies were minimized with MM2 Force Field of Chem3D application interface and the ligands were saved as pdb files. The command line version of the obabel program, installed on Ubuntu LTS operating system, was used to convert the pdb files into pdbqt files. The pdbqt files for the protein and prepared ligands were used for the docking calculation with AutoDock Vina. The resulting binding poses and interactions were visualized and processed using the Pymol program. The binding energy of the compounds as well as interacting residues are presented in Table The coordinates of the tubulin (protein) were downloaded from the protein databank (PDB code = 5LYJ). The beta tubulin portion (B chain) that consists of the colchicine site (Fig. Supplementary Information.


Saccharomyces carlsbergensis RU01, by using tannic acid. High-pressure freezing and transmission electron microscopy (HPF-TEM) revealed that the yeast cell wall obtained from yeast malt (YM) medium supplemented with 0.1% w/v tannic acid was thicker than that of yeast cultured in YM medium alone. The production of β-glucan from S. carlsbergensis RU01 was optimized in 3% w/v molasses and 0.1% w/v diammonium sulfate (MDS) medium supplemented with 0.1% w/v tannic acid. The results showed that MDS medium supplemented with 0.1% w/v tannic acid significantly increased the dry cell weight (DCW), and the β-glucan production was 0.28±0.01% w/v and 11.99±0.04% w/w. Tannic acid enhanced the β-glucan content by up to 42.23%. β-Glucan production in the stirred tank reactor (STR) was 1.4-fold higher than that in the shake flask (SF) culture. Analysis of the β-glucan composition by Fourier transform infrared (FTIR) spectroscopy showed that the β-glucan of S. carlsbergensis RU01 cultured in MDS medium supplemented with 0.1% w/v tannic acid had a higher proportion of polysaccharide than that of the control. In addition, β-glucans from brewer’s yeast can be used as prebiotic and functional foods for human health and in animal feed.In this study, we increased β-glucan production from brewer’s yeast,  Saccharomyces cerevisiae cell wall, which is composed of approximately 55–65% β-glucan [S. cerevisiae. They achieved high productivity and purity with clean and mild treatment of β-glucan from the yeast cell wall. A method has been developed to produce a high level of yeast glucan from agricultural waste such as molasses and corn steep liquor (CSL) with fed-batch culture fermentation [β-Glucans are polysaccharide molecules composed of glucose units and can be found in bacteria, algae, yeasts, mushrooms, molds, and higher plants. Their structure depends on the source of the β-1,3 linkage, and each such molecule possesses many novel properties and can improve human and animal health and the immune system. β-1,3-Glucans are classified as biological response modifiers. The molecular mass, shape, structure, and source of β-glucans that provide the most significant therapeutic benefit are highly diverse . Many reβ-glucan . Kim andβ-glucan  and Liu β-glucan  studied entation .S. cerevisiae are commonly used to produce ales in the temperature range of 16–25 °C. On the other hand, Saccharomyces carlsbergensis strains are industrially used to produce lagers in the temperature range from 8 to 15 °C [S. carlsbergensis is commonly used for beer production. The waste production from this beer fermentation process is high. S. carlsbergensis, however, is rich in protein, vitamin B, chitin, and β-glucans, which possess several physiological functions. Interestingly, breweries can generate additional revenue by isolating β-glucan from spent beer yeast as a high-value product. Tian et al. [Tannins are water-soluble polyphenols found in plant species used in the brewing industry, e.g., grape, malts, and hops, while mashing the boiling wort . Tanninsto 15 °C . The yean et al.  used alkn et al. , 24. Alkn et al. . A benefn et al. .S. carlsbergensis RU01. The cultivation mode of β-glucan production in molasses and diammonium sulfate (MDS) medium supplemented with tannic acid was studied.This study aimed to investigate the effect of tannic acid on cell morphology, dry cell weight (DCW), and β-glucan production in S. carlsbergensis strain RU01 was isolated from beer beverage waste. For long-term maintenance, the strain was stored at −20 °C in yeast malt  