Spodoptera frugiperda has imposed a serious impact on global food security. Matrine is a botanical pesticide with a broad spectrum of insecticidal activity which was recommended for controlling Spodoptera frugiperda. In order to discover effective insecticide for Spodoptera frugiperda, two matrine derivatives modified with carbon disulfide and nitrogen-containing groups were systhesized. And their inhibition activities on Sf9 cell were evaluated. The structural configuration of compounds were characterized by IR, HPLC, MS, NMR and XRD, with yields of 52% and 65%, respectively. The IC50 of the two newly synthesized compounds on Sf9 cell reduced to 0.648 mmol/L and 1.13 mmol/L, respectively, compared with that of matrine (5.330 mmol/L). In addition, microscopic observation of Sf9 cell treated with the compounds showed that the number of adherent cells decreased, the cells shrunk, vacuolated and apoptotic bodies appeared. The two newly synthesized compounds exhibited better inhibitory effect on Sf9 cell than that of the parent matrine, suggesting that the positive effect of the introduction of 1-pyrrolidinecarbodithioate and diethylcarbamodithioate groups to matrine. The morphological observation of Sf9 cell induced by derivatives indicated that apoptosis induction may be a mechanism that inhibits insect cell proliferation and exerts insecticidal effect.The invasion of  Spodoptera frugiperda (S. frugiperda) with strong resistance and migration ability has imposed a serious impact on global food security. Relevant reports and studies have shown that it is an omnivorous pest with great appetite which may result in reduced or lost harvest1. With the help of air currents and monsoons, S. frugiperda which were divided into corn-strain and rice-strain, can spread across continents4. Therefore, it is extremely difficult to make the prevention and control of S. frugiperda due to its severe harm, fast mobility, wide spreading range, strong drug resistance, etc.The invasion of S. frugiperda were used5. Due to the resistance of S. frugiperda, many traditional synthetic insecticides have not been recommended for use, including organophosphorus, pyrethroids, carbamates and so on, among which its resistance to pyrethroid pesticide has reached hundreds of times7. Researchers have begun exploring natural ingredients as active compounds for the prevention and control of S. frugiperda. Studies on the insecticidal activities of natural compounds and their derivatives against S. frugiperda have been reported, such as botanical essential oils and sesquiterpenoids. It was found that methylchavirol has an effect on development and metabolism of the insect, while pepper oil has an effect on embryo development9. And the examination of 18 sesquiterpenoids showed that the drimanic family with C-9 carbonyl and C-8 and C-9 epoxides have significant antifeedant activity against S. frugiperda and Epilachna paenulata, suggesting the important of structure–activity relationship for exploring pesticides10. However, a few active ingredients have been investigated to effectively control S. frugiperda, so there is an urgent need to develop highly effective biological pesticides.The severity of the insect pests has aroused great attention from the people, and many effective methods for controlling S. frugiperda13. As a biological pesticide with lactam structure, matrine has been recommended as a biological pesticide for controling S. frugiperda. However, the promotion of matrine as a biological pesticide is severely hampered by insignificant activity, slow efficacy and low bioavailability. It is a common method to develop new pesticides by modifying the structure of natural compounds14. A series of novel quinolinomatrine derivatives which were designed and synthesized from natural product insecticide exhibited good insecticidal and acaricidal activities15. The introduction of trifluoroethoxy-containing carbonyl thiourea, trifluoroethyl ether, propargyl ether and diacylhydrazine bridge groups can significantly improve the insecticidal activity of anthranilamide19. According to the special insecticidal mechanism of pyridalyl against lepidopterous larvae, 1,1-dichloropropene derivatives bearing structurally diverse substituted heterocycle rings that replace the pyridine ring of pyridalyl have been designed and synthesized, and the compounds displayed significant insecticidal activity against prodenia litura and diamondback moths20. In addition, our previous studies showed that introducing carbon disulfide and cyclohexylamine groups can improve the toxicity of matrine to Lipaphis erysimi and Mulberry Root-Knot Nematode, while the introduction of indole and cyclohexylamine groups can increase the inhibition activity of matrine on Sf9 cell22.It has been reported that new amides and Bt toxins have a potential to control Sf9 cell through apoptosis induction23. Vip3Aa protein showed a high toxicity against lepidopteran insect larvae and promoted apoptosis of Sf9 cell through mitochondrial dysfunction25. And synthesized proVip3Aa toxin caused cell disruption and death via apoptosis26. Moreover, recombinant virus AcMNPV-Ac34-EGFP could activate the JNK apoptotic signaling pathway to inhibit the proliferation of Sf9 cell, and curcumin could induce nucleophagy by blocking the activation of PI3K/AKT/TOR pathways28. Research on the insecticidal mechanism is of great significance to the development of insecticides.Studying the cytotoxicity of insecticides is helpful to further study insecticidal mechanism. For example, β-Asarone extracted from Acorus calamus Linn exhibited inhibitory activity on 30. Particularly, nitrogen-containing heterocycles