All the synthesized compounds efficiently inhibited both enzymes. Compound 5g was the most potent derivative in the series, and powerfully inhibited both α-glucosidase and α-amylase. The IC50 of 5g against α-glucosidase was 0.35917 ± 0.0189 µM (standard acarbose IC50 = 6.109 ± 0.329 µM), and the IC50 value of 5g against α-amylase was 0.4379 ± 0.0423 µM (standard acarbose IC50 = 33.178 ± 2.392 µM). The Lineweaver-Burk plot indicated that compound 5g is a competitive inhibitor of α-glucosidase. The binding interactions of the most active analogues were confirmed through molecular docking studies. Docking studies showed that 5g interacts with the residues Trp690, Asp548, Arg425, and Glu426, which form hydrogen bonds to 5g with distances of 2.05, 2.20, 2.10 and 2.18 Å, respectively. All compounds showed high mutagenic and tumorigenic behaviors, and only 5e showed irritant properties. In addition, all the derivatives showed good antioxidant activities. The pharmacokinetic evaluation also revealed promising resultsA series of symmetrical salicylaldehyde-bishydrazine azo molecules,  In partGlyset®) , can revR)-methyl 2-benzamido-3-((tert-butyldimethylsilyl)oxy)propanoate. Moreover, these AGIs also show some serious adverse effects, including causing flatulence and diarrhoea  mM−1. The EI dissociation constant, Ki, was determined by the secondary plot of 1/V versus the inhibitor concentration.Based on the ICwww.rcsb.org). The energies of the target proteins were minimized with an Amber force field by using a conjugate gradient algorithm in UCSF Chimera 1.10.1 . The overall protein architecture and statistical percentages of helices, β-sheets, coils and turns were retrieved from the online server VADAR 1.8 . The Discovery Studio 4.1 Client  was used as a visualizing tool to generate graphical depictions of the target proteins.The three-dimensional (3D) structures of α-amylase and α-glucosidase, with PDBIDs of 1DHK and 4J5T, respectively, were retrieved from the Protein Data Bank (PDB) , molar refractivity density, polarizability and drug likeness score were evaluated using the online computational tools Molsoft (http://www.molsoft.com/) and Molinspiration (http://www.molinspiration.com/). Moreover, Lipinski’s rule of five was also considered to evaluate the efficacy of the ligand structures. Furthermore, the ligand efficiency (LE), lipophilic ligand efficiency (LLE) and lipophilicity-corrected ligand efficiency (LELP) values of all the ligands were calculated using the Data Warrior tool. Moreover, the mutagenic and irritant risks were assessed using the Data Warrior tool.The synthesized ligands were sketched using the ACD/ChemSketch tool and minimized using UCSF Chimera 1.10.1. The basic biological properties, such as molecular weight (g/mol), numbers of hydrogen bond acceptors and donors (HBA/D), LogMolecular docking experiments were conducted on all the synthesized ligands against α-amylase and α-glucosidase using the PyRx docking tool . The grid box-centred parametric values for α-amylase were adjusted as x = −18.44, y = −20.91, z = 8.22, while the size values for all coordinates were adjusted as x = 77.93, y = 68.98, and z = 103.65, respectively. Similarly, for α-glucosidase, the grid values were adjusted as center_x = 102.22, center_y = 40.10, center_z = 18.02, while the size values were adjusted as size_x = 57.60, size_y = 59.83 and size_z = 72.29, respectively. A default exhaustiveness value = 8 was used in both docking studies to maximize the binding conformational analysis. The docking poses (100 numbers of runs) for each docking study were adjusted to obtain the best docking results. The selected compound was docked separately and evaluated based on the lowest binding energy  and the structure-activity relationship. Graphical depictions of all the docking complexes were prepared using Discovery Studio (2.1.0).5a–5h) were synthesized and characterized through elemental and spectroscopic techniques. The abilities of the synthesized compounds to inhibit α-glucosidase and α-amylase were evaluated. Compounds 5a–5h all significantly inhibited both of these enzymes. Derivatives 5a–5h also showed good antioxidant activities. Kinetic studies were performed to elucidate the modes of inhibition of α-amylase and α-glucosidase, and the most potent derivative, 5g, was a dual inhibitor of these enzymes via competitive inhibition. The binding modes of ligands with the target proteins were explored by molecular docking studies. Pharmacokinetics and chemo-informatics evaluations revealed that the properties of the molecules were similar, and a few of the derivatives in this series can serve as lead molecules in the design of drugs for the treatment of DM.A series of new bis-azo dyes based on Schiff bases (


Bletilla striata is a traditional Chinese medicine used to treat hemorrhage, scald, gastric ulcer, pulmonary diseases and inflammations. In this study, we investigated bioactivity of the effective fraction of B. striata (EFB) in reducing the inflammatory cytokine production induced by water or organic extracts of PM2.5.2.5 extracts were collected and analyzed by chromatographic system and inductively coupled plasma mass spectrometer. Cell viability was measured using MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, and cell supernatant was analyzed by flow cytometry, ELISA, and qRT-PCR in cultured mouse macrophage cell line RAW264.7 treated with EFB and PM2.5 extracts. Expressions of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway were measured by Western blot.PM2.5 composition is complex and the toxicity of PM2.5 extracts were not noticeable. The treatment of EFB at a wide dose-range of 0–40 μg/mL did not cause significant change of RAW264.7 cell proliferation. EFB pretreatment decreased the inflammatory cytokines in the macrophage. Further analysis showed that EFB significantly attenuated PM2.5-induced proinflammatory protein expression and downregulated the levels of phosphorylated NF-κBp65, inhibitor of kappa B (IκB)-α, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38.PMB. striata extracts for treating PM2.5-triggered pulmonary inflammation.Our study demonstrated the potential effectiveness of  Epidemiological studies have shown that exposure to PM2.5 is strongly related to chronic bronchitis, asthma, chronic obstructive pulmonary disease (COPD), emphysema, lung cancer, and other respiratory diseases [2.5 are complicated and they have been reported to include water-soluble inorganics, toxic metals, polycyclic aromatic hydrocarbons (PAHs), and bacterial endotoxins [2.5-induced pulmonary dysfunction [2.5-treated macrophages [2.5-induced pulmonary inflammation and dysfunction [Airborne fine particulate matter (PMdiseases –3. The cdotoxins . Pulmonafunction . The trafunction . The NF-function . Upon strophages –11. Actifunction .Bletilla striata (Thunb.) Rchb. f., which is a traditional Chinese medicine, has been widely used for its pulmonary protective, hemostasis, analgesic, antiulcer, anti-fibrosis, and anti-inflammatory effects [B. striata, including bibenzyls, phenanthrenes, polysaccharides, anthocyanidins, dihydrophenanthrenes, steroids, triterpenes, and glycosides [B. striata polysaccharide was reported to reduce the levels of pro-inflammatory cytokines and suppress MAPK/NF-κB signaling pathway activity in rats with gastric ulcer induced by ethanol [B. striata, studied the pharmacological activities of B. striata extract [B. striata (EFB) and efficacy components [B. striata, which can induce HepG2 cells apoptosis in a dose-dependent manner [2.5 could trigger pulmonary inflammation and oxidative stress, which result in pulmonary fibrosis [B. striata exhibited a variety of therapeutic effects including anti-inflammatory, and anti-fibrosis by significantly down regulated serum levels of IL-1β, TNF-α, transforming growth factor-(TGF-β) and other inflammatory factors [B. striata [B. striata decreased the inflammatory cytokine levels of IL-6 and TNF-α to protect IEC-18 cells from lipopolysaccharide (LPS)-induced injury. Hence, we speculated that the extract of B. striata can impact PM2.5-induced injury. effects –15. Numeycosides , which e ethanol . In our  extract , explorimponents , and obtmponents . At the t manner . Studiesfibrosis , 23, our factors , 25, and striata . Besides striata  showed tB. striata and extracts have anti-inflammatory and anti-oxidative effects [2.5 induced inflammation by B. striata has not been reported. 2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene (Coelonin), as one of the main active components from B. striata total ethanol-extract (BTE) was separated and can significantly down regulated IL-1β and IL-6 expression on LPS-induced RAW264.7 cells [B. striata. The study was to explore the protective effects of BTE and Coelonin on PM2.5-induced inflammatory cytokine expression in macrophages and investigate the underlying mechanism of the prevention and treatment effects of B. striata regarding PM2.5-related inflammatory disease.In recent years, a large number of scientific studies, including clinical trials, have been conducted in the field of traditional Chinese medicine, and it has been found that  effects , 29. The.7 cells . Therefo2.5 were collected on glass fiber filters using a Thermo Anderson G-2.5 large-volume sampler  with a flow rate of 1.13 m3/min. The sampling location was set on the rooftop of laboratory building 4 at the Zhejiang Chinese Medical University, which was 20 m higher above the ground, without obvious nearby source of pollution, from October 1, 2016 to March 3, 2017.The particles were deposited on 203 mm × 254 mm glass fiber filters purchased from Whatman , which had been prebaked at 300 °C for 5 h before use. Samples of PM2.5 (WEP) were then diluted to 10 mg/mL using phosphate-buffered saline (PBS) buffer, while organic extracts of PM2.5 (OEP) were diluted in dimethyl sulfoxide (DMSO)/sterile PBS . The stock solution was sonicated for 45 min before the experiment and then further diluted to the desired concentration with Dulbecco’s Modified Eagle Medium .For water extraction, small pieces of the glass fiber filter (0.5 × 0.5 cm) were cut and immersed in ultrapure water. For organic extraction, small pieces of the glass fiber filter (0.5 × 0.5 cm) were cut and immersed in dichloromethane. These samples were sonicated for 6 × 30 min. Water extracts were filtered through 0.45 μm filters and freeze-dried, while organic extracts were rotary-evaporated, and stored at − 80 °C for later use. Water extracts of PM+, K+, Mg2+, Ca2+, NH4+, SO42−, NO3−, and Cl− in the PM2.5 water extract was analyzed using an ICS-2000 ion chromatography system  equipped with an AS3000 autosampler. Ion standard materials were purchased from the National Standards Center . A total of 20 kinds of soluble metals were detected in the water extracts using an inductively coupled plasma mass spectrometer . The analytical model involved a full quantitative analysis; the oxide level was < 2%; the double charge formation was < 3%. The samples were analyzed under the above optimized conditions, and rhenium were used as internal standards. A 7890A-5975C gas chromatography-mass spectrometer  equipped with an electron ionization (EI) ion source was used to conduct PAHs analysis in the organic extracts [Water-soluble inorganic ions, including NaB. striata was collected from Meichuan Town in Hubei Province, People’s Republic of China, and authenticated as a purple orchid with medicinal properties by Prof. Zhi-Shan Ding (one of the authors). A voucher specimen (LA-20161103) has already been deposited in Zhejiang Chinese Medical University. The dry tubers of B. striata were processed, smashed, and filtered through a 40-mesh sieve. 