medium supplemented with 0.1% w/v tannic acid (Sigma) and 20% w/v glycerol (Sigma). Before the experiment, the strain was propagated twice in YM medium supplemented with 0.1% w/v tannic acid.S. carlsbergensis RU01 was available as a cryofixed (by the high-pressure freezing (HPF) method) unit that exhibits reliable physical performance in yeast cells [S. carlsbergensis strain RU01 were grown in YM supplemented with 0.1% w/v tannic acid and in YM as a control at 30 °C for 48 h. Some of the cultures were starved in a refrigerator at 4 °C, and some were suspended in 20% v/v glycerol for at least 4 h. S. carlsbergensis RU01 was cryofixed by HPF using an HPM 010  at −193 °C and 210 MPa. The cells were observed after cryocutting at −175 to −185 °C. Yeast cell sections with a thickness of 100 nm were prepared. The samples were imaged with a Philips CM10 transmission electron microscope. The structures of yeast in near-native state were studied.st cells . The celS. carlsbergensis RU01 was cultured in 3% w/v molasses and 0.1% w/v diammonium sulfate (MDS) medium supplemented with 150 mL of 0.1% w/v tannic acid in a 250-mL Erlenmeyer flask at 30 °C, pH 5.0, and 200 rpm for 36 h. The growth rate was measured at 600 nm. The dry weight was determined. Then, 2 mL of culture was diluted to 50 mL, filtered through a predried filter (0.45 μm pore size), and washed twice with 50 mL of normal saline solution. Cells on the filters were dried at 100 °C in an oven until constant weight and were subsequently cooled at room temperature in a desiccator for 2 h before weighing. The DCW was the difference between the filter weights with or without yeast.S. carlsbergensis RU01 was cultured in MDS medium supplemented with 0.1% w/v tannic acid at 30 °C and pH 5.0 for 48 h. The medium was inoculated with an initial concentration of approximately 6.0×107 CFU/mL (250 mL). Three agitation rates  and two aeration rates (0.5 and 1.0 vvm) were used.Scale-up of the culture was performed in a 7.5-L bioreactor  with a 5-L working volume. S. carlsbergensis RU01 was extracted by a modified alkaline extraction method [The cell wall of n method . The celAccording to the manufacturer’s instructions, the 1,3/1,6-β-glucans were determined in quadruplicate using an assay kit . The β-g−1. Spectra were preprocessed with OPUS-TOUCH software. The curve fitting used to quantify the ratio of polysaccharides, proteins, and lipids was based on a least-square method using Gaussian bands.Powdered glucan was analyzed by FTIR spectroscopy . FTIR spectra were obtained in absorption mode at room temperature. Spectra were recorded from 400 to 4000 cmp<0.05) was reported. The significance of the results was validated using SPSS (version 18.0).All experiments were performed in triplicate, and analysis of variance (ANOVA) with a confidence interval of 95% , proteins (1500–1700 cm−1), and lipids (2800–3000 cm−1), in yeast cells [S. carlsbergensis RU01 was performed. The polysaccharide, protein, and lipid levels of the glucan were calculated from the band area (%). The alkaline extract of S. carlsbergensis RU01 cell walls was designated β-glucan because it contained this polysaccharide as the main component, mixed with proteins and lipids  and %polysaccharide than the control. The results showed that the polysaccharide content was higher than that in the control, while the protein content was slightly lower than that in the control. It was suggested that tannic acid enhanced the β-glucan content and decreased the protein content in the cell wall of S. carlsbergensis RU01.The FTIR spectrum was used to study three main regions, corresponding to polysaccharides (925–1190 cmst cells , 40. Curst cells , 42. FTIS. carlsbergensis RU01 was enhanced by tannic acid. The cell wall of S. carlsbergensis RU01 became thick, and β-glucan production increased significantly. The β-glucan content was enhanced 42.23% by tannic acid and 1.4-fold upon changing the mode of cultivation. The extracted β-glucans were mainly polysaccharides, mixed with proteins and lipids. In addition, β-glucans from brewer’s yeast can be used as a prebiotic and functional foods for human health and in animal feed.β-Glucan production in