proved to have strong systemic conductivity and unique biological activity32. This research aims to improve the inhibitory activity of matrine on S. frugiperda cells by modifing the structure of matrine. Two new compounds were designed and synthesized by introducing 1-pyrrolidinecarbodithioate and diethylcarbamodithioate groups, and their insecticidal effects on Sf9 cell were also explored in vitro.Carbon disulfide is used as an intermediate for pesticides, and nitrogen-containing substances are used as active modification groupsThe synthesis of two compounds were carried out by two nucleophilic additions in one-pot Fig. . Firstly−1 and 1639 cm−1 were assigned to carbonyl group absorption. Additionally, due to the introduction of the groups, the peak at about 1600 cm−1 which was assigned to carbon–carbon double bond of sophocarpine disappeared. And the characteristic bands at about 1068 cm−1 and 1067 cm−1 can be assigned to the ν(C=S) absorption. It indicates that the active groups were introduced into matrine.The IR spectra of compounds 1 and 2 are shown in Fig. HPLC chromatograms of sophocarpine, compounds 1 and 2 are shown in Fig. 20H31N3OS2 and C20H33N3OS2 on the basis of ESI–MS ion peaks at m/z 393.19  and 395.21  respectively.The mass spectrum of compounds 1 ~ 2 are showed in Supplementary Figs. 1H NMR spectroscopy, and a singlet at about 42 ppm in the 13C NMR spectroscopy. The chemical shift peak at about 166 ppm was attributed to the carbon atom of carbonyl from matrine. Even if the chemical structure difference is very small, the peak can be separated in carbon and hydrogen spectrum. The chemical shift of carbon–sulfur double bond (C=S) in compounds 1 and 2 were observed at 190.91 ppm and 193.53 ppm, respectively. And the multiplets at 1.94–2.01 ppm in the 1H NMR spectroscopy and singlet at 20.89 ppm in the 13C NMR spectroscopy can be assigned to the C24, 25 of the compound 1. The C24, 25 of compound 2 showed a triplet at 1.27 ppm in 1H NMR spectrum, and a singlet at 11.71 ppm and 12.66 in the 13C NMR spectroscopy, respectively. This is because the pyrrolidinyl group is cyclic, while the diethylamine group is chain. Thus, the FT-IR and LR–ESI–MS analysis results both demonstrate the successful synthesis of matrine derivatives.The composition of compounds 1 and 2 are determined by NMR spectroscopy, as shown in Supplementary Figs. X-Ray diffraction patterns of compounds 1 and 2 were collected by a XtaLAB PRO MM007HF diffractometer. The crystals were kept at 100.00 (10) K during data collection. Table 28. Therefore, the single crystal diffraction analysis will be compared with the previous data. After the Michael addition reaction, the carbon–carbon double bonds of sophocarpine disappeared, causing the bond length in the lactam ring to stretch, and the bond angle also changed to varying degrees which also indicated that the pyrrolidine and diethylamine groups were successfully introduced.The crystallographic data of compounds 1 and 2 have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1576162 and 1576161, respectively. Some key crystallographic data of compounds 1 and 2 were provided in Tables In compound 1, matrine and carbon disulfide were linked by S19-C13 with the bond length of 1.812 (2) Å, forming the bond angle of C12-C13-S19 110.41 (17)°. In addition, carbon disulfide and pyrrolidine were linked by C20-N22 with the bond length of 1.332 (3) Å, forming the bond angle of C20-N22-C23 [123.1(2)°]  showed that the Sf9 cell treated with 1.0% DMSO were in a good condition at 24, 48, and 72 h, showing complete adherence, vigorous cell proliferation, and in a long or fusiform shape  of matrine to Sf9 cell was 8.586, 5.402 and 5.336 mmol/L at 24 h, 48 h and 72 h, respectively  showed a low inhibitory effect on Sf9 cell, suggesting that the introduction of 1-pyrrolidinecarbodithioate and diethylcarbamodithioate groups can effectively enhance the inhibitory effect of matrine on Sf9 cell. Although the structures of the compounds 1 and 2 are similar, differing only in the nitrogen function groups introduced, i.e., heterocyclic nitrogen ring in compound 1 and a chain structure of amine in compound 2, the activity of the compound 1 was found to be much higher than that of compound 2, likely owing to the unique biological activity of the nitrogen-containing heterocycles32.In summary, compared with matrine, both compounds 1 and 2 showed significant inhibitory effects on Sf9 cell, suggesting the introduction of 1-pyrrolidinecarbodithioate and diethylcarbamodithioate groups to matrine molecular structure could greatly increase the insecticidal activity of matrine significantly. The results of this work provides a new direction for controlling S. frugiperda and a method for improving pesticide activity of matrine through structural modification.Two matrine derivatives containing carbon disulfide and nitrogen-containing groups were successfully synthesized, and their structures were confirmed by various structure characterization techniques. The in vitro activity tests indicated that two matrine derivative compounds 1 and 2 exhibited significant cytotoxic activity against Sophocarpine (purity ≧ 98%) and matrine (purity ≧ 98%) were purchased from Baoji Fangsheng Biological Development Co., Ltd. Pyrrolidine was purchased from Shanghai Macklin Biochemical Co., Ltd. Carbon disulfide, petroleum ether, and anhydrous methanol were purchased from Tianjin Damao Chemical Reagent Factory. Diethylamine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Absolute ethanol, ethyl acetate and dichloromethane were purchased from Tianjin Kaitong Chemical Reagent Co., Ltd., Tianjin Beilian Fine Chemical