100 g powder was refluxed and extracted three times with 1 L of 95% ethanol. The filtrate was allowed to cool, filtered, and dried in the reduced pressure distillation. The obtained semi-solid B. striata total ethanol-extract (BTE) was stored at − 20 °C until used.18 column  was used with the column temperature set at 30 °C, and elution solvents were acetonitrile with 0.1% formic acid. Analytes were monitored at 280 nm. The peaks with a retention time of 12.5~15.5 min was collected to obtain the extract of BTE, and analyzed by HPLC fingerprints and SYNAPT G2-Si HD Mass Spectrometer  to identify the extract as Coelonin  ethanol. The active fraction eluted with 40% ethanol in water were dried in vacuum and analyzed using a Dionex Ultimate3000 high-performance liquid chromatography , then, separation and purification were carried out by Dionex Ultimate3000 semi-preparative high-performance liquid chromatography . A Welch Ultimate XB-C2 atmosphere. The cells were passaged every 2–3 days, after reaching 70–80% confluency.The mouse macrophage cell line RAW264.7 was purchased from Shanghai Institutes for Life Science, Chinese Academy of Sciences . The RAW264.7 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), which was obtained from CellMax, supplemented with 100 units/mL penicillin and 100 mg/mL streptomycin at 37 °C in a humidified 5% CO4 cells per well. After overnight growth, the culture medium was removed. To assess the effects on cell viability, 100 μL of cell culture medium containing either PM2.5 water or organic extracts at 12.5, 25, 50, 100, and 200 μg/mL were added to each well, and either BTE or Coelonin at 1.25, 2.5, 5, 10, 20, and 40 μg/mL were added. After 24 h of incubation, 20 μL MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); Promega, Madison, WI, USA) was added to each well for 2 h of incubation at 37 °C in a humidified 5% CO2 incubator. The absorbance at 490 nm of the formazan was assessed using a BioTek Epoch2 microplate reader .RAW264.7 cells were plated in a 96-well plate at a density of 5 × 105 cells per well in 100 μL of culture media and incubated for 12 h. Subsequently, the differentiated RAW264.7 cells were treated with PM2.5 at different concentrations of 12.5, 25, 50, 100, and 200 μg/mL for an additional 12 and 24 h. The supernatant of the PM2.5 extract-treated cells was collected to assess TNF-α secretion using an enzyme-linked immunosorbent assay (ELISA) obtained from eBioscience .RAW264.7 cells were plated in a 96-well plate at a density of 1 × 102.5 water extract, in another group, same treatments by BTE and Coelonin were repeated and followed by treating with 100 μg/mL organic extract. After 18 h treatment, the supernatant of these cells was then collected for the analysis of TNF-α, IL-6, and MCP-1 secretion using an ELISA and a BD Biosciences cytometric bead array assay . Each immunoassay was performed in accordance with the manufacturer’s instructions.Further, other cells were pretreated with BTE at 0, 2.5, 5, 10, and Coelonin at 0, 1.25, 2.5, 5, and 10 μg/mL for 2 h before treating with 200 μg/mL PM2O. The total RNA was reverse-transcribed to cDNA using a PrimeScript™ RT Master Mix kit . Relative expression levels of IL-6 and TNF-α were quantified using SYBR® Premix Ex Tap™ II . All primer sequences  are listed in Table -ΔΔCt method following normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Total RNA was extracted with RNAiso Plus  according to the manufacturer’s instruction, and dissolved in 30 μL diethylpyrocarbonate (DEPC)-H2.5 water extract at a concentration of 200 μg/mL was used and the PM2.5 organic extract at a concentration of 100 μg/mL was used. Total proteins were isolated using a protein extraction kit . Harvested proteins were denatured at 95 °C for 10 min, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a polyvinylidene fluoride (PVDF) membrane . The membranes were blocked with 5% bovine serum albumin  for 1 h. They were subsequently probed at 4 °C overnight with the primary antibodies, comprising anti-β-actin (110000); anti-IκBα (1:1000), anti-phospho-IκBα (1:1000), anti-NF-κB p65 (15000), anti-phospho-NF-κBp65 (1:2000), anti-p38 (1:2000), anti-phospho-p38 (1:1000), anti-JNK1 + JNK2 + JNK3 (1:2000), anti-phospho-JNK1 + JNK2 + JNK3 (1:1000), anti-ERK1 + ERK2 (1:2000) and anti-phospho-ERK1 + ERK2  purchased from Abcam . Subsequently, the membranes were incubated for 1 h with a horseradish peroxidase (HRP)-conjugated secondary antibody  at room temperature. Enhanced chemiluminescence (ECL) substrate  was added to detect the proteins using a chemiluminescence imaging system .For the western blot analysis, the PMP values < 0.05 were considered significant.All data are presented as mean ± standard deviation. A statistical comparison of the between-group differences was performed using one-way analysis of variance (ANOVA) in SPSS17.0 software . 2.5 water extract, and the concentrations of PAHs in the PM2.5 organic extract were measured, and results are shown in Table 2.5 in the atmosphere is very complex due to many factors, such as the combustion source, industry pollution, climate, and season [The concentrations of metal elements in the PMd season .Table 22.5 water and organic extracts was evaluated using MTS assays. After 24 h exposure of RAW264.7 cells to a wide dose range of PM2.5 water and organic extracts from12.5 μg/mL to 200 μg/mL, the cell viability slightly decreased at the high doses (100 and 200 μg/mL) as presented in Fig. 2.5 was not noticeable. Figure The viability of RAW264.7 cells exposed to PM2.5. In vitro, PM2.5 has been shown to induce inflammatory responses in various human and animal cells with time and dose dependent increases in gene expression [2.5 extracts at 12.5, 25, 50, 100, and 200 μg/mL for 12 and 24 h, were evaluated using ELISA. Figure 2.5 water extract in a concentration-dependent manner after comparing with the untreated cells. Figure 2.5 organic extract. A significant injury was found in treated cells with 200 μg/mL WEP or 100 μg/mL OEP, and the dose was used in further experiments.Inflammation played a major role in cell damage caused by PMpression , and it pression . In the 2.5 water extract or 100 μg/mL PM2.5 organic extract. The IL-6, TNF-α, and MCP-1 levels were elevated after exposure to PM2.5 extracts. The ability of BTE and Coelonin to inhibit TNF-α, IL-6, and MCP-1 secretion induced by PM2.5 water or organic extract is illustrated in Fig. 2.5 extract-only group) occurred for PM2.5 water and organic extract-treated cells treated with 20 μg/mL BTE and 10 μg/mL Coelonin (the highest doses tested)  Fig.  b,c. Fornin Fig. . These r2.5 water and organic extracts  and Coelonin  significantly decreased the TNF-α mRNA expression induced by PMact Fig. . In addiner Fig. , and theect Fig. . BTE or 2.5 extracts. IκBα, NF-κBp65, ERK, JNK, and p38 phosphorylation were significantly increased in PM2.5 water extract (200 μg/mL) and organic extract (100 μg/mL)-treated cells compared with untreated cells, and expression level was related to PM2.5 extracts treatment time.Based on previous literature , NF-κB p2.5 extracts activated NF-κB and MAPK pathway, the mechanistic studies were conducted. BTE and Coelonin acted differently on the pathway activated by the PM2.5 water and organic extracts, but both significantly attenuated PM2.5 extracts induced phosphorylation. Specifically, the levels of NF-κB family protein were significantly elevated after the treatment of PM2.5 extracts, and decreased after the treatment of BTE and Coelonin at some tested concentrations  levels, mediated by NADPH on the cell membrane [Macrophages can phagocytose PMmembrane . Howevermembrane . Cathepsmembrane . The ROSmembrane .B. striata, a well-known traditional Chinese herb, has been widely used to treat hematemesis, ulcers and skin chapping. Previous studies in our laboratory showed that the chloroform and ethyl acetate extract further separated from the B. striata ethanol extract has strong anti-free radical activity, while the chloroform extract also has strong tyrosinase inhibition activity and anti-tumor activity [B. striata and its extracts can interfere with lung injury induced by PM2.5. In this study, we explore the effects of BTE or Coelonin on PM2.5 water extract or organic extract induced cellular inflammatory injury in cultured the mouse macrophage cell line RAW264.7 and further to investigate the molecular mechanism of anti-inflammatory. An elevated level of proinflammatory cytokines such as TNF-α and IL-6 suggests the establishment of local inflammatory response. Our results showed that the increased level of TNF-α, IL-6 and MCP-1 expression in RAW264.7 cells were attributable to the damaging effects of PM2.5 water or organic extract (Figs. 2.5 water and organic extract-treated cells, indicating that the components of PM2.5 water and organic extracts induce inflammatory cytokine secretion independently of NF-κB, MAPK pathway, and inflammasomes (Fig. 2.5 extracts induced macrophages. Furthermore, ERK, JNK, and p38 protein phosphorylation levels were assessed by Western blotting (Figs. B. striata ethanol extract which were in accordance with previously published evidences [2.5 may be related to the inhibition of NF-κB activation and MAPK signals. The secretion of inflammatory cytokine was effectively suppressed by BTE and Coelonin, suggesting that BTE and Coelonin can potentially become effective components of anti-inflammatory medicine. Their specific mechanisms for anti-inflammatory effects are to be further investigated.activity  and the activity . It is ict Figs.  and 4. Pmes Fig. . The resng Figs. . Resultsng Figs. . These rvidences . Its pro2.5 extracts can induce inflammatory responses and BTE and Coelonin may protect mouse macrophage through anti-inflammatory, possibly via the involvement of NF-κB/MAPK signaling pathways. Based on the protective effects of BTE and Coelonin observed in pretreated cells, the regulation of BTE and Coelonin can be a fascinating approach to prevent/treat macrophage injury in inflammation response. B. striata, a well-known traditional Chinese medicine, alleviates PM2.5 extract-induced pulmonary inflammation. BTE and Coelonin inhibited the expression of inflammatory cytokines in RAW264.7 cells. Thus, B. striata may be useful as a pharmacological agent to protect against PM2.5-induced inflammatory disease.In summary, the present study indicates that PMAdditional file 1. Isolation, purification and identification of Coelonin.