Purpose: The purpose of the present study was to estimate the effect of shionone (SHI) on sepsis-induced acute lung injury (ALI).Methods: The cecal ligation and puncture (CLP) surgery was performed to induce sepsis in mice. Pulmonary hematoxylin and eosin staining, the wet/dry ratio, myeloperoxidase (MPO) activity, and the survival rate were detected. The RAW264.7 cells were treated with SHI and stimulated with lipopolysaccharide (LPS). The cells were also overexpressed by extracellular mechanism protein 1 (ECM1) adenovirus. The relative levels of granulocyte–macrophage colony-stimulating factor, IL-6, IL-1β, TNF-α, IL-10, and TGF-β in the serum and supernatant were measured by ELISA. The protein expressions of ECM1, p-STAT5, signal transducer and activator of transcription 5 (STAT5), p-NF-κB, nuclear factor kappa-B (NF-κB), Arg1, CD206, CD16/32, and iNOS in the CLP-induced lung tissues and LPS-induced cells were detected by western blot. The cell counts of Ly6G, F4/80, CD16/32, and CD206 were evaluated by flow cytometry. The ECM1 expression was also observed by immunohistochemistry and immunofluorescence staining.Results: As a result, the histopathological change, pulmonary edema, and the MPO activity were relieved by SHI. SHI treatment increased the percentage of neutrophil and macrophage in the bronchoalveolar lavage fluid. Besides, SHI administration inhibited pro-inflammatory cytokines and M1 phenotype indices, as well as augmented the anti-inflammatory cytokines and M2 phenotype indices. SHI also attenuated the ECM1/STAT5/NF-κB pathway both in vivo and in vitro. The overexpression of ECM1 confirmed that the regulated effect of SHI was due to ECM1 signaling.Conclusion: In conclusion, the present study suggests that SHI ameliorated sepsis-induced ALI by screwing M1 phenotype to M2 phenotype macrophage via the ECM1/STAT5/NF-κB pathway. Sepsis is a severe disorder featured by overwhelming systemic inflammatory reaction and causes various organ failures which are even lethal. Cecal ligation and puncture (CLP) is the commonly used method to induce polymicrobial sepsis containing gram-negative bacteria–related sepsis. The cecum ligated area and puncture number are responsible for the intensity of sepsis . The perAs one of the major complication of sepsis, acute lung injury (ALI) is the devastating disease characterized by respiratory failure, alveolar–capillary membrane barrier, pulmonary edema, and immune/inflammatory reaction. ALI and its severe form the acute respiratory distress syndrome are the important factors contributing to the death of sepsis patients. The neutrophils and macrophages are major cells located in the alveoli and are frequently found in the bronchoalveolar lavage fluid (BALF). The activation of neutrophil and macrophage is the pathological hallmark of the immune/inflammatory response in ALI . DespiteUpon the initiation of inflammation, the macrophage polarizes to classically activated phenotype (M1) and alternatively activated type (M2) in different stimuli conditions. M1 microphage is induced by LPS and releases the pro-inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6. The biomarkers of the M1 macrophage include CD16/32 and iNOS. It is universally acknowledged that M1 phenotype aggravates the pathophysiology of inflammatory dysfunctions. The M2-type macrophage, secreting anti-inflammatory cytokine IL-10, is marked by CD206 and arginase (Arg1). In the normal situation, most tissue resident macrophages exert M2-like characteristics. The M2 macrophage is also induced by harmful stimuli and repairs the tissue damage. The promotion of the M2 macrophage and the suppression of the M1 macrophage has been reported to restrain the symptom of sepsis-induced ALI . CLP andAster tataricus is distributed in eastern Asia and exhibits a protective effect on respiratory disease, such as pharyngitis, cough, and asthma. Shionone (SHI) is the triterpenoid ingredient extracted from the dried root and rhizome of A. tataricus (in vivo and in vitro.The Chinese herb ataricus . It reprataricus . It has ataricus . SHI hasataricus . It seemEscherichia coli 055:B5) was produced by Sigma . Mouse TNF-α, IL-1β, IL-6, IL-10, and TGF-β were purchased from Elabscience . The myeloperoxidase (MPO) commercial kit was provided by Jiancheng . The primary antibodies ECM1, Ly6G, F4/80, p-STAT5, STAT5, p-NF-κB, NF-κB, CD16/32, iNOS, CD206, Arg1, and GAPDH were produced by Cell Signaling Technology (CST) .SHI, with purity over 98%, was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. Dexamethasone (DXM) was supplied from Xiansheng Drug Store . The drugs were dissolved by DMSO [with the concentration of DMSO less than 0.1% (w/v)]. LPS  were purchased from Qinglongshan Animal Farm . The animals were housed in standard laboratory at 24 ± 1°C with 40–60% and 12 h/12 day/night cycle. The mice were allowed to adapt to the environment 1 week before the experiment. All experiments were conducted according to the guide for the Care and Use of Laboratory Animals of NIH. Every effort was achieved to reduce the sacrifice and suffering of the mice.The mice were randomly assigned to the sham, CLP, CLP + DXM (5 mg/kg), CLP + SHI (300 mg/kg), and CLP + SHI (600 mg/kg) groups. CLP surgery was employed to induce the murine sepsis model. After anesthesia, midline laparotomy was carried out on the mice, and the cecum was revealed. The feces were pushed to the cecum and fixed by a 5-0 propylene line. Any intestinal obstruction was avoided. The cecum was punctured 10 times to leak a small amount of faex. After that, the cecum was replaced in the abdominal cavity, and the wound was sutured. The sham mice suffered the same surgical process without puncturing. The mice were intragastrically treated with drugs 2 h before the surgery, and at 0, 2, and 12 h after the surgery. The sham and CLP mice were intragastrically treated with the vehicle at the same time. Their survival condition was recorded during the 48 h after the surgery. Then, the mice were sacrificed. After the sacrifice, the mice were lavaged with a tracheal cannula three times. A total volume of 1.3 ml of the BALF was obtained. The wet-to-dry weight (W/D) ratio was determined. The lung tissues were harvested and fixed in 4% paraformaldehyde and stored at −80°C for the pending test.The lungs of the sacrificed mice were removed and weighted as wet weight (W). The lungs were then dried at 60°C for 48 h and weighted as dry weight (D). Consequently, the W/D ratio was measured to examine edema of the lungs.The lungs were homogenized and the protein concentration was examined by using the BCA commercial kit. The pulmonary MPO activity was assessed in accordance with the instrument. The absorbance change was determined at 460 nm using a full wavelength microplate reader.The lung tissues were fixed in 4% paraformaldehyde, imbedded with paraffin, and sliced. The sections were deparaffinized, rehydrated, and then stained by hematoxylin and eosin (H&E). The pulmonary histopathological alteration was observed under a light microscope.2 and 95% air. The passage was carried out every 2–3 days. Only the cells in the logarithmic growth period were applied for the experiment in the present research.Murine RAW264.7 macrophage cells were supplied by American Type Culture Collection. The cells were cultured in Dulbecco's Modified Eagle Medium containing 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C atmosphere with 5% CO4 cells/ml were seeded onto six-well or 96-well plates. About 24 h later, the culture medium was renewed. The RAW264.7 cells were assigned into the control, LPS, LPS + SHI (0.5 μg/ml), LPS + SHI (1 μg/ml), and LPS + SHI (2 μg/ml) groups. The cells were treated with SHI . About 2 h later, the cells were stimulated with LPS (5 μg/ml) for 24 h. The supernatant and cells were collected for further experiments.A total of 5 × 102 cells/ml were seeded onto six-well plates. About 24 h later, the culture medium was renewed. The ECM1 adenovirus was added for 24 h. Afterward, the cells were treated with SHI (2.0 μg/ml) for 2 h and then stimulated with LPS for 24 h. The cells were collected for pending experiments.The overexpressed adenovirus of ECM1 was provided by Heyuan Bio-Technology Co., Ltd. The RAW264.7 cells were divided to the control, SHI, overexpression (OE), and OE + SHI groups. A total of 5 × 104 RAW264.7 cells/ml were seeded onto 96-well plates for 24 h. The cells were incubated with SHI  for 2 h, and then with LPS (5 μg/ml) for another 24 h. The MTT solution (5 mg/ml) was added for 4 h. Thereafter, the culture medium was removed, and DMSO was added to dissolve formazan. The optical density value was detected at 570 nm using a full wavelength microplate reader.A total of 5 × 10The concentrations of