Development Co., Ltd., and Tianjin Best Chemical Co., Ltd., respectively. Fetal bovine serum (FBS), antibiotics and phosphate buffer solution (PBS) were purchased from Gibco. Dimethyl sulfoxide (DMSO) and Thiazole blue (MTT) were purchased from SIGMA and Guangzhou Qiyun Biotechnology Co., Ltd., respectively.1H NMR and 13C NMR spectra were obtained on a Bruker AVANCE III HD 500 MHz spectrometer (Bruker). X-Ray diffraction patterns of compounds were collected using an XtaLAB PRO MM007HF diffractometer. An Agilent HPLC 1200 was used for HPLC analysis under the main chromatographic conditions, including a CNW Athena C18-WP  column, a detection wavelength of 220 nm, and a mobile phase of 10:80:10 (v/v/v) ethanol/Acetonitrile/KH2PO4 (ϕ = 0.3%) at a flow rate of 1.0 mL min−1.Infrared spectra were measured by Spectrum 100 (PerkinElmer) with KBr disk. Mass spectral data were collected by Bruker amaZon speed. Anhydrous methanol (2.00 mL) and carbon disulfide (1.00 mL) were added into a 50 mL three-neck flask equipped with a condensation and reflux device, and stirred vigorously at 30 °C. Pyrrolidine (0.42 mL) was slowly added into the mixture. The reaction was violent, and white smoke was observed. White smoke disappeared after 0.5 h. Sophocarpine (0.31 g) and anhydrous methanol (2.00 mL) were added with stirring and continued reacting for 24 h at 45 °C. Finally, a slightly black and yellow viscous product was obtained. The reaction mixture was collected after adding 10 mL of anhydrous methanol and filtered by Buchner funnel. The concentrated filtrate was separated by column chromatography and eluted with ethanol: ethyl acetate . After the initial product was obtained, it was purified by crystallization with mixture of ethanol-petroleum ether .Purified water (2.00 mL) and of diethylamine (1.00 mL) were added into a 50 mL three-necked flask equipped with a condensing and refluxing device, and stirred vigorously at 45 °C. Carbon disulfide (0.60 mL) was slowly added into the mixture. A large amount of white smoke and a large amount of white solids were observed in the flask after 2 h. Sophocarpine (0.615 g) and purified water (2.00 mL) were added into the three-neck flask to react at 60 °C for 23 h. A yellow viscous material was obtained. The reaction mixture was obtained after adding 10 mL of dichloromethane and filtered on a Buchner funnel. The concentrated filtrate was separated by column chromatography and eluted with ethyl acetate: methanol . The target product obtained by column chromatography was evaporated and concentrated, and crystallized in petroleum ether.−1): ν  1635 s; ν  1068 s. 1H NMR  δ 1.22–1.53 , 1.53–1.76 , 1.94–2.01 , 2.05 , 2.14–2.27 , 2.63 , 2.78 , 3.10 , 3.59 , 3.86–4.02 , 4.37 . 13C NMR  δ 20.89, 21.31, 24.43, 26.18, 27.84, 31.97, 35.75, 37.80, 41.97, 42.57, 50.94, 52.12, 55.04, 57.47, 64.17, 166.87, 190.91. LR–ESI–MS: calculated for C20H31N3OS2 [M + H]+: 393.2, found: 394.8. HPLC: 7.894 min.Yield: 52%, Colorless. Selected IR data : ν  1639 s; ν  1067 s. 1H NMR  δ 1.27 , 1.39–1.55 , 1.55–1.78 , 1.99 , 2.05–2.29 , 2.66 , 2.81 , 3.11 , 3.71 , 3.95–4.05 , 4.38 . 13C NMR  δ 11.71, 12.66, 20.90, 21.33, 26.73, 27.85, 31.88, 35.75, 37.74, 41.97, 42.70, 46.97, 49.39, 52.17, 57.45, 64.09, 166.92, 193.55. LR–ESI–MS: calculated for C20H37N3O3S2 [M + H]+: 395.4, found: 396.7. HPLC: 7.618 min.Yield: 65%, Colorless. Selected IR data , and the stock liquid was configured by Grace’s Insect Medium with 0.35 g/L sodium bicarbonate. The growth status of Sf9 cell in good condition were selected to prepare a cell suspension with a density of 6 × 104–8 × 104 cells/mL. And then the Sf9 cell were incubated in a 96-well plate with 100 µL cell suspensions in each well at 27 °C for 24 h. Different concentrations of test compound were added to the test group, and 1.0% DMSO was added to the control group. The experiment was set up with 6 repetitive experiments, and the data of 24, 48, 72 h were recorded. Adding 10 μL of MTT solution (prepared in PBS) to the corresponding wells, followed by incubation in the dark for 4 h in an incubator at a constant temperature. All liquid was discarded and 150 μL of DMSO was added to dissolve the crystalline formazan. After the crystals were completely dissolved, the 96-well plate was removed from the bench. The absorbance  of the corresponding well was detected at 429 mm with a microplate reader. Cell viability was calculated by the following equation: Inhibition rate (%) = (1 − ODtrest)/(ODcontrol) × 100%.1.5 ~ 2.0 mL of cell suspension was added to each well in a 6-well plate, and the cells entered the logarithmic growth phase after 24 h of culture. One well was set as the blank control group, and the test compound was added to the remaining wells. The 6-well plate was placed in a biochemical incubator to continue culturing the cells, and the cell morphology was observed and recorded with IPCM after 24, 48, and 72 h, respectively.Sf9 cell induced by different concentrations of compounds at 24, 48, and 72 h. And a linear fit was performed on the inhibition rates at 24 h, 48 h, and 72 h induction time to determine the IC50 of the compounds on the Sf9 cell.The chemical structures were drawn by Chemdraw. Infrared spectras were processed by Origin 2019. Mass spectrums were processed by Qualitative Analysis of Mass Huter Acquisition Date. The NMR spectroscopys were analyzed by MestReNova. Single crystal diffraction data were obtained by Olex 2. The inhibition rates were performed using origin 9.0 and SPSS statistics software, and the standard deviation (SD) of six independent experiments were calculated. Histograms were drawn from the inhibition rates of Supplementary Figures.


More antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance. Therefore, the elimination of antimicrobial crossover between human and veterinary medicine is of great interest. Unfortunately, the development of new antimicrobials is an expensive high-risk process fraught with difficulties. The repurposing of chemical agents provides a solution to this problem, and while many have not been originally developed as antimicrobials, they have been proven safe in clinical trials. PBT2, a zinc ionophore, is an experimental therapeutic that met safety criteria but failed efficacy checkpoints against both Alzheimer’s and Huntington’s diseases. It was recently found that PBT2 possessed potent antimicrobial activity, although the mechanism of bacterial cell death is unresolved. In this body of work, we show that PBT2 has multiple mechanisms of antimicrobial action, making the development of PBT2 resistance unlikely. Streptococcus uberis. In this work, we show that PBT2 functions as a Zn2+/H+ ionophore, exchanging extracellular zinc for intracellular protons in an electroneutral process that leads to cellular zinc accumulation. Zinc accumulation occurs concomitantly with manganese depletion and the production of reactive oxygen species (ROS). PBT2 inhibits the activity of the manganese-dependent superoxide dismutase, SodA, thereby impairing oxidative stress protection. We propose that PBT2-mediated intracellular zinc toxicity in S. uberis leads to lethality through multiple bactericidal mechanisms: the production of toxic ROS and the impairment of manganese-dependent antioxidant functions. Collectively, these data show that PBT2 represents a new class of antibacterial ionophores capable of targeting bacterial metal ion homeostasis and cellular redox balance. We propose that this novel and multitarget mechanism of PBT2 makes the development of cross-resistance to medically important antimicrobials unlikely.Globally, more antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance (AMR). The development of novel ionophores, a class of antimicrobials used exclusively in animals, holds promise as a strategy to replace or reduce essential human antimicrobials in veterinary practice. PBT2 is a zinc ionophore with recently demonstrated antibacterial activity against several Gram-positive pathogens, although the underlying mechanism of action is unknown. Here, we investigated the bactericidal mechanism of PBT2 in the bovine mastitis-causing pathogen, IMPORTANCE More antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance. Therefore, the elimination of antimicrobial crossover between human and veterinary medicine is of great interest. Unfortunately, the development of new antimicrobials is an expensive high-risk process fraught with difficulties. The repurposing of chemical agents provides a solution to this problem, and while many have not been originally developed as antimicrobials, they have been proven safe in clinical trials. PBT2, a zinc ionophore, is an experimental therapeutic that met safety criteria but failed efficacy checkpoints against both Alzheimer’s and Huntington’s diseases. It was recently found that PBT2 possessed potent antimicrobial activity, although the mechanism of bacterial cell death is unresolved. In this body of work, we show that PBT2 has multiple mechanisms of antimicrobial action, making the development of PBT2 resistance unlikely. The widespread emergence of antimicrobial resistance (AMR) threatens the security of modern medicine. Antimicrobial use in food-producing animals is recognized as an important driver of AMR, raising concerns in light of the considerable overlap of antimicrobials used in both humans and animals . With foStreptococcus uberis is a common environmental cause of bovine mastitis  scaffold, has been demonstrated to act as a zinc and copper ionophore in mammalian cells . PBT2 fa20–20–in vitro and in vivo in a murine wound infection model, highlighting the potential of PBT2 to be repurposed as a topical antimicrobial in veterinary medicine  and dysregulates manganese homeostasis, causing cells to become hypersensitive to oxidative stress. Our work builds on the current knowledge of the coordinated relationship between zinc and manganese in bacteria and illustrates how it can be exploited for antimicrobial potential.While PBT2 was shown to increase cytosolic zinc and disrupt metal ion homeostasis in bacteria , a definS. uberis. A checkerboard assay was undertaken to determine the MIC and combined inhibitory concentrations (CICs) of PBT2 and zinc. Both PBT2 and zinc individually displayed antibacterial activity against S. uberis, with MICs of 5.0 mg/liter (14.5 μM) and 800 μM, respectively  was determined by spectrometric measurement on a microplate reader. Data represent the means from biological triplicates. Download FIG S1, TIF file, 0.3 MB.Checkerboard analysis of PBT2 and Zn synergy. Growth of Copyright © 2020 Harbison-Price et al.2020Harbison-Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00157-20.9TABLE S1S. uberis. CIC, combined inhibitory concentration; FIC, fractional inhibitory concentration; FICI, fractional inhibitory concentration index. Download Table S1, DOCX file, 0.01 MB.PBT2 and zinc exhibit antibacterial synergy against Copyright © 2020 Harbison-Price et al.2020Harbison-Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. uberis to develop resistance to PBT2+Zn and two medically important antibiotics, rifampin and the fluoroquinolone antibiotic ciprofloxacin, by serial passaging through progressively increasing drug concentrations over a course of 30 days. While the ciprofloxacin and rifampin