C-methyluridine and 2′-C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking group. Substitution of a 2-(methylthio) ethyl phosphoramidate for the aryloxyl phosphoramidate ProTide group of 2′-C-methyluridine completely abolished antiviral activity of the compound. The aryloxyl phosphoramidate ProTide of 2′-C-methyluridine outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and protection from cytopathic effect, while the former compound’s triphosphate active metabolite was better incorporated by purified ZIKV NS5 polymerase over time. These findings suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue ProTides in a disease-relevant cell model.Zika virus (ZIKV), an emerging flavivirus that causes neurodevelopmental impairment to fetuses and has been linked to Guillain-Barré syndrome continues to threaten global health due to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) have been employed to improve the bioavailability of ribonucleoside analogues. Here, we synthesized and tested a small library of 13 ProTides against ZIKV in human neural stem cells. Strong activity was observed for 2′- The explosive spread of Zika virus (ZIKV) during the 2015–2016 epidemics in Latin America attracted worldwide attention to this previously neglected disease. The lack of effective vaccines or small molecules to prevent or treat this infection remains a cause for concern and emphasizes the urgent need for new therapeutic options -l-alanine 1-methylethyl ester and to afford 2′-C-ethenyluridine aryloxy phosphoramidate 5.All nucleoside analogue ProTides, including McGuigan aryloxy phosphoramidite ProTides and 2-(methylthio)ethyl tryptamine ProTides, were synthesized using established phosphorus chemistry ,26. The ic route  27]. Th. ThC-eth/benzene . Ribosyl1 was synthesized by following a previously reported procedure -L-alanine 1-methylethyl ester  was added. The reaction mixture was stirred for 18 h as the temperature was allowed to warm to room temperature. The solvent was removed by rotary evaporation. The reaction mixture was purified first using flash chromatography (a gradient from 0 to 30% methanol in dichloromethane) and then using preparative, normal-phase HPLC (10 to 40% MeOH in dichloromethane gradient) to afford 25.5 mg of the product (54.3%) in ≥ 95% purity. 1H NMR : δ= 7.65 , 7.40–7.34 , 7.27 , 7.20 , 6.04 , 5.61 , 4.97 , 4.49 , 4.36 , 4.16 , 4.08 , 3.92 , 3.08 , 1.35 , 1.22 . 31P NMR  δ= 3.78.To a stirred suspension of 2′-1  in benzene (2 mL) and ethanol (2 mL) under H2 (g), we added 5% palladium on barium sulfate  followed by quinolone (22 µL) and stirred at room temperature for 2 h. The mixture was diluted in ethyl acetate, washed 3 times with water, and dried over anhydrous sodium sulfate. The reaction mixture was purified using flash chromatography (0 to 30% ethyl acetate in hexane gradient) to afford purified product;  1H NMR : δ = 8.23–8.09 , 8.07–8.03 , 7.92–7.88 , 7.69–7.39 , 7.18–7.12 , 6.46 , 6.25 , 4.54 , 4.81 , 4.73 , 4.54 To a stirring solution of 2  were dried under high vacuum in separate round bottom flasks for 2 h. Under N2 (g) and stirring, we added dry acetonitrile (2 mL) to uracil followed by the addition of bis(trimethylsilyl)acetamide . The reaction mixture was refluxed at 80 °C for 1 h then cooled to 0 °C. Then, compound 2 in dry acetonitrile (2 mL) was added to the reaction mixture followed by tin (IV) chloride  and heated to 60 °C for 3 h. The reaction mixture was poured into a separatory funnel containing ice cold water, extracted 3 times with ethyl acetate, and the combined organic layer was dried over anhydrous sodium sulfate. The reaction mixture was purified using flash chromatography (0 to 100% ethyl acetate in hexane gradient) to afford purified product;  1H NMR : δ= 9.22 , 8.09 , 7.86–7.82 , 7.63–7.56 , 7.63–7.56 , 7.29–7.21 , 6.65 , 6.12 , 6.04 , 5.64 , 5.46–5.40 , 4.94 , 4.81 , 4.66 Uracil  and 3  was dried overnight on high vacuum. Under N2 (g), methanol (1.5 mL) was added, then the reaction mixture was cooled to 0 °C followed by the dropwise addition of sodium methoxide . The reaction mixture was raised to room temperature and stirred for 1.5 h. The reaction mixture was cooled to 0 °C followed by the addition of formic acid until pH = 4. The reaction mixture was dried in vacuo then purified using flash chromatography (0 to 40% methanol in dicholoromethane gradient) to afford a purified product;  1H NMR : δ= 8.13 , 5.95 , 5.74–5.65 , 5.44 , 5.26 , 4.22 , 4.03–3.97 , 3.84–3.79 Compound 4  in dry tetrahydrofuran (1 mL) at 0 °C was added tert-butyl magnesium chloride . The reaction mixture was raised to room temperature and allowed to react for 30 min. The reaction mixture was cooled to 0 °C, then N-[(S)-phenoxyphosphinyl]-L-alanine 1-methylethyl ester  was added and gradually warmed to room temperature overnight. The reaction mixture was purified using flash chromatography (0 to 30% methanol [MeOH] in dichloromethane gradient) to afford the purified product;  1H NMR : δ= 7.76 , 7.38 , 7.31–7.25 , 7.25–7.16 , 5.94 , 5.68 , 5.60 , 5.48 , 5.44 , 5.27 , 4.96 , 4.58–4.49 , 4.45–4.38 , 4.17 , 4.00–3.87 , 1.38 , 1.21 . 31P NMR  δ = 3.78.To a stirring solution of C-methyluridine  and the triethylammonium 2-(methylthio)ethyl phosphonate  were added to an oven-dried schlenk tube and dried overnight on the high vacuum. To the flask under N2 (g) was added dry pyridine (2 mL) followed by the dropwise addition of trimethylacetyl chloride , and the reaction was stirred for 3 h at room temperature. The reaction was quenched by saturated sodium bicarbonate and extracted 3 times with dicholormethane. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo at room temperature and dried for 2 h on high vacuum. The dried residue was dissolved in pyridine (2 mL) under N2 (g). To the reaction mixture was simultaneously added trimethylamine , tryptamine , and carbon tetrachloride  and stirred for 25 min at room temperature. The reaction mixture was diluted with methanol and purified using flash chromatography (0 to 30% methanol in dichloromethane gradient) to afford a purified product as a mixture of two epimers at phosphorus;  1H NMR : δ= 7.72 , 7.55–7.50 , 7.33–7.29 , 7.06 , 6.97 , 5.95 , 5.64 , 4.37–4.29 , 4.17 , 4.04 , 4.00–3.90 , 3.77 , 3.21 , 2.96 , 2.66 , 2.07 , 1.13 . 31P NMR  δ= 10.52 and 10.41.The 2′-2.Human fetal NSCs were purchased from Clontech . Maintenance of hNSCs was performed in Neurobasal medium without phenol red  and in the presence of B-27 supplement , N-2 supplement , 20 ng/mL fibroblast growth factor , GlutaMAX , and sodium pyruvate. Cells were prepared in 6-well plates precoated with laminin  and were grown to near confluence (80 to 90%) prior to passage. For passaging, cells were rinsed gently with phosphate-buffered saline (PBS)  and then treated with TryPLE for 5 min at 37 °C for cell detachment . The GSC 387 cell line was prepared and maintained similarly to what has been previously described using the media and detachment conditions used for hNSCs described above  supplemented with 1% DMSO (Sigma) and 5% fetal bovine serum (FBS) (Gibco) was used to resuspend concentrated viral stocks, and aliquots were stored at −80 °C. Plaque assay on Vero cells was used to determine viral titers, which were greater than or equal to 2 × 1050 and CC50 curves.NSCs (5000 cells/well) were seeded into 1536-well black, clear bottom plates containing 20-point, 2-fold serial dilutions of ProTides or DMSO vehicle controls and were either mock-infected, infected with ZIKV H/PAN/2016/BEI-259634 at the multiplicity of infection (MOI) of 10, or infected with ZIKV PRVABC59/Human/2015/Puerto Rico at MOI of 10. Cell viability was measured using the CellTiter-Glo assay  72 h post-infection. An EnVision plate reader (PerkinElmer) was used for readouts of luminescence intensity. All data were normalized to DMSO vehicle controls and were expressed as % relative luminescence intensity. Normalized activity and toxicity data were plotted against ProTide concentration and fit to a sigmoidal dose-response curve with variable slope using Prism 6 (GraphPad Software) to generate ECC-methyluridine aryloxyl phosphoramidate ProTide, or DMSO vehicle controls, and were either mock-infected or infected with ZIKV H/PAN/2016/BEI-259634 at MOI of 10. Cell foci images were acquired 72 h post-infection in bright-field using an ImageXpress Micro automated microscope .GSC 387 cells (5000 cells/well) were seeded into 1536-well black, clear bottom plates with wells containing either 10 or 30 µM sofosbuvir, 10 or 30 µM 2′-2 at 37 °C in high-glucose DMEM with 1% FBS and 1% penicillin/streptomycin for 24 h before the addition of the viral inoculum from ZIKV-infected hNSCs. Inocula were collected from ZIKV H/PAN/2016/BEI-259634-infected NSCs (MOI 0.1) in the presence of 10 μM or 30 μM 2′-C-methyluridine aryloxyl phosphoramidate ProTide, 10 μM or 30 μM sofosbuvir, mock-infected cultures, infected cultures without compound treatment, and infected cultures with the DMSO vehicle at 48 h post-infection. Collected inocula were diluted 10-fold in high-glucose DMEM containing 1% FBS and 1% penicillin-streptomycin in 96-well plates. These inocula were added to the pre-prepared Vero cells for 1 h in a volume of 100 µL and at a temperature of 37 °C, covered with an agarose overlay, and lastly incubated for 72 h at 37 °C and 5% CO2. Fixation with 4% formaldehyde  for 24 h was then performed for the samples. Following this, the overlay was removed, and crystal violet staining was performed to count the ZIKV plaques.Vero cells were used for the titering experiments for drug-treated and vehicle-treated hNSC cultures infected with ZIKV H/PAN/2016/BEI-259634. Vero cells were seeded at a density of 30,000 cells per well in 96-well plates and incubated in 5% COEscherichia coli. Cultures transfected with the RdRP construct were grown at 37 °C in LB media containing 1% ethanol and 100 µg/mL amplicillin until an OD600 of 0.6–0.7 was reached. Protein expression was then induced with 100 µM IPTG prior to an additional 5 h incubation at 20 °C and lower speed shaking (130 RPM). Cells were then harvested by centrifugation at 9000 RPM for 30 min at 4 °C, and pellets were stored at −80 °C. For purification, pellets were resuspended in lysis buffer  supplemented with a protease inhibitor tablet (Thermo Fisher). Cells were lysed using a microfluidizer  and clarified via centrifugation at 12,000 RPM for 30 min at 4 °C. The supernatant was loaded onto TALON cobalt affinity beads  at 4 °C. The unbound protein was washed with 50 mL each of the following buffers: wash buffer  containing 100 mM NaCl and 10 mM imidazole, wash buffer containing 500 mM NaCl and 20 mM imidazole, and finally wash buffer containing 200 mM NaCl and 30 mM imidazole. The protein was eluted in wash buffer containing 200 mM NaCl and a linear imidazole gradient of 30–300 mM. Protein fractions were pooled and dialyzed overnight against 50 mM HEPES at pH 7.0, 100 mM NaCl, 10% glycerol, 0.01% CHAPS, and 5 mM dithiothreitol at 4 °C. The protein was concentrated to ≥6 µM and stored in single-use aliquots at −80 °C.The RdRP domain of ZIKV NS5 (amino acids 2772-3423 of accession number AMB18850) was fused to an N-terminal 6×-histidine tag containing a TEV cleavage site, and the cDNA was codon-optimized and synthesized by IDT . This construct was then inserted into a pET-17b vector (Invitrogen) and heterologously expressed in the Rosetta strain of 32P-labeled at the 5′ position upon incubation with [γ-32P] ATP  and T4 polynucleotide kinase  at 37 °C for 30 min. Excess [γ-32P] ATP was removed with a Bio-Spin 6 column . After heat inactivation, the 5′-32P labeled primer was combined with an excess template (5′-AUUCACUCAGAUCAACAACU-3′). The primer and the template were annealed via step incubation at 95 °C (5 min), 55 °C (15 min), and 37 °C (10 min) to generate the primer/template substrate (P/T substrate), where the next correct nucleotide to be incorporated was UTP.To prepare the double-stranded RNA substrate, primer (5′-AGUUGUUGAUC3′) was 2, and 10 mM dithiothreitol. At specified time points, reaction aliquots were quenched with ethylenediamine tetra-acetic acid (EDTA) to a final concentration of 0.3 M.Single nucleotide