GM-CSF, IL-1β, IL-6, TNF-α, IL-10, and TGF-β were measured by using commercial ELISA kits by following the manufacturer’s instruction .g for 5 min at room temperature. To measure the cell surface markers of macrophage polarization, the cells were stained using anti-Ly6G-PE and anti-F4/80-FITC at 4°C for 30 min in the dark environment. The stained samples were detected using FACSCalibur  and analyzed using the FlowJo software (Tree Star Inc.).The cells were collected and centrifuged at 6,000 The lung tissues and RAW264.7 cells were lysed by RIPA. The protein concentration was determined by using the BCA Protein Assay Kit . The extract of protein was boiled at the equal amount. The sample was electrophoresed by 8–12% SDS-PAGE and electrotransferred onto the polyvinylidene diflouride membrane. The membrane was blocked at room temperature for 2 h by 3% bovine serum albumin (BSA) and then incubated with specific antibody at 4°C overnight. After washing with TBST, the blot was incubated with horseradish peroxidase–labeled secondary antibody at room temperature for 2 h on the shaker. Consequently, the immunoreactivity was visualized using enhanced chemiluminescence reagent.The lung tissues were immersed into 4% paraformaldehyde. The slides were dewaxed by xylene and exposed to gradient concentration ethanol. After antigen retrieval using citrate buffer at 95°C, the sections were quenched by endogenous peroxidases and blocked by 2% BSA for 30 min. The slides were incubated with primary antibody ECM1 followed by secondary antibody. The sections were stained with DAB and then with hematoxylin. An Olympus BX53 microscope  was used for visualization. The relative ECM1 expression was determined by ImageJ software.The cells were stained with 4% paraformaldehyde and then blocked using 3% BSA supplemented with 0.1% Triton X-100. The samples were incubated with anti-Arg1 antibodies at 4°C overnight. After washing, the cell was incubated with Alexa Fluor® 488 secondary antibody  in dark condition at room temperature for 1 h. The nuclei were stained with DAPI for 10 s. The samples were exposed to anti-fluorescence quenching sealing solution , and Arg1 expression was observed under the fluorescence microscope. Finally, the fluorescence intensity was calculated by using ImageJ and the representative image was observed.p < 0.05 was considered as statistically significant.Our data are depicted as mean ± SD. The results were analyzed by one-way ANOVA and followed up with Tukey’s multiple comparison test. p < 0.01) (p < 0.01), which were more potent than in the SHI (50 mg/kg) group (p < 0.05). Besides, CLP surgery significantly increased the wet/dry ratio compared with that in the sham group (p < 0.01). The administration of SHI and DXM markedly reduced the pulmonary wet/dry ratio compared with the CLP group (p < 0.01) (p < 0.01). While SHI and DXM treatments were capable of reducing these indices (p < 0.01) (p < 0.01), which were evidently decreased in the SHI and DXM groups (p < 0.01) (p < 0.01). CLP stimulation elevated the expression of TGF-β when compared with the sham group  group all died within 24 h. The survival percent in the SHI (100 mg/kg) and DXM groups was effectively reversed compared with that of the CLP group ( < 0.01) . The his < 0.01) . The tre < 0.01) . The CLP < 0.01) , the CLP < 0.01) . The SHIam group . Our datp < 0.01). Whereas, the SHI and DXM administrations all reduced the cell population percent of the neutrophils and macrophages in BALF (p < 0.01).The ratio of the neutrophils and macrophages were detected using flow cytometry by anti-Ly6G PE and anti-F4/80 Alex Fluor488 (FITC), respectively. As presented in p < 0.01), as well as CD206 and Arg1  expressions. The treatments with SHI (100 mg/kg) and DXM markedly downregulated CD16/CD32 and iNOS expressions (p < 0.01), while SHI (50 mg/kg) also inhibited the expressions of CD16/CD32 and iNOS . Nevertheless, SHI (100 mg/kg) and DXM promoted the CD206 and Arg1 expressions when compared with the CLP group, and SHI (50 mg/kg) showed augmented effect on CD206 and Arg1 levels  . The administration of SHI (100 mg/kg) and DXM inhibited the ECM1 expression (p < 0.01). SHI (100 mg/kg) and DXM elevated the phosphorylated levels of STAT5 compared with those of the CLP group (p < 0.01). SHI (50 mg/kg) also augmented the