MICs increased by 72-fold and 4,608-fold, respectively, the PBT2+Zn MIC increased by only 4-fold .Because antimicrobial activity can be negatively affected by proteins and lipids present in milk , assessiS. uberis cells in response to PBT2 treatment. Consistent with the ionophore activity of PBT2, a concentration-dependent increase in whole-cell zinc accumulation was observed in response to increasing concentrations of PBT2 (P = 0.006)  . Additio= 0.006) . This su= 0.006) . Howeverreatment . This isP = 0.0001) compared to those in untreated cells  or zinc had significantly lower cellular iron than untreated cells   . CollectS. uberis cells in response to PBT2-zinc by using the radioisotopes [14C]methyltriphenyl phosphonium iodide ([14C]TPP+) and [7-14C]benzoate, respectively. No observable differences in either component of the PMF occurred in response to treatment with PBT2-zinc at either 1× or 10× the CIC (0.5 mg/liter PBT2 + 10 μM zinc)   or transmembrane pH gradient (ΔpH)  components of the proton motive force (PMF) . To examinc) see .10.1128/mSphere.00157-20.2FIG S2S. uberis cells following treatment with PBT2-Zn at 1× CIC (0.5 mg/liter PBT2 + 10 μM zinc) or 10× CIC (5.0 mg/liter PBT2 + 100 μM zinc) for 20 min. As a control, cells were treated with toluene (0.4% [vol/vol]) to permeabilize cell membranes and dissipate the ΔpH and ΔΨ. In panels A and B, cells were suspended in THB at pH 5.2 to establish a large ΔpH, while cells in panel C were suspended in THB at pH 7.5. The ΔpH was calculated from the distribution of [14C]benzoate using the Henderson-Hasselbalch equation, and the ΔΨ was determined from the uptake of [14C]TPP+ according to the Nernst relationship. Internal pH was determined from the ΔpH. Error bars represent the standard deviations of the mean from a biological triplicate . Download FIG S2, TIF file, 0.3 MB.Effect of PBT2-zinc on components of the protonmotive force (PMF). Internal pH (A), transmembrane pH gradient (ΔpH) (B), and membrane potential (ΔΨ) (mV) (C) of Copyright © 2020 Harbison-Price et al.2020Harbison-Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the As we observed an increase in cellular zinc, but no effect on the membrane potential of whole cells , we hypoStreptococcus (GAS), vancomycin-resistant Enterococcus (VRE), or methicillin-resistant Staphylococcus aureus (MRSA)  of the liposome internal pH . As PBT2s (MRSA) , we attrs (MRSA) , suggests (MRSA)  36). Co. CoStreps (MRSA) . This sut pH 8.5 . Given t10.1128/mSphere.00157-20.3FIG S3FIG S3, TIF file, 0.4 MB.Effects of PBT2-zinc on internal pH of empty liposomes. Pyranine-containing liposomes with an internal pH of 6.5 (A), 7.7 (B), or 8.5 (C) were suspended in MES-MOPs-Tris buffer of matching pH and treated with PBT2 and zinc. Actual measured internal pH values of liposomes, including untreated and vehicle (DMSO)-treated controls, are shown on the left. The relative change in internal pH of liposomes treated with PBT2 is shown on the right and is normalized for the effect of zinc alone on internal pH. Error bars represent the standard deviations from triplicate measurements. Download Copyright © 2020 Harbison-Price et al.2020Harbison-Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00157-20.4FIG S4FIG S4, TIF file, 0.2 MB.Effect of PBT2 on internal pH of liposomes in the absence of zinc. Pyranine-containing liposomes with an initial internal pH of 6.5, 7.7, or 8.5 were suspended in buffer of matching pH and treated with various concentrations of PBT2. Untreated and vehicle (DMSO) controls are indicated. Error bars represent the standard deviations from triplicate measurements. Download Copyright © 2020 Harbison-Price et al.2020Harbison-Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00157-20.5FIG S5−6 ± 3.12 × 10−5 M and ΔH = −7.48 ± 0.14 kcal/mol. Download FIG S5, TIF file, 0.1 MB.PBT2 binds zinc in a 2:1 stoichiometry. Titration curve and binding isotherm of 0.3 mM zinc injected into 0.035 mM PBT2 at pH 7.7 and 37°C, with best-fit thermodynamic parameter estimates of K = 1.97 × 10Copyright © 2020 Harbison-Price et al.2020Harbison-Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00157-20.6FIG S6−3 M) following suspension in buffer at pH 6.5, 7.7, or 8.5 with various concentrations of PBT2. (C) Change in internal pH of zinc-containing liposomes suspended in buffer of pH 6.5, 7.7, or 8.5 with various concentrations of PBT2, relative to internal pH of untreated controls. Error bars represent the standard deviations from triplicate measurements. Download FIG S6, TIF file, 0.2 MB.Effects of PBT2 on internal pH of zinc-containing liposomes. (A) Internal pH of untreated zinc-containing liposomes following suspension in buffer at pH 6.5, 7.5, or 8.5. Initial internal pH of liposome preparations was pH 7.5. (B) Internal pH of pyranine-containing liposomes loaded with zinc  permease AdcABC, a conserved zinc acquisition system among streptococci (adcA (CGZ53_03345), was downregulated 4.5 log2-fold in response to PBT2-zinc  was upregulated 3.7 log2-fold in response to PBT2-zinc (czcD zinc efflux pump (CGZ53_05790) increased 1.8 log2-fold  was strongly downregulated (3.5 log2-fold) in response to PBT2-zinc treatment  for the conversion of accumulated O2·− to H2O2 and O2. To determine if oxidative stress contributes to the lethality of PBT2 and zinc, we tested whether an antioxidant could prevent cell killing. Pretreatment with reduced glutathione (GSH) (50 mM) added extracellularly, an antioxidant capable of neutralizing ROS in bacteria  (P = 0.0179), but zinc alone had no effect , and when in combination with PBT2-zinc, manganese partially restored SodA function (P = 0.0345)  . Treatmeo effect . Transcrtion see . Provisi 0.0345) .10.1128/mSphere.00157-20.7FIG S7ahpF, ahpC, and sodA genes in mid-log-phase S. uberis cells (OD600 of 0.3) after 1 h treatment with or without PBT2 (1.0 mg/liter) and zinc (100 μM), individually or in combination. Relative expression (expressed as log2-fold change) was calculated relative to the untreated control and normalized to the reference gene (pflC) using the TΔΔC method. Error bars represent the standard deviations of the means from biological triplicates. Download FIG S7, TIF file, 0.1 MB.Expression of oxidative-stress response genes following PBT2 and zinc treatment. Relative expression of Copyright © 2020 Harbison-Price et al.2020Harbison-Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2O2. We found that PBT2 or zinc alone had no effect on H2O2 levels compared to that in untreated cells (2O2 (P = 0.0380)  for the detoxification of H2O2, yet no changes in expression of either ahpC or ahpF were observed in response to PBT2-zinc    . S. uberBT2-zinc . As a cr 0.0001) . This fi 0.4742) . This suS. uberis was unable to generate significant resistance to PBT2+Zn in comparison to the single-target antibiotics ciprofloxacin  protects against zinc toxicity. In these previous studies, the proposed mechanism of zinc-mediated manganese starvation is through zinc competitively binding to the extracellular PsaA, the manganese SBP of the PsaABC manganese importer, thereby limiting manganese uptake. Given the identical compositions of the metal-binding sites in the S. uberis PsaA homolog (MtuA) and S. pneumoniae PsaA  is reduced 2.3 log2-fold in response to PBT2-zinc. The molecular target of this zinc mismetallation was not identified in our study; however, mismetallation of metalloregulators, which sense specific metal ions and modulate transcription of regulated genes in accordance with physiological needs, was previously observed under conditions of cytosolic metal intoxication  does not affect intracellular zinc content nor SodA activity yet depletes manganese to similar levels as PBT2 alone, PBT2-mediated zinc accumulation may inhibit SodA function through zinc mismetallation. Reduced SodA activity as a result of the zinc ionophoric activity of PBT2 may therefore result from a combination of depletion of the essential manganese cofactor and mismetallation of SodA with zinc. Mismetallation of the GAS SodA with iron reduces the enzyme activity ; however2O2 in PBT2-zinc-treated cells indicates a bactericidal mechanism whereby intracellular zinc intoxication leads to the generation of ROS, in addition to impairing the ability to manage oxidative stress. Zinc lacks biological redox activity but may perturb the intracellular redox status indirectly through protein mismetallation and inactivation. Indeed, heme accumulation following zinc mismetallation of PerR causes toxic superoxide generation as a result of redox cycling between heme and menaquinone  may potentiate this oxidative stress by reacting with H2O2 in the Fenton reaction, generating damaging hydroxyl radicals (·OH). Extensive oxidative damage to DNA by ·OH and to cellular proteins by O2·− and H2O2  (Sigma-Aldrich) or on THB agar (THA) containing 1.5% (wt/vol) agar for all experiments. Bacteria were routinely grown at 37°C with agitation (200 rpm). All inoculations were performed from overnight cultures (16 h) to a final optical density at 600 nm (OD600) of 0.05.ain NZ01  was isol4·7H2O (zinc) and MnSO4·H2O (manganese) stocks were prepared in filter-sterilized distilled water (dH2O). The following radiochemicals were obtained from Moravek Inc.: [7-14C]benzoate (54.1 mCi/mmol), [14C]methyltriphenyl phosphonium iodide (TPP+) (57 mCi/mmol), [14C]polyethylene glycol 4000 (PEG), and [3H]water (1 mCi/g).PBT2 was synthesized as described previously . All stoS. uberis NZ01 (OD600 of 0.05) at a final volume of 200 μl. After 24 h of incubation, cell growth (OD600) was determined by a Varioskan Flash microplate reader (Thermo Scientific). The PBT2 and zinc MIC and CIC were determined as the lowest concentrations that showed no visible growth. The interaction effects were derived from calculation of the FICI, the sum of the FIC for each compound, which is in turn defined as the quotient of the MIC of the inhibitor in combination (CIC) and the MIC of the inhibitor alone, as follows  and zinc (0 to 800 μM) were arrayed in a 96-well flat-bottom microtiter plate and resuspended in THB inoculated with  follows :FICI=FICFICI analysis was interpreted according to threshold values described previously , wherebyS. uberis NZ01 were harvested by centrifugation and washed twice in phosphate-buffered saline (PBS)  before inoculation into pasteurized whole-fat (3.8%) cow milk at an OD600 of 0.1. S. uberis cells were then inoculated 1:2 into milk containing 2-fold serial dilutions of PBT2 with various concentrations of Zn in a 96-well flat-bottom microtiter plate. After a 24-h incubation at 37°C, 10-μl aliquots were spot plated on THA plates according to the Miles-Misra drop plate method  or zinc (800 μM), or THB containing a CIC of PBT2 and zinc (0.5 mg/liter PBT2 and 10 μM zinc) in biological triplicates. Aliquots of cultures were taken at 0, 2, 4, 6, and 24 h postchallenge, serially diluted in 1× phosphate-buffered saline (PBS), and spot plated on THA plates. The surviving CFU/ml of cultures was determined after a 24-h incubation at 37°C.S. uberis NZ01 at an OD600 of 0.05. Cells from the highest PBT2+Zn or antibiotic concentrations that showed growth after 24 h of incubation were subcultured in a new microtiter plate with 2-fold dilutions of PBT2+Zn or