incororation assays were modeled from previous work . BrieflyPrior to loading equi-volume samples on a 20% polyacrylamide, 8 M urea, TBE  denaturing gel, the quenched samples were combined with dye  and 1 µL of 5 µM D10 (5′-AGTTGTTGAT-3′) trap, followed by incubation at 95 °C for 5 min. The trap was used to bind to RNA template to reduce gel smearing . Oligos 2+, and sofosbuvir-DP  using PPDB 4WTD : 1) a moP (gc-o3 ) generatP (gc-o3  (160 atoP (gc-o3 ) (154 atP (gc-o3 )  and CC50 (cytotoxic concentration 50) values from these data  and the 2′-C-ethynyluridine aryloxyl phosphoramidate ProTide . These outperformed sofosbuvir in terms of anti-ZIKV activity . Activity was observed for the 2′-C-methyladenosine aryloxyl phosphoramidate ProTide  and 2′-C-methylcytidine aryloxyl phosphoramidate ProTide against PRVABC59 strain , albeit at lower levels. None of the other ProTides tested displayed activity. Interestingly, the 2′-C-ethenyluridine aryloxyl phosphoramidate ProTide was completely inactive and quite toxic to the host cells, suggesting that this functional group may be more promiscuous in terms of target binding or reactive with cellular components [C-methyluridine ProTide completely abolished activity. This result was not expected because the classic McGuigan ProTides are unmasked with the involvement of both a carboxyesterase and HINT1, whereas the methylthioethyl tryptamine phosphoramidate requires only HINT1 in addition to spontaneous, chemical steps [To profile the activity and toxicity profiles of our library of ProTides, we screened compounds in a 20-point, two-fold dose response with a highest concentration of 50 µM against ZIKV PRVABC59/Human/2015/Puerto Rico and ZIKV H/PAN/2016/BEI-259634 in neural stem cells (MOI of 10) using a luminescent cell-viability assay (CellTiter Glo) and determined ECese data . We obseal steps . ClearlyC-methyluridine aryloxyl phosphoramidate, to protect against ZIKV-induced cytopathic effect, we treated the glioblastoma stem cell line GSC 387 with either 10 or 30 µM 2′-C-methyluridine aryloxyl phosphoramidate and compared this to 10 or 30 µM sofosbuvir or the DMSO vehicle control treatment. Samples were prepared in both ZIKV-infected (MOI of 10) and uninfected conditions. After 72 h post-infection, bright-field microscopy images were obtained for each sample and are shown in C-methyluridine aryloxyl phosphoramidate ProTide was better able to protect against ZIKV-induced cytopathic effect at the lower concentration of 10 µM than sofosbuvir, which is in line with our EC50 data in neural stem cells. These data suggest that monosubstituted 2′-C-modified uridylate aryloxyl phosphoramidate ProTides may be an attractive chemical series to pursue for more potent and selective ZIKV polymerase inhibitors than sofosbuvir.We and others have previously shown that certain lines of brain tumors with stem cell-like genetic programs, such as glioblastomas, are highly susceptible to ZIKV infection ,47,48,49C-methyluridine aryloxyl phosphoramidate ProTide against ZIKV, we performed plaque assays at 48 h post-infection to titer ZIKV  levels during treatment with either 10 or 30 µM of 2′-C-methyluridine aryloxyl phosphoramidate ProTide or 10 or 30 µM of sofosbuvir in human fetal neural stem cells   . Both anreported ,31,50. Iporation ,20. ImpoA previously prepared model (gc-o3 ) of ZIKVC-modified uridylate ProTides (with the exception of the ethenyl derivative) was observed, suggesting a possible nucleobase bias to compound activity. Recent biochemical data examining levels of off-target incorporation of nucleoside analogue triphosphates by the human mitochondrial RNA polymerase may help explain the phenotypic relationship we observed; both 2′-C-methyluridine triphosphate and 2′-C-methyl-2′-C-fluorouridine triphosphate (the active metabolite of sofosbuvir) show much lower levels of incorporation by this host enzyme than 2′-C-modified analogues with other nucleobases [C-modified compounds that were tested in clinical trials. This may also suggest that there is greater selectivity for 2′-C-modified uridylate incorporation by flavivirus polymerases over host polymerases, though the exact mechanism by which this occurs should be examined in future studies.In this work, we demonstrated that ProTides other than sofosbuvir have promise as potential antiviral drugs to treat ZIKV infections. Interestingly, an apparent bias towards 2′-leobases . We propC-methyluridine from an aryloxyl phosphoramidate masking group to a 2-(methylthio)ethyl phosphoramidate resulted in a complete loss of activity, suggesting that the local metabolic environment of the host cell is critical for processing of the ProTide functionality. Further studies examining the relationship between different ProTide functionalities and the cell type dependence of their antiviral activity as well as testing the impact of these effects using in vivo animal model testing are planned in our research groups.We also observed that changing the ProTide functionality of 2′-We have demonstrated here for the first time that ProTide technology can be broadly applied to anti-ZIKV drug development with nucleobase and ProTide group selectivity observed. These data will guide future work to design highly selective, safe, and bioavailable compounds for the treatment of ZIKV infections.


Here, we investigated whether voltage-gated chloride channel-3 (ClC-3) impacts the effects of 17β-estradiol (E2) on spared nerve injury (SNI)-induced neuropathic pain in ovariectomized (OVX) female Sprague Dawley rats that were divided into OVX, OVX + SNI, OVX + SNI + E2, OVX + SNI + E2 + DMSO , or OVX + SNI + E2+Cltx (ClC-3-blocker chlorotoxin) groups. Changes in ClC-3 protein expression were monitored by western blot analysis. Behavioral testing used the paw withdrawal threshold to acetone irritation and paw withdrawal thermal latency (PWTL) to thermal stimulation. Immunofluorescence indicated the localization and protein expression levels of ClC-3. OVX + SNI + E2 rats were subcutaneously injected with 17β-estradiol once daily for 7 days; a sheathed tube was implanted, and chlorotoxin was injected for 4 days. Intrathecal Cltx to OVX and OVX + SNI rats was administered for 4 consecutive days (days 7–10 after SNI) to further determine the contribution of ClC-3 to neuropathic pain. Patch clamp technology in current clamp mode was used to measure the current threshold (rheobase) dorsal root ganglion (DRG) neurons and the minimal current that evoked action potentials (APs) as excitability parameters. The mean number of APs at double-strength rheobase verified neuronal excitability. There was no difference in behaviors and ClC-3 expression after OVX. Compared with OVX + SNI rats, OVX + SNI + E2 rats showed a lower paw withdrawal threshold to the acetone stimulus, but the PWTL was not significantly different, indicating increased sensitivity to cold but not to thermal pain. Co-immunofluorescent data revealed that ClC-3 was mainly distributed in A- and C-type nociceptive neurons, especially in medium/small-sized neurons. 17β-estradiol administration was associated with increased expression of ClC-3. 17β-estradiol-induced increase in ClC-3 expression was blocked by co-administration of Cltx. Cltx causes hyperalgesia and decreased expression of ClC-3 in OVX rats. Patch clamp results suggested that 17β-estradiol attenuated the excitability of neurons induced by SNI by up-regulating the expression of ClC-3 in the DRG of OVX rats. 17β-estradiol administration significantly improved cold allodynia thresholds in OVX rats with SNI. The mechanism for this decreased sensitivity may be related to the upregulation of ClC-3 expression in the DRG. Animal use was approved by the Committee of Animal Experimental Ethics of the First Affiliated Hospital of Medical College, Shihezi University, China. Animals were housed in plastic boxes with controlled temperature (24 ± 2°C), humidity (40–50%), and a 12:12 h light:dark cycle. We selected rats with relatively uniform and stable baseline responses to cold and hot stimuli for the experiment. Rats were OVX bilaterally, and the sham OVX (ShamOVX) group underwent operations as previously described  through the catheter to verify the intraspinal location. Immediate bilateral hindlimb paralysis (within 15 s) lasting 20–30 min confirmed correct catheterization. Animals without these features were excluded from subsequent experiments. DRGs for patch clamps were incubated with Cltx in vitro. The 7-day procedure of 17β-estradiol  administration was performed as previously described , OVX + SNI + E2 + DMSO, and OVX + SNI + E2 + chlorotoxin (Cltx). For intrathecal delivery  , Cltx waescribed .Rats were deprived of food overnight, and serum 17β-estradiol levels were assessed according to a previously described protocol . BrieflyThermal hyperalgesia was assessed according to a previously described protocol . The theCold sensitivity was measured by applying a drop of acetone to the plantar surface of the hind paw as previously described . Rats we4–6 DRGs tissues were collected. Samples for RT polymerase chain reaction (RT-PCR) and western blot experiments were snap-frozen in liquid nitrogen and stored at −80°C. Samples used for immunofluorescence imaging were perfused through the ascending aorta with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer  as previously reported . Rats were sacrificed after behavioral testing was performed, and ipsilateral Lreported .4–6 DRG on the surgical side was removed and fixed in 4% paraformaldehyde overnight, followed by dehydration in 20% or 30% sucrose in phosphate buffer at 4°C. The tissue was cut into 5-μm thick sections with a cryostat . The sections were blocked with 20% bovine serum albumin (BSA) for 1 h in a 37°C incubator , washed with phosphate-buffered saline (PBS), and incubated with primary antibody  overnight at 4°C. After washing with PBS, the sections were incubated with secondary antibody  for 1 h at 37°C. For double immunofluorescence staining, tissue sections were incubated with a mixture of anti-ClC-3 antibody and antibodies against neurofilament-200 , calcitonin gene related peptide  for 2 nights at 4°C, or IB4 . Except for IB4-treated tissue sections, the other sections were treated with a mixture of FITC- and TRITC-conjugated secondary antibodies at a 1:100 dilution for 1 h at 37°C. IB4 was 1:750 mixed with TRITC-conjugated secondary antibody. The sections were rinsed with 0.01 M PBS three times, mounted on gelatin-coated slides, and air dried. Immunoreactivity was visualized by fluorescence microscopy, and a negative control was used by omission of the primary antibody to confirm the specificity of the immunoreaction. Sections were observed at 200× magnification using a confocal laser scanning microscope . Optical density measurements and data analysis of CLCN3-positive cells for the two types of DRG neurons were performed using Image-Pro Plus 6.0 . The percentage fluorescence results of positive neurons of three independent experiments were recorded.The Ltris-buffered saline Twenty three times for 10 min and incubated with the secondary antibodies (anti-mouse immunoglobulin G against the primary antibodies). Staining was visualized using enhanced chemiluminescence . Band intensities were quantified by ImageJ software .Frozen tissues were homogenized, and proteins were extracted using a nucleoprotein and cytoplasmic protein extraction kit  and 30 μg of protein was mixed with sodium dodecyl sulfate sample buffer. Proteins were separated on standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8–10% gels) and transferred onto 0.45-μm nitrocellulose membranes . Membranes were blocked in 5% milk for 1 h and incubated overnight at 4°C with the following primary antibodies: mouse anti-ClC-3  and anti-β-actin . The next day, the membranes were rinsed with 4–6 DRGs of rats using Trizol (Invitrogen) and reverse-transcribed to cDNA using a qRT-PCR kit (Invitrogen) according to the manufacturer’s instructions (Ct)– and used for electrophysiological recordings 6–24 h after plating.Lescribed . The dru2, 2 MgCl2, 10 D-glucose, 10 HEPES; the pH was adjusted to 7.4 with NaOH. The pipette solution contained (in mM): 30 KCl, 100 K-aspartate, 5 MgCl2, 2 