phosphorylation of STAT5 (p < 0.05). Both DXM and SHI administration reduced the protein levels of phosphorylated NF-κB (p < 0.01)  . Further < 0.01) . Our resp < 0.01), which indicated that SHI could promote macrophage function (p < 0.01) (p < 0.01), which exhibited stronger efficacy than SHI (0.5 μg/ml) treatment (p < 0.05)  compared with the control group. The incubation with SHI  downregulated CD16/CD32 expression and upregulated CD206 expression (p < 0.01) (p < 0.01) (The SHI treatment increased the levels of GM-CSF (function . The inc < 0.01) . On the  < 0.05) . The CD1 < 0.01) . Arg1 ex < 0.01) . The resp < 0.01). SHI (1 and 2 μg/ml) treatment enhanced Arg1 and CD206 expressions compared with those of the LPS group, which were stronger than SHI (0.5 μg/ml group) (p < 0.05) (p < 0.01) (p < 0.01). The treatment with SHI (1 and 2 μg/ml) further strengthened the STAT5 phosphorylation (p < 0.01), which was more efficient compared with that of the SHI (0.5 μg/ml) group (p < 0.05). The incubation with SHI  prominently decreased the protein levels of p-NF-κB (p < 0.01)  . Additio < 0.01) . LPS ind < 0.01) . The resp < 0.01). It was noteworthy that the SHI-governed upregulations on p-STAT5, Arg1, and CD206 were hampered by ECM1 overexpression (p < 0.01) (p < 0.01) (p < 0.01), while the co-treatment with ECM1 adenovirus and SHI elevated the supernatant levels of these pro-inflammatory cytokines compared with the LPS + SHI group (p < 0.01) (p < 0.01). By contrast, the supernatant contents of the anti-inflammatory cytokines in the LPS + ECM1 overexpression + SHI group were obviously downregulated compared with those in the LPS + SHI group (p < 0.01)  . On the  < 0.01) . Additio < 0.01) . The sup < 0.01) . The expThe high concentrations of pro-inflammatory cytokines in the serum result in multi-organ damage or even “systemic inflammatory response syndrome” in sepsis patients and animals. Sepsis also led to the accumulation of neutrophils and phagocytic cells. The neutrophil influx from the vessel to lung tissue and induce pulmonary inflammation in sepsis. Excessive neutrophils even elaborate sepsis-caused tissue damage. The transmigration of the neutrophils is reflected by the MPO activity . BronchoMacrophages are critical phagocytic cells in the immune system and are distributed in multiple tissues including the lungs. The macrophage handles danger stimuli, maintains homeostasis, modulates inflammatory reaction, and accelerates wound healing. The macrophage polarization is responsible for the occurrence and development of immune disorder. CD16/32 and CD206 are the surface markers of M1 type and M2 type macrophages, respectively. Arg1 is promoted in the alternative activated macrophage. Former literatures identified the upregulation of Arg1 in the submucosal inflammatory cell of asthma patients. IL-10 is not only regarded as the specific M2 macrophage marker but also the indicative biomarker for deactivated macrophage. The elevation of M2 macrophage increased the anti-inflammatory reaction and the survival rate in septic mice . The shiNF-κB, a critical cytoplasmic transcription factor driving immune and inflammatory process, is stimulated by TNF-α, LPS, and other tissue inflammatory injury . NF-κB cin vivo and in vitro. Furthermore, the overexpression of ECM1 hampered the SHI treatment–mediated upregulation of M2 phenotype biomarkers and downregulation of M1 phenotype biomarkers, indicating that ECM1 was involved in the regulatory effect of SHI on macrophage polarization.ECM1 is highly expressed in macrophages and the tissues infiltrated with inflammatory cells. The specific downregulation of ECM1 in macrophages caused the promotion of Arg1 and prevented the screwing to the M1 phenotype. As the extracellular matrix factor, ECM1 controls the expression of the inflammatory lymphocytes in the respiratory system . The recIn conclusion, the present research demonstrated that ECM1 attenuated sepsis-induced ALI by screwing M1 phenotype to M2 phenotype polarization in CLP-induced mice and LPS-induced macrophage RAW264.7 cells, which was mediated by the ECM1/STAT5/NF-κB pathway. It was beneficial to perform the experiment with transgenic mice and alveolar macrophages. Besides, the activation and chemotaxis mechanism of macrophages could be explored in further investigation.