antibiotics. Serial passaging was repeated for 30 days in biological duplicates.Serial passaging experiments to generate resistance to PBT2+Zn/antibiotics were undertaken as described in Bohlmann et al. . Broth (S. uberis were diluted to an OD600 of 0.05 in THB and grown to mid-log phase (OD600 of 0.3). Aliquots (45 ml) of mid-log-phase cells were separated into individual flasks and challenged for 1 h with PBT2  with or without zinc (100 μM) or untreated. Samples were prepared for ICP-MS analysis according to the protocol used by Bohlmann et al.  and internal cell volume (3.76 ± 0.78 μl/mg protein). Protein content was determined by a bicinchoninic acid (BCA) assay, and the internal cell volume was estimated from the difference between the intracellular and extracellular partitioning of 3H2O and the nonmetabolizable [14C]PEG, as in a previously described method  of samples were converted to molar units, whereby the intracellular metal ion concentrations of bacterial samples were calculated from the d method .S. uberis were diluted to an OD600 of 0.1 in THB (50 ml) at pH 7.5 and grown to an OD600 between 0.5 and 1.0. For ΔpH experiments, cells were harvested by centrifugation , washed twice in THB (pH 5.0), and resuspended to an OD600 of 1.0. For ΔΨ experiments, cells were harvested under the same centrifugation conditions and then resuspended to an OD600 of 1.0 in THB (pH 7.5). In both experiments, cultures were then energized by incubating with glucose (20 mM) for 1 h at 37°C and 200 rpm. Aliquots of energized cells were either untreated or treated with membrane-permeabilizing toluene (0.4% [vol/vol]) or PBT2+Zn at 1× or 10× the CIC (0.5 mg/liter PBT2 + 10 μM zinc) for a further 20 min . Following this, 1-ml aliquots were incubated in glass tubes with either [7-14C]benzoate  or [14C]TPP+  for 5 min at 37°C. Radioisotope measurements and calculations of the ΔpH and ΔΨ were performed as previously described  and measured for change in internal pH. Error bars represent the standard deviations from triplicate measurements. Download Copyright © 2020 Harbison-Price et al.2020Harbison-Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 4·7H2O (zinc) and PBT2 were dissolved in 100 mM MOPS-Tris buffer . The zinc sample (0.3 mM) was injected (30 × 10 μl) into PBT2 (0.035 mM), and the data were analyzed using Origin 7 software and fitted to a single site-binding model.ITC  experiments were performed at 30°C with continuous stirring using a VP-ITC . ZnSOS. uberis NZ01 cells were grown to mid-log phase (OD600 of 0.3) in THB (45 ml) and treated for 1 h with either PBT2 (1.0 mg/liter), zinc (100 μM), PBT2 and zinc combined, or untreated. Total RNA was isolated using TRIzol-chloroform extraction as described previously  according to the manufacturer’s instructions, and RNA was quantified on a NanoDrop instrument (Thermo Scientific).Biological triplicates of eviously  and puripflC using the threshold cycle (ΔΔTC) method (cDNA for each sample was synthesized using a SuperScript III reverse transcriptase kit (Invitrogen). Primers used for quantitative PCR (qPCR) were designed on Primer-BLAST and are detailed in ) method .10.1128/mSphere.00157-20.10TABLE S2Table S2, DOCX file, 0.1 MB.Real-time qPCR primers used in this study. Download Copyright © 2020 Harbison-Price et al.2020Harbison-Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 600 of 0.05 in THB and grown to an OD600 of 0.3. Cultures were split into 50-ml aliquots and treated for 1 h with PBT2 (1.0 mg/liter), zinc (100 μM), or manganese (800 μM), individually or in combination, or were untreated. Cultures were then harvested by centrifugation , washed with HEPES buffer , resuspended in 1 ml of HEPES buffer, and lysed mechanically by bead beating three times . Lysates were centrifuged  and assayed for SOD activity using a SOD assay kit (Invitrogen) according to the manufacturer’s instructions. Protein concentrations in the lysates were determined by a BCA assay, and SOD activity was normalized for total protein content.Overnight cultures were inoculated to an OD2O2 was measured using the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen) according to the manufacturer’s instructions. An overnight culture of S. uberis cells was diluted to an OD600 of 0.05 in THB and grown to mid-log phase (OD600 of 0.3). Mid-log-phase cells were separated into aliquots (10 ml) and were treated for 1 h with PBT2 (1.0 mg/liter), zinc (100 μM), or manganese (800 μM), individually or in combination, H2O2 (1 mM), or untreated. Cells were then harvested by centrifugation . Cell pellets were washed twice in PBS under the same centrifugation conditions and resuspended in 1× reaction buffer at an OD600 of ∼3.5. For analysis, samples were diluted to an OD600 of ∼0.4 in 1× reaction buffer and Amplex Red reagent/horseradish peroxidase (HRP) working solution in a black-walled, clear-bottomed 96-well microplate. Amplex Red fluorescence was then measured using a Varioskan Flash microplate reader at excitation and emission wavelengths (λex and λem) of 560 and 590 nm, respectively.H