Mg-ATP, 0.1 Na- GTP, 40 HEPES; the pH was adjusted to 7.2 with KOH. All chemicals were obtained from Sigma.All recordings were performed on small and medium diameter (20–35 μm) neurons as previously described . Coverslpost hoc tests. Regarding the western blot, PCR, and patch-clamp data, analysis among multiple groups was carried out by one-way analysis of variance followed by Tukey’s post hoc tests. Student’s t-test was used for two-group comparisons. P < 0.05 was considered statistically significant.All data are expressed as mean ± SEM of three independent experiments. The normal distribution hypothesis of the test data and the homogeneity of variance were examined before further statistical analysis. Statistical analysis was performed using SPSS 10.0 . PWCL and PWTL were analyzed using repeated-measures analysis of variance, and multiple comparisons between groups at each time point were conducted using Bonferroni’s P < 0.001; n = 6 in each group).Normal female rats underwent OVX 2 weeks before SNI . BehavioP < 0.001; day 7, 21.53 ± 1.142 vs. 2.283 ± 0.7183, P < 0.001; day 10, 20.13 ± 0.8730 vs. 2.505 ± 0.5909, P < 0.001; day 14, 17.34 ± 1.156 vs. 2.503 ± 0.5914, P < 0.001; day 21, 15.24 ± 0.8483 vs. 2.117 ± 0.6256, P < 0.001; n = 6 in each group). There was no significant change in thermal stimulation . Behavioral tests showed that OVX + SNI rats developed significant cold hyperalgesia. The increased sensitivity to cold stimulation started on the 3rd day after SNI and lasted until the end of behavioral testing . ClC-3 expression, mainly in medium/small-sized as well as in large DRG neurons, indicated that ClC-3 may be involved in the regulation of superficial sensations such as pain.Immunofluorescent double staining experiments showed that ClC-3 protein colocalized with IB4, CGRP, and NF-200 . The per4–6 DRGs at different time points after SNI showed high distribution of ClC-3, and the positive cells in the ipsilateral DRGs decreased in a time-dependent manner after SNI . A significant change in ClC-3 protein was detected after SNI . These changes began on the 3rd day after SNI and reached the lowest point on day 7. The ClC-3 mRNA level was also downregulated on the 10th day after SNI .Immunofluorescent staining in rat ipsilateral Lfter SNI , 5A. QuaP < 0.01; day 7, 7.017 ± 0.5443 vs. 21.53 ± 1.142, P < 0.001; day 10, 6.580 ± 0.9755 vs. 20.13 ± 0.8730, P < 0.001; day 14, 6.867 ± 0.7654 vs. 17.34 ± 1.156, P < 0.001; day 21, 6.807 ± 0.9490 vs. 15.24 ± 0.8483, P < 0.001; n = 6 in each group). The 17β-estradiol injection did not affect PWTL , cold hyperalgesia was partially reversed, and the effect persisted from day 3 until the end of behavioral testing. For thermal hyperalgesia, the analgesic effect was not observed . The qRT-PCR results revealed that 17β-estradiol regulated the expression of ClC-3 at the mRNA level. The OVX + SNI + E2 group had higher ClC-3 mRNA levels compared to the OVX + SNI group .After 17β-estradiol administration, LP < 0.001; day 14, 23.77 ± 0.9978 vs. 5.700 ± 0.7425, P < 0.01; on day 10 of SNI, OVX + SNI + E2 + Cltx vs. OVX + SNI, 25.33 ± 1.113 vs. 20.13 ± 0.8730, P < 0.001, on day 14, 23.77 ± 0.9978 vs. 17.34 ± 1.156, P < 0.001; n = 6 in each group). Vehicle solution had no effect (n = 6 per group). L4–6 DRG tissues were harvested on day 10 of SNI after behavioral testing, and ClC-3 protein and mRNA levels were measured. Western blot analysis revealed that 17β-estradiol could not upregulate the expression of ClC-3 after Cltx was administered . The qRT-PCR and immunoblotting results were not consistent with the western blot analysis results, as Cltx administration did not regulate the expression of ClC-3 mRNA  or 10% DMSO as vehicle, 20 μL, was administered intrathecally to SNI rats for 4 consecutive days . There was no significant change in thermal stimulation .To further determine the contribution of ClC-3 to neuropathic pain, Cltx 1 μM/day or 10% DMSO as vehicle, 20 μL, was administered intrathecally to OVX and OVX + SNI rats for 4 consecutive days . Cold hyperalgesia appeared significantly altered in OVX rats on days 10 and 14 . The mean number of APs at double-strength rheobase (2 rheobase) was higher in the OVX + SNI group . Other action potential parameters such as membrane capacitance, resting membrane potential, and magnitude of APs were not significantly different between the groups  in 50-pA step increments under the current clamp mode to measure the current threshold (rheobase), i.e., the minimal current that evoked an action potential, which was used as a parameter for excitability . All DRGNI group . When 17e groups . Furthere groups . AdminisThis study reported that ClC-3 expression in DRG neurons was not significantly changed 2 weeks after OVX. However, according to the literature, mechanical pain was observed 5 weeks after simple OVX and there were also observed changes in pain-related proteins . We can Neuropathic pain is a worldwide health concern with poor treatment outcomes . IncreasClC-3 mRNA levels were not significantly decreased compared to those in the 17β-estradiol-administered group (+, K+, and Ca2+ channels (When 17β-estradiol was administered, the increase in excitability was attenuated. Conversely, excitability increased after administration of both 17β-estradiol and Cltx, a ClC-3 specific blocker. The most likely ion channel internalization by Cltx in gliomas is ClC-3 . Cltx, wed group . This sued group . Furthered group . Patholoed group . Evidenced group ; it can ed group . Many sted group . Furthered group . The pated group . A previchannels ; however–/H+ reverse transporters (A recent report indicated that ClC-3 is a member of the voltage-gated chloride channel family; its deletion caused increased excitability of DRG cells and decrease in the mechanical pain threshold in rats and mice . ClC-3 bsporters . Accordisporters . It has sporters . In neutsporters . Howeversporters . Future sporters . Reportesporters . Future In conclusion, our results showed the complex interactions involved in estrogen-induced pain regulation and revealed the potent role of 17β-estradiol in neuropathic pain, which was altered in female OVX rats. Estrogen may decrease sensitivity to cold stimulation through increased ClC-3 expression in rats experiencing chronic neuropathic pain 2 weeks after OVX.The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the Medical College of Shihezi University.J-QS, Z-ZX, and L-CZ conceived and designed the experiments. Z-ZX conducted the experiments. Q-YC, S-YD, MZ, and C-YT helped with the experiments. Z-ZX and YW analyzed the data. Z-ZX and J-QS wrote the manuscript. All authors discussed and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Clostridium ljungdahlii are unaffected by cyanide at concentrations of up to 1.0 mM with fructose as a carbon source and up to 0.1 mM with syngas as a carbon source. After the culture is adapted to cyanide it shows no growth inhibition. While the difference in growth is an increasing lag-phase with increasing cyanide concentrations, the product spectrum shifts from 97% acetic acid and 3% ethanol at 0 mM cyanide to 20% acetic acid and 80% ethanol at 1.0 mM cyanide for cultures growing on (fructose) and 80% acetic acid and 20% ethanol at 0.1 mM cyanide (syngas).Cyanide is a minor constituent of crude syngas whose content depends on the feedstock and gasification procedure. It is a known poison to metal catalysts and inhibits iron-containing enzymes like carbon monoxide dehydrogenase of acetogenic organisms. Therefore, it is considered a component that has to be removed from the gas stream prior to use in chemical synthesis or syngas fermentation. We show that the growth rate and maximum biomass concentration of  In addition to these main constituents, crude syngas also contains variable amounts of methane (CH4) and C2-compounds such as ethane (C2H6), ethylene (C2H4), and acetylene (C2H2); tar components like benzene, toluene, xylene, and naphthalene; halogens such as hydrogen chloride (HCl) and hydrogen fluoride (HF); sulfur compounds like hydrogen sulfide (H2S), carbonyl sulfide (COS), and carbonyl disulfide (CS2); and nitrogen species such as nitrogen oxides (NOx), ammonia (NH3), and hydrogen cyanide (HCN) as well as oxygen (O2) and reactive oxygen species (ROS)  . The amoes (ROS)  to 2500 es (ROS) .3 are usable substrates for these microorganisms. Since gas conditioning makes for about 22% of the total investment costs of biomass to liquid (BTL) plants and one-third of the investment costs for gasification alone   and hydrogenase. CODHs are found in various organisms and some inhibitory effects are well described in the literature as reviewed below. Among the above mentioned impurities cyanide is a common inhibitor of CODH. at 10 μM . Other rrmophila , Methano barkeri , Moorellceticum) , Clostriaceticum , Rhodospm rubrum , Acetobam woodii , and C. oformans . The com- and CO . AndersoFe CODHs . Cell-frR. rubrum is completed in less than 5 s and hints toward a rapid-equilibrium inhibition. The inhibition kinetic seems to follow a ping-pong mechanism and CODH has to be in an oxidized state before COS can bind and the whole inhibition process is more complex than with cyanide. Nevertheless, the inhibition is fully reversible when COS is removed from the atmosphere and CODH is incubated in the presence of CO . Although COS, SO2, and CS2 are able to reverse cyanide inhibition, the latter two have proved ineffective as inhibitors for CO oxidation  containing Ni–Fe hydrogenases are less sensitive to acetylene than Ni–Fe hydrogenases without Se. The same tendency can be found for Ni–Fe and Ni–Fe–Se hydrogenases from Methanococcus voltae and M. thermophila . In cell-free extracts of as phase . It has th phase . 40 ppm petitive .C. ljungdahlii under heterotrophic and autotrophic conditions.In literature about the usage of syngas or waste gases for syngas fermentation, the removal of gas impurities is named as a crucial point. As can be seen from the mentioned literature, studies of effects of syngas impurities usually focus on isolated enzymes or cell extracts. So far only few reports can be found in literature about the effects of syngas impurities on growing acetogens. To our knowledge, no reports about the effects of cyanide on growing acetogens exist. Therefore, this work investigates the effect of cyanide ions on growth and product formation of C. ljungdahlii DSM13528. Medium for batch cultivation of C. ljungdahlii in flasks was based on N-morpholino)ethanesulfonic acid (MES), 0.5 g yeast extract , 2 g NaCl, 0.33 g NH4Cl, 0.25 g KCl, 0.25 g KH2PO4, 0.5 g MgSO4⋅7H2O, 0.1 g CaCl2⋅2H2O, 10 mL trace element solution (composition see below), 10 mL vitamin solution (composition see below), and 0.001 g resazurin. Trace element solution contained per liter: 2 g nitrilotriacetic acid, 1 g MnSO4⋅H2O, 0.567 g FeSO4⋅7H2O, 0.2 g CoCl2⋅6H2O , 0.2 g ZnSO4⋅7H2O, 0.02 g CuCl2⋅2H2O, 0.02 g NiCl2⋅6H2O, 0.02 g Na2MoO4⋅2H2O, 0.02 g Na2SeO3⋅5H2O, and 0.022 g Na2WO4⋅2H2O. Vitamin solution contained per liter: 0.002 g biotin, 0.002 g folic acid, 0.01 g pyridoxine , 0.005 g thiamine-HCl, 0.005 g riboflavin, 0.005 g niacin, 0.005 g Ca-pantothenate, 0.005 g cobalamin, 0.005 g 4-aminobenzoic acid, and 0.005 g lipoic acid . The medium was prepared using strict anaerobic techniques and the pH was adjusted to 5.9 using KOH before bottling. Bottles were made anaerobic using a gas mixture containing 20 vol% carbon dioxide in nitrogen . After autoclaving at 121°C, 1 g cystein–HCl⋅H2O per liter was added.If not stated differently, all chemicals were purchased from Carl-Roth (Germany). The strain used in this work was - and degassing of HCN during sterilization and storage. All buffers were prepared in sealed serum bottles. C. ljungdahlii DSM13528 was grown in 50 mL complex media with either 10 g L-1 fructose or 2.02 bar absolute pressure of synthesis gas. The gas atmosphere from anaerobic and autoclaved bottles was replaced with syngas prior to inoculation of syngas experiments by evacuation of the bottles and pressurization with syngas. Fructose experiments were conducted in 125 mL serum bottles and syngas experiments in 250 mL serum bottles. Figure 1 gives an overview of the experiments. The following final cyanide concentrations were investigated : 0 (only phosphate buffer is added), 0.025, 0.05, 0.1, and 1.0. To achieve the desired KCN concentrations for every flask 1 mL of potassium phosphate buffer with or without cyanide was added per 50 mL of culture media. Cultures with 1 mL sterile anaerobic water per 50 mL of culture media were used as controls for growth and product formation. All experiments were conducted in triplicates. Cultures were cultivated at 37°C without shaking. Headspace pressure in cultures growing with syngas was used as an indicator of substrate consumption and was measured before and after collection of liquid samples as well as before and after collection of gas samples using a digital manometer . The sample size for liquid samples was 2 and 5 mL for gaseous samples. Gas samples were analyzed with a 3000 micro-GC  to determine the consumption of the individual components of the syngas used. The micro-GC was equipped with one 10 m molsieve module for CO, H2, and N2 detection and a 10 m PoraPlot Q module for CO2 detection. All modules were equipped with a thermal conductivity detector and analytics used isothermal conditions at 80°C.Due to the toxicity of cyanide, it was added in liquid form. Cyanide was prepared as anaerobic potassium cyanide (KCN) solution in 100 mM potassium phosphate buffer at pH 11 to prevent hydrogenation of CNg for 10 min and OD of the supernatant was measured. The difference of both values gave the OD of the sample. This procedure was necessary because OD values of the supernatant changed over the course of cultivation. For correlation between OD and cell dry weight (CDW), 5 mL samples were taken at the end of cultivation and transferred to dry, pre-weight 15 mL screwing cap reaction tubes. The tubes were centrifuged at 4816 ×g and 4°C for 15 min. The supernatant was discarded and pellets were washed two times with a 9 g L-1 NaCl solution. The tubes with the washed pellets were dried at 60°C for 48 h before they were weighed again and the BDM was calculated. Using the final OD of the cultures resulted in an average correlation coefficient of 0.3 g OD-1.Cell concentrations were determined using an Ultrospec1100pro spectrophotometer  at a wavelength of 600 nm. The optical density (OD) of 1 mL of a liquid sample was measured, then cells were removed via centrifugation at 16,100 ×-1 up to an end temperature of 180°C. Total analysis time was 20 min. Due to incompatibility of available cyanide assays with the medium, analytics of cyanide content of samples could not be conducted.Fructose-containing samples were analyzed for fructose, acetic acid, and ethanol using enzymatic assays of Roche Yellow line . Samples from syngas grown cultures were analyzed for products using a 6890N GC  equipped with auto-sampler, ROTICAP-FFAP capillary column , and flame ionization detector. The carrier gas was helium with a pressure of 1 bar and a split ratio of 7.5:1. The analytical standard mixture consisted of 5 mM ethanol, 5 mM sodium acetate, and 9.09 mM isobutanol in 0.18 M HCl. Samples were prepared by acidifying 500 μL of the sample with 50 μL internal standard solution consisting of 100 mM isobutanol in 2 M HCl. Injection volume was 1 μL of sample or standard. The temperature profile of the column oven started with initial 60°C for 2 min followed by a temperature increase of 10°C minFigure 2. With increasing cyanide concentration, lag-phase increases and maximum growth rate decreases from 0.1 h-1 without cyanide to 0.08 (0.025 mM cyanide), 0.05 (0.05 mM cyanide), 0.02 (0.1 mM cyanide), and 0.05 h-1 (1.0 mM cyanide). Substrate consumption and product formation show this tendency as well. However, since the exponential growth phase in 0.1 mM cyanide happened between 96 and 168 h, where no samples were collected, it is likely that the maximum growth rate is closer to 0.05 h-1. While cultures without cyanide reach a maximum CDW of 0.73 ± 0.03 g L-1, all cyanide containing cultures reach, within the margin of standard deviation, the same maximum CDW of approximately 0.49 g L-1. The exception is the culture with 1.0 mM cyanide, where the maximum CDW is 0.73 ± 0.03 g L-1 the same as at cyanide-free conditions. At this cyanide concentration, cells do not start to grow until 168 h but from then on they consume the same amount of fructose as the other cultivations within the following 96 h. Another effect of increasing cyanide concentrations can be found with the formed products. C. ljungdahlii converts consumed fructose into acetic acid and ethanol. The yields for those two are 0.76 (acetic acid) and 0.03 g g-1 (ethanol) when growing without cyanide. Those yields shift with increasing cyanide concentrations. For acetic acid values of 0.61 (0.025 mM cyanide), 0.65 (0.05 mM cyanide), 0.48 (0.1 mM cyanide), and 0.06 g g-1 (1.0 mM cyanide) are obtained while the yields for ethanol are 0.05 (0.025 mM cyanide), 0.11 (0.05 mM cyanide), 0.14 (0.1 mM cyanide), and 0.48 g g-1 (1.0 mM cyanide).First experiments on the influence of cyanide used concentrations of 0, 0.025, 0.05, 0.1, and 1.0 mM KCN in cultures with either fructose or synthesis gas as a substrate. The effects of cyanide on growth and product formation are shown in C. ljungdahlii, once it starts growing at 1.0 mM cyanide, reaches CDW concentrations of the same value as without cyanide, the next step is to use the grown culture from 1.0 mM cyanide containing medium (aka induced strain) to inoculate fresh cyanide-containing medium. Therefore, experiments with 0, 0.1, and 1.0 mM cyanide were conducted using non-induced C. ljungdahlii and the induced strain. Figure 3 shows the results of that comparison. Without cyanide present, the strains reach comparable maximum CDW of 0.72 ± 0.03 (non-induced strain) and 0.78 ± 0.11 g L-1 (induced strain). However, with increasing cyanide concentrations the induced strain shows no significant delay in growth at 0.1 mM cyanide and a lag-phase of about 24 h at 1.0 mM cyanide. The non-induced strain on the other side has a lag-phase of about 24 h at 0.1 mM cyanide and of about 260 h at 1.0 mM. Notably, the induced strain reaches a maximum CDW of 0.83 g L-1 at 0.1 mM cyanide, which is above the values found in cultures of both strains without cyanide. As for the maximum growth rates, the non-induced strain reaches values of 0.11 h-1 (0 mM cyanide), 0.05 h-1 (0.1 mM cyanide), and 0.04 h-1 (1.0 mM cyanide) while the induced strain reaches 0.10 (0 and 0.1 mM cyanide) and 0.06 h-1 (1.0 mM cyanide).Since -1 ethanol at this cyanide concentration, the induced strain produces a maximum of 1.43 g L-1. For cyanide concentrations of 0.1 mM, the induced strain produces more ethanol than the non-induced strain. Maximum acetic acid concentrations in non-induced and induced strains cultures are comparable to the values found in cultures without cyanide. At the highest cyanide concentrations, both the non-induced and induced strains produce equal amounts of ethanol with the difference that the induced strain reaches the maximum concentration after 159 h while the non-induced strain takes 358 h. At 1.0 mM cyanide, the non-induced strain produces 1.3 ± 0.16 g L-1 acetic acid while the induced strain produces 2.85 ± 0.10 g L-1.Substrate consumption of the induced and non-induced strains follows the same pattern as the growth at different cyanide concentrations. Fifty percent of fructose is consumed after 30, 48, and 96 h for the induced and 50, 120, and 324 h for the non-induced strain at 0, 0.1, and 1 mM cyanide, respectively. When looking at product formation, the induced and non-induced strains yield the same concentration of acetic acid at 0 mM cyanide and while the non-induced strain only produces 0.06 g LC. ljungdahlii at the same concentrations of cyanide as for heterotrophic growth. Figure 4 shows the development of CDW and headspace pressure for cultures growing in the presence of 0, 0.025, 0.05, 0.1, and 1.0 mM KCN with a gas atmosphere of 21.1 ± 3.13 vol% H2, 23.1 ± 3.74 vol% CO, 10.4 ± 1.70 vol% CO2, and 39.5 ± 6.85 vol% N2. Cultures were seeded from 5% inoculated cultures grown for 7 days with syngas as carbon and energy source. The control culture (without phosphate buffer) and the culture with 0 mM cyanide show the same growth behavior. For cyanide concentrations of 0.025, 0.05, and 0.1 mM the lag-phase increases with increasing cyanide concentration. However, despite that, cultures at 0.05 and 0.1 mM cyanide reach the same maximum CDW of about 0.20 g L-1 while the 0.025 mM culture reaches with 0.16 ± 0.01 g L-1 a slightly lower value. Maximum growth rates are 0.08 h-1 (control), 0.08 h-1 (0 mM cyanide), 0.03 h (0.025 mM cyanide), 0.04 h-1 (0.05 mM cyanide), and 0.04 h-1 (0.1 mM cyanide). No growth can be observed within 497 h in the presence of 1.0 mM cyanide.After the effects of cyanide on heterotrophic growth on fructose were investigated, we studied the effects of cyanide on autotrophic metabolism of The development of headspace pressure, representing the available substrate, shows a similar trend as the CDW development. Final pressure in cyanide-free cultures is 0.86 ± 0.01 bar. At this pressure, the gas atmosphere consists only of carbon dioxide and nitrogen (data not shown). With their lag-phase reflecting time delay, cultures with 0.025 and 0.05 mM cyanide reach the same pressure as cyanide-free cultures. At 432 h (18 days), the 0.1 mM cyanide containing culture reaches a pressure of 0.90 ± 0.04 bar but has only completely consumed carbon monoxide. Hydrogen partial pressure in that culture after 18 days is 34.42 ± 3.31 mbar. The pressure drop in 1.0 mM cultures is due to liquid and gaseous samples taken from the bottles since no growth or product formation occurred (data not shown).C. ljungdahlii and a strain induced on fructose in 1.0 mM cyanide-containing medium. Pre-cultures for this experiment were grown for 48 h with syngas as carbon and energy source. Syngas composition was 27.1 ± 0.6 vol% H2, 29.0 ± 0.5 vol% CO, 13.5 ± 0.2 vol% CO2, and 28.3 ± 0.4 vol% N2. Figure 5 shows the results for CDW, headspace pressure, and concentrations of acetic acid and ethanol. Cultures of the non-induced strain without cyanide show the shortest lag-phase and reach maximum CDW of 0.12 ± 0.02 g L-1. Non-induced cultures at 0.1 mM cyanide do not start to grow before 188 h and reach a maximum CDW of 0.24 ± 0.01 g L-1. As for the induced cultures, the ones with 0 and 0.1 mM cyanide show the same growth pattern in the first 259 h. The cyanide-free cultures of induced C. ljungdahlii reach a maximum CDW of 0.11 ± 0.02 g L-1 while the ones at 0.1 mM cyanide reach 0.13 ± 0.01 g L-1. No increase in CDW is measured for 1.0 mM cyanide for both non-induced and induced strain. Maximum growth rates for non-induced cultures are 0.04 (0 mM cyanide) and 0.03 h-1 (0.1 mM) and for the induced strain 0.03 (0 mM cyanide) and 0.05 h-1 (0.1 mM cyanide). However, exponential growth could only be found during the first 48 h after growth started. Initial headspace pressure is 2.03 ± 0.01 bar. For each cyanide concentration, the non-induced cultures reach the lowest final pressure with the exception of 1.0 mM where no growth occurred.Similar to the approach with fructose growing cultures, bottles with 0, 0.1, and 1.0 mM were seeded with non-induced -1. At 353 h maximum concentration of acetic acid in 0.1 mM cultures of the induced strain are measured with 1.54 ± 0.24 g L-1. Final ethanol concentrations are below 0.3 g L-1 for all cultures except the non-induced cultures in 0.1 mM cyanide containing medium which have 0.53 ± 0.24 g L-1 ethanol.Final acetic acid concentrations are within the same margin of standard deviation for non-induced and induced strain at 0 mM cyanide and the non-induced at 0.1 mM cyanide. On average they reach 2.06 g LTable 1 summarizes the results of experiments under heterotrophic conditions. Our results show that for cyanide concentrations of 0.025–0.1 mM the resulting maximum CDW is lower in cyanide-free cultures but at 1 mM cyanide concentration it is within the same range as the control cultures. Although when comparing the non-induced with the induced strain at 1.0 mM cyanide in Figure 3, the latter grows to a