We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs.The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC In tota9 μg/mL) , E. Live9 μg/mL)  revealed8 μg/mL) –E. All a8 μg/mL) .Since VOCs have been reported to contain mutations that confer resistance to RBD-directed therapeutic antibodies such as LY-CoV555 –20, we e50 and IC80 of each experimental antibody 1.4 to 6.3-fold lower than that seen for the WA-1 (IC50 of 0.0008–0.0203 μg/mL and IC80 of 0.0026–0.0435 μg/mL)  .Next, we assessed antibody binding to eleven cell surface expressed spike variants that have appeared subsequent to D614G , 23. We 50 of A23–58.1 was 5-fold lower (IC50 <0.0006 μg/mL)  for all variants and showed more than 4-fold improved potency for 6 of the 10 variants tested (IC50 <0.0008 μg/mL)   . Consistent with published data, REGN10933 did not neutralize Var4 (Y453F/D614G) or Var11 (K417N/E484K/N501Y/D614G) , 27, 28;6 μg/mL) . In cont8 μg/mL) . Neutral7 μg/mL) ,C. Final8 μg/mL) . Togethe50 0.0001–0.0401 μg/mL) but LY-CoV555, CB6, REGN10989 and REGN10933 were not able to neutralize either B.1.351 variant (IC50 >10 μg/mL) (50 <0.0006–0.0115 μg/mL) against B.1.1.7 relative to WA-1. A19–46.1 maintained potency against B.1.351.v2 and ~2.5-fold lower neutralization against B.1.351.v1 , while A23–58.1, B1–182.1 and A19–61.1 maintained high potency against both B.1.351 variants (IC50 <0.0006–0.0108 μg/mL)  , 11 and 0 μg/mL)   . In comp8 μg/mL) . These rThe two most potent antibodies, A23–58.1 and B1–182.1, shared highly similar gene family usage in their heavy and light chains, despite being from different donors . Both us2 surface area from the antibody and 607 Å2 from the spike  (The A23–58.1 epitope is composed of residues between β5 and β6 at the tip of RBD  and 3D. 2) . Lys417 2) . Overall2)  confirmi2)  and 3D. 2) . The lac2)  and the We next sought to understand the structural mechanisms by which A23–58.1 overcomes mutations that cause reduced antibody potency against virus variants. To do this, we superposed the antibody-RBD complex structures of CB6 (PDB ID 7C01) , REGN109We next used the structural analysis to investigate the relative contribution of predicted contact residues on binding and neutralization . Cell suNext, based on structural modeling of the negative stain EM density , we chosin vitro resistance against A23–58.1, B1–182.1, A19–46.1 or A19–61.1  expressing the WA-1 SARS-CoV-2 spike (rcVSV-SARS2)  to identA19–61.1 . rcVSV-SA19–61.1 , G446V (freq. 24%) and G593R (freq. 19%)  and did Finally, a single mutation, F486S (freq. 91%) was positively selected for when virus was incubated in the presence of A23–58.1. This is in agreement with our structural analysis  that shocov.lanl.gov) in whichlanl.gov). For A19lanl.gov). Finallyde novo mutations may be occurring throughout multiple rounds of passaging in the presence of increasing concentrations of antibodies. In each round, the well with the highest concentration of antibody with at least 20% CPE was carried forward into the next round. We found that wells with A19–61.1 or A19–46.1 single antibody treatment reached the 20% CPE threshold in their 50 μg/mL well after 3 rounds of selection (Next, we determined the rates of election . Similarelection . Converselection . While fin vitro time to escape experiments suggest that combinations of these antibodies may have a lower risk for loss of neutralizing activity, these antibodies represent a potential means to achieve both breadth against current VOCs and to mitigate risk against those that may develop in the future.Worldwide genomic sequencing has revealed the occurrence of SARS-CoV-2 variants that increase transmissibility and reduce potency of vaccine-induced and therapeutic antibodies –16. ReceSupplement 1


Aeribacillus pallidus BTPS-2 was isolated from Bhurung geothermal spring of Nepal. The 16 s rRNA sequence showed 99.8 % similarity with the type strain Aeribacillus pallidus DSM 3670. The morphological, physiological and biochemical properties were similar to the type strain. Alpha-amylase from A. pallidus BTPS-2 was purified to 19-fold purification by DEAE-Cellulose ion exchange chromatography. The Km value of amylase on starch was 0.51 ± 0.05 mg/mL. The optimum pH and temperature were 7.0 and 70 °C. SDS-PAGE analysis showed a single band at 100 kDa. The half-life of the enzyme at 80 °C was 2.81 h. The enzyme showed an inhibitory effect in the presence of Fe2+, Pb2+, Sn2+ and Hg2+ at 10 mM concentrations. TLC analysis showed that the enzyme is a liquifying alpha-amylase. The enzyme reduced the viscosity of algal biomass suspension up to 74.2 ± 0.17 % which was more efficient than Bacillus amyloliquefaciens alpha-amylase (80.5 ± 0.2 %).A thermophilic strain,  The supernatant is collected. Ammonium sulphate was added to the culture supernatant to get 75 % saturation level. All precipitation work was performed at 4 °C. The precipitated solution was centrifuged at 15,000 rpm for 15 min at 4 °C. The pellet was dissolved in 1 mL of 0.01 mM phosphate buffer, pH 7.0. Overnight dialysis was performed using 12,000 MW cut-off dialysis bags against 0.01 M phosphate buffer with three changes of the same buffer and dialysate is centrifuged at 15,000 rpm at 4 °C. The dialyzed solution was purified in a DEAE-Cellulose mini-column. The column was washed with 0.01 M phosphate buffer. Enzyme solution was added slowly and again washed with the same buffer. The enzyme was eluted with 1 M NaCl with a flow rate of 1 mL per min and fractions of 1 mL each was collected. All the fractions were analyzed for amylase assay by DNS assay at 55 °C and pH 7.0. Three fractions with the highest amylase activity were collected and dialyzed against 0.1 M phosphate buffer overnight and freeze-dried (Telstar Lyoquest). Denaturing SDS-PAGE of the purified enzyme was performed according to Laemmli  for dete2.82.8.1The effect of temperature on amylase activity was measured from 30 °C to 80 °C at pH 7.0. The amylase activity of appropriately diluted stock enzyme solution was measured as mentioned in section 2.8.2The effect of pH on the alpha-amylase activity was performed in the pH range of 3.0−9.0. The activity of diluted stock enzymes was measured as explained in Section 2.