maximum CDW lower than both strains without cyanide while the non-induced strain, after 261 h lag-phase, reaches a CDW comparable to the control cultures. The reason for this remains unknown. Although the lag-phase of the induced strain in the presence of 1.0 mM cyanide is prolonged compared to 0.1 mM, the cultures start to grow after 20 h. This is less than a 10th of the time the non-induced strain needs to start growing under these conditions, which shows that C. ljungdahlii can be conditioned to grow in the presence of cyanide. Once induced, growth happens at the same rate as without cyanide induction (0.1 h-1) up to CN- concentrations of 0.1 mM.For acetogenic bacteria cyanide is known to reversibly inhibit the enzyme CODH  but avai+ and ferredoxin by capturing the CO2 from decarboxylation of pyruvate. CODH is the central enzyme in the WLP. If it is inactivated by cyanide, capturing of CO2 is no longer possible and the next possibility of recycling NAD+ and ferredoxin is by converting acetic acid to ethanol -1) in both sets of experiments with fructose as a carbon source. It is evident that with increasing cyanide concentration more ethanol is formed while the amount of acetic acid decreases. This indicates that with increasing cyanide concentration CODH activity is increasingly inhibited and NAD+ and ferredoxin are recycled by conversion of acetic acid to ethanol. The overall product yield is supporting this as well. With no CODH activity, the maximum possible product yield is 0.51 g g-1. In our experiments, the YP/S decreases from 0.80 ± 0.08 g g-1 at 0 mM cyanide to 0.55 g g-1 and 0.47 g g-1 in the non-induced cultures and 0.58 g g-1 in the induced cultures at 1.0 mM cyanide.At heterotrophic growth conditions, the purpose of the Wood–Ljungdahl pathway (WLP) is to recycle NAD ethanol . This shTable 2 summarizes the results for CDW, maximum growth rate, and lag-phase of the experiments under autotrophic conditions. Similar to the results with fructose as a carbon source, the lag-phase is prolonged with increasing levels of cyanide, but no increase in CDW or consumption of substrates is measurable at 1.0 mM cyanide. Even the induced strain does not grow at that concentration. 1.0 mM cyanide seems to be a concentration at which no autotrophic growth of C. ljungdahlii is possible. This result is in accordance with the findings from fructose grown cultures, where product spectrum and product yield indicate a loss of most of the CODH activity at 1.0 mM cyanide. Autotrophic growth on CO or CO2 is only possible with the WLP functional. Inhibitory effects of cyanide on CODH will directly result in reduced growth and product formation. However, up to 0.1 mM cyanide the final CDW and maximum growth rate seem unaffected by cyanide. Figure 7 shows the development of the partial pressures of hydrogen, carbon monoxide, and carbon dioxide in the headspace of the culture bottles. At 0.1 mM cyanide, the CO consumption in the induced strain cultures starts after a lag-phase of 48 h and the non-induced strain after 192 h. Both show no other signs of cyanide inhibition when compared with the cyanide-free cultures but completely consume the available carbon monoxide. This indicates that either cyanide is actively degraded or the organism adapts to it in another way. Whatever the mechanism behind the adaption is, it seems to influence hydrogenase activity. Usually, in cultures growing on CO and H2, hydrogenases are inhibited by CO until the liquid CO concentration is low enough to revoke the inhibition  whereas the non-induced strain consumes 260 mbar H2. Hydrogenases are usually not inhibited by cyanide (-, an inhibitory effect of cyanide on hydrogenase activity in the induced strain cultures seems unlikely.hibition . While f cyanide  and sincC. ljungdahlii is adapting to cyanide could not be identified during this study. Hydrocyanic acid in aqueous solution is known to slowly disintegrate into NH3 and formic acid (C. ljungdahlii upregulates enzymes like cyanase, cyanidase, nitrilase, or rhodanese to neutralize HCN (C. ljungdahlii DSM 13528 (C. ljungdahlii adapts to cyanide.How mic acid . While alize HCN . HoweverSM 13528  and the C. ljungdahlii has prolonged lag-phases when grown in the presence of cyanide with fructose or syngas as carbon and energy source. But once the growth starts, the growth rate and maximum CDW are similar to those in cultures without cyanide. Even more, our results are the first to show that C. ljungdahii can be adapted to cyanide. Literature commonly states that syngas used as a substrate for fermentation has to be as clean as possible to avoid inhibitory effects (The work presented here shows that  effects . This ma effects . On the Reported cyanide contents of crude syngas can reach values up to 2500 ppm  which woFO designing of cyanide inhibition experiments, supervision of experiments and evaluation of results, and writing of the manuscript. MZ designing of cyanide inhibition experiments and supervision of experiments, input on the evaluation of results, and critical revision of the manuscript. OO performance of experiments with non-induced strains. EH performance of comparison experiments with induced and non-induced strains. AN substantial input on experimental design and evaluation of results, and critical revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


E. multilocularis causes the disease alveolar echinococcosis (AE) in humans. The continuously proliferating metacestode  of the parasite infects mostly the liver and exhibits tumor-like growth. Current chemotherapeutical treatment options rely on benzimidazoles, which are rarely curative and have to be applied daily and life-long. This can result in considerable hepatotoxicity and thus treatment discontinuation. Therefore, novel drugs against AE are urgently needed. The anti-malarial mefloquine was previously shown to be active against E. multilocularis metacestodes in vitro, and in mice infected by intraperitoneal inoculation of metacestodes when administered at 100 mg/kg by oral gavage twice a week for 12 weeks. In the present study, the same dosage regime was applied in mice infected via oral uptake of eggs representing the natural route of infection. After 12 weeks of treatment, the presence of parasite lesions was assessed in a liver squeeze chamber and by PCR, and a significantly reduced parasite load was found in mefloquine-treated animals. Assessment of mefloquine plasma concentrations by HPLC and modeling using a two-compartment pharmacokinetic model with first-order absorption showed that >90% of the expected steady-state levels  were reached. These levels are close to concentrations achieved in humans during long-term weekly dosage of 250 mg . In vitro structure-activity relationship analysis of mefloquine and ten derivatives revealed that none of the derivatives exhibited stronger activities than mefloquine. Activity was only observed, when the 2-piperidylmethanol group of mefloquine was replaced by an amino group-containing residue and when the trifluoromethyl residue on position 8 of the quinoline structure was present. This is in line with the anti-malarial activity of mefloquine and it implies that the mode of action in E. multilocularis might be similar to the one against malaria.The cestode   •E. multilocularis.Tested anti-malarial mefloquine against mice infected orally with •Reduced liver lesion numbers upon bi-weekly treatment with 100 mg/kg mefloquine.•Similar mefloquine levels reached in mice as in human malaria prophylaxis.•Efficacy of mefloquine is dependent on two molecular residues. ABZalbendazoleAEalveolar echinococcosisPGIphosphoglucose isomeraseSHsodium-hypochloriteTLCthin-layer chromatography1Echinococcus multilocularis causes alveolar echinococcosis (AE) in humans and a variety of mammals, such as dogs, captive monkeys, beavers, and others  as well as in Central and Eastern Europe , and other mammals, may accidentally acquire eggs containing an infectious oncosphere orally, and be infected with the parasite. Following infection, the oncosphere differentiates into the metacestode stage, primarily in the liver, where it infiltrates the adjacent tissue by asexual proliferation of vesicles. Metacestodes exhibit an unlimited reproductive potential, gradually forming cancer-like lumps, often with necrotic areas in the centre. Thus, human AE is a chronic disease with extensive morbidity and mortality if untreated  harbor the adult stage of ntreated . The onlntreated . Benzimintreated . Howeverntreated . Howeveridazoles . A drawbidazoles . ABZ treidazoles . With inidazoles . Two druidazoles .P. falciparum, and over 20′000 hits with activity in the low μM range have been identified. Over the past years, several anti-malarials were shown to exhibit activity against E. multilocularis metacestodes . In addition, ten derivatives of the molecule were tested 22.1All chemicals were purchased from Sigma-Aldrich , if not stated otherwise.2.2E. multilocularis eggs were obtained from naturally infected foxes during the regular Swiss hunting season in spring 2017 according to E. multilocularis worms from the small intestines of foxes were collected and kept in 0.9% NaCl, the worms were squashed and the suspension was first passed through a 105 μm and a 41 μm diameter mesh, followed by a 21 μm mesh . The suspension was stored at 4 °C in PBS with 100 U penicillin and 100 μg streptomycin  (PBS-P/S). The egg suspension was centrifuged every second to third week , the supernatant was removed and replaced by fresh PBS-P/S. Integrity (maturity) of Echinococcus eggs was assessed by sodium hypochlorite resistance test (multilocularis egg suspension (500–1000 eggs/mL). The total number of eggs was determined in a McMaster-chamber. Few minutes after the addition of sodium-hypochlorite solution, oncospheres with intact membranes were counted. Sodium hypochlorite resistance was calculated from triplicate counts as percentage of intact oncospheres.nce test . In brie2.3E.multilocularis eggs (corresponding to 46 viable eggs) suspended in 100 μL PBS. An additional 9 female BALB/c mice received oral gavage of 100 μL PBS only. After two weeks, animals were transferred back to a conventional biosafety level 2 facility.All manipulations with animals followed the guidelines of the Swiss legislation on experimental animal procedures and the experiment was approved by the Bernese cantonal authorities under the license number BE112/14. Eight-week old female BALB/c mice  weighing 20.4 ± 0.8 g at the beginning of the experiment were housed in temperature- and humidity-controlled animal facilities (biosafety level 2) with day/night cycle (12/12 h) and free access to water and food. Prior to egg infection, 35 mice were transferred to a biosafety level 3 animal facility and were infected by oral gavage of approximately 200 2.42, livers were resected and cut into single liver lobes. Each liver lobe was placed into a squeeze chamber and presence of lesions was assessed in a blinded way using a stereo microscope. Lesion numbers of the three infected groups were analyzed by one-sided exact Wilcoxon rank-sum test using the R package coin version 1.2.2  mefloquine treatment (n = 9); (II) albendazole (ABZ) treatment (n = 8); (III) placebo treatment (n = 9). Group (IV) consisted of the non-infected control group (n = 9). Based on power analysis in G*Power (version 3.1.9.2), a power of 0.8 and a p-value of 0.05, the minimal group size was calculated to n = 8. In groups I, II, and IV, we increased this number to n = 9, as for these groups plasma concentration assessments in three times three animals were planned . The samples were digested in 720 μL lysis buffer and 30 μL Proteinase K solution for 2 h at 65 °C. One glass bead of 5 mm in diameter was added prior to digestion and the samples were homogenized just before the start of the digestion and after 1 h of digestion in a FastPrep 24 Tissue lyser  at 4 m/s for 60 s. DNA extraction was then continued according to the manufacturer's protocol with 160 μL of the digested samples. The extracted DNA was subsequently quantified in triplicates using the QuantiFluor dsDNA System  according to the manufacturer's manual. Polymerase chain reaction (PCR) was performed according to The presence or absence of 2.7g at 4 °C. Each plasma sample was then spiked with the internal standard quinine (0.1 g/L in methanol) to 31.25 mg/L. All samples were immediately frozen on dry ice and stored at - 80 °C until analysis by HPLC. At every time point, an internal standard sample with quinine only was frozen in order to follow stability of the standard over time.At least 60 μL blood-samples were taken during the 12-week course of treatment from the tail vein of mice for subsequent analysis of mefloquine plasma concentrations. Blood samples were taken 1 and 5 weeks after treatment initiation (after doses 3 and 11 respectively). 12-week blood samples were taken by heart puncture (after dose 23) after euthanasia. At each of these time points, blood samples were retrieved 6, 24, and 48 h after mefloquine dosage from 3 mice in each group. Blood was taken with heparin-coated microvette tubes and plasma was retrieved by centrifugation for 15 min, 10′000×g. Supernatants were collected and were dried for 2.5 h at 30 °C in an Eppendorf Concentrator 5301. Dried samples were reconstituted in 110 μL acetonitrile/potassium dihydrogen phosphate buffer . After centrifugation , samples were transferred into conical HPLC cuvettes  and were immediately subjected to HPLC.Plasma extraction and determination of mefloquine concentrations in mouse plasma were largely performed according to 2.8A calibration curve was established by spiking of plasma from non-treated mice with a 1:2 dilution series of mefloquine from 25 to 0.098 mg/L (stock in acetonitrile/potassium dihydrogen phosphate), covering the range of the plasma samples. All calibration samples included the internal quinine standard as described above. Three standard curves were prepared independently. All standard samples were extracted exactly as stated for the plasma samples above.2.9HPLC was performed as described previously with adaptations . Mefloqu2.10a, the apparent clearance after extravascular administration CL/F, the intercompartment clearance CLd/F, and the apparent volumes of the central and peripheral compartment V1/F and V2/F. A secondary parameter was the terminal elimination half-life T½. Expected steady-state minimum (Cmin) and maximum (Cmax) concentrations were derived by simulating continued mefloquine dosing. Pharmacokinetic calculations were done using Phoenix WinNonlin 7.0  and Figures prepared in Microsoft Excel (2010) and in Adobe Illustrator 2015.1.0.Mefloquine concentrations were modeled using a standard two-compartment pharmacokinetic model with first-order absorption. Mean mefloquine concentrations and a mean dose of 2.04 mg were used for calculations. Primary parameters were the absorption rate constant k2.11Melting points were determined with a MQAPF-302 Micro Química apparatus and are uncorrected. NMR spectra were determined using 400 or 500 MHz Bruker AC spectrometers using tetramethylsylane as internal standard. Splitting patterns are as follows: s, singlet; d, duplet; t, triplet; quin, quintet; m, multiplet; Brl, broad signal. Infrared spectra were obtained using a Thermo Nicolet 6700 spectrometer. Mass spectra were recorded on Agilent 122 5532 GC/MS column by electron impact and high resolution spectra on a Bruker compact-TOF. The progress of the reactions was monitored by thin-layer chromatography (TLC) on 2.0 × 6.0 cm aluminum sheets  with a thickness of 0.25 mm, ultraviolet light irradiation. For column chromatography, a Merck silica gel (70–230 mesh) was used. Solvents and reagents were used without further purification.in vitro activity against E. multilocularis. The synthesis of six of them  has been described elsewhere (see 10 derivatives of mefloquine were synthesized to be compared to mefloquine here see  (Barbosahere see . The oth2.11.11) was taken as such for the next step without further purification. Melting point (m.p.) 205–207 °C (lit.: 208–210 °C). Phenol 1 was not tested against E. multilocularis, but was needed for further synthesis.Polyphosphoric acid  was added to an equimolar solution of aniline  and ethyl 4,4,4-trifluoroacetoacetate . The reaction mixture was stirred at 150 °C for 2 h. Reaction completion was monitored by TLC. The reaction mixture was slowly poured into ice water (500 mL) with vigorous stirring and stirred at room temperature during 30 min. The precipitated solid was filtered, washed with 50 mL water and dried in a vacuum oven for 4 h to get the crude product as white solid. The crude product  was dissolved in acetone (70 mL) by stirring at room temperature. K2CO3 (6 eq. (11.66) g, 84.48 mmol) and methyl iodide  were then added. The reaction was stirred overnight at room temperature. Then, the solvent was removed under reduced pressure and 70 mL water was added. The aqueous phase was extracted with ethyl acetate (3 × 60 mL) and the combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure to leave a solid. This was purified by flash chromatography  to furnish the compound 4-methoxy-2-(trifluoromethyl)quinoline δ: 8.27 , 8.08 , 7.85 , 7.67 , 7.28 , 4.17 . 13C NMR  d: 164.47, 148.83 , 147.72, 131.95, 131.00, 128.42, 127.49, 121.63, 121.54, 96.08, 55.83. (IR v cm−1: 1315 (C-O-C). Theoretical mass calculated for [C11H8F3NO + H]: 228.0636; Found: 228.0634.The phenol 2.11.31  was dissolved in acetone (70 mL) by stirring at room temperature. K2CO3 (6 eq. (11.66) g, 84.48 mmol) and ethyl bromide  was added. The reaction was stirred overnight. Then, the solvent was removed under reduced pressure and 70 mL water was added. The aqueous phase was extracted with ethyl acetate (3 × 60 mL) and the combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure to leave a solid which was purified by trituration in hot n-Hexane. The compound 4-ethoxy-2-(trifluoromethyl)quinoline  δ: 8.29 , 8.07 , 7.84 , 7.67 , 7.25 , 4.42 , 1.60 . 13C NMR  δ: 165.08, 150.23 , 149.22, 132.40, 129.82, 128.84, 123.15, 123.02, 121.54, 97.96, 66.55, 14.65. IR v cm−1: 1340 (C-O-C). Theoretical mass calculated for [C12H10F3NO + H]: 242.0793; Found: 228.0785.The phenol 2.11.43  and 5 mL of ethanolamine was heated to 130 °C under stirring for 4 h when TLC analyses indicated total consumption of the starting material. Water (15 mL) was added to the reaction mixture and it was extracted with ethyl acetate (3 × 25 mL). The organic phase was dried and evaporated under reduced pressure to yield an oil which was submitted at chromatographic purification on silica gel . The product 2-((2-(trifluoromethyl)quinolin-4-yl)amino)ethanol  in 10 mL of CH2Cl2. After 2 h of reaction at reflux, TLC indicated a total consumption of the starting material. 10% NaOH solution (20 mL) was slowly added and extracted with ethyl acetate (3 × 20 mL). The organic phase was dried with Na2SO4 and evaporated for yield a residue which was submitted to chromatographic purification on silica gel . The product N-(2-chloroethyl)-2-(trifluoromethyl)quinolin-4-amine (compound 5 in 0.3 mL of SOCl2.12In vitro culture of E. multilocularis (isolate H95) metacestodes in co-culture with Reuber rat hepatoma cells was performed as described previously , including penicillin/streptomycin . Parasites were distributed into a 48 well-plate at 1 mL per well and mefloquine or derivatives were added to a final concentration of 10, 20, 30, and 40 μM. 0.1% Tx-100 served as a positive control, DMSO only as a solvent control. Samples were prepared in triplicates. PGI-assays were carried out after 5 and 12 days , as well as the non-infected mice (group IV) underwent treatment for 12 weeks. No adverse effects were observed in any of the animals. Thereafter, all animals were euthanized. The number of liver lesions was assessed by stereo microscopical examination in a squeeze chamber (E. multilocularis lesions in 5 of 9 mice. In ABZ-treated mice (group II), lesions were observed in 3 out of 8 mice, which was not significantly different from the placebo group (p = 0.591). In the mefloquine-treated group (group I), only 1 out of 9 mice exhibited a parasite lesion in the liver, and compared to the placebo group, the difference was statistically significant (p = 0.044)  A. Howevecroscopy .Fig. 2Me3.2E. multilocularis-infected and corresponding non-infected animals at weeks 2, 6, and 12 of treatment. A representative HPLC chromatogram is shown in During the 12 weeks mefloquine-treatment, mefloquine plasma concentrations were periodically assessed by HPLC in a = 0.3 h−1, CL/F = 0.014 L/h, CLd/F = 0.027 L/h, V1/F = 1.04 L, V2/F = 7.39 L. The terminal half-life of mefloquine was calculated as 580 h. After 12 weeks of treatment, the predicted steady-state trough level (Cmin 1.15 mg/L) was reached to 91.4% and the predicted steady-state peak level (Cmax 2.63 mg/L) was reached to 96.6%.The limited number of blood samples that could be drawn from a single mouse did not allow for individual pharmacokinetic calculations. However, to get a more comprehensive picture on the evolution of mefloquine concentrations, a pharmacokinetic analysis using average concentrations was performed B. Parame3.3E. multilocularis. The in vitro activities of 10 structural mefloquine derivatives against E. multilocularis metacestodes were assessed by PGI-assay. As shown in in vitro activity by PGI-assay. With the exception of PASALR-01-144, which was the least active of these compounds, all other active derivatives contain a trifluoromethyl-group in the R1 residue  mg/L and Cmin 1.14 (±0.34) mg/L in one study (max 1.68 (±0.24) mg/L and Cmin 1.12 (±0.29) mg/L in a different study , which includes confusion and disorientation (23.2%), dementia and amnesia (7.2%), and seizures (7.8%), as well as prodromal symptoms such as anxiety (11.3%), depression (17.4%), sleep disturbance (23.3%), and other neurological symptoms . SeriousE. multilocularis lesion whereas in mice without treatment five out of nine had multiple lesions. The egg-infection model of murine AE is not as well developed as the secondary murine model of AE, and up to date assessments of parasite burden at the endpoint have relied solely on morphological observation of parasite lesions in squeezed livers by stereo microscopy. In this study, we have applied an additional and more objective assessment, by using whole-liver PCR based on the method described by Trachsel and colleagues . This is as also the case in dogs with AE, where sensitivity of Em2-serology ranges between 0.52 and 0.92 , is needed to improve the efficacy and safety-profile of mefloquine. In vitro efficacy studies on 10 mefloquine-derivatives against E. multilocularis metacestodes showed that the trifluoromethyl group of mefloquine at the R1 position (position 8 of the quinoline structure) seems to be essential, as it is described against Plasmodium spp. (in E. multilocularis (Plasmodium spp. (Schistosoma mansoni (E. multilocularis metacestodes.To date, little is known regarding the mode of action of mefloquine against ium spp. . The othium spp. , but thiocularis . Furtherium spp. , as well mansoni . Upon su mansoni . For ant mansoni . Accordi5E. multilocularis eggs at 100 mg/kg, is at least as active as 200 mg/kg ABZ applied 5 days per week. In the present study, analytical assessment of plasma levels showed that oral application of mefloquine by gavage led to plasma levels that are slightly above the described levels reached in humans taking the compound for malaria prophylaxis. Thus, there is a promising opportunity that potentially might be exploited also for the treatment of human AE. This could be of particular interest for patients that suffer from severe benzimidazole toxicity. However, due to the inherent variability of the biological material used for such infections in this model, and the limited numbers of mice that got successfully infected, further confirmatory studies need to be carried out in the future. In vitro structure-activity relationship studies show that the efficacy of mefloquine is highly dependent on the presence of two residues, both of which are also essential for its anti-malarial activity. Further studies will be needed to elucidate the precise mode of action of mefloquine against E. multilocularis.We here provide first evidence that bi-weekly mefloquine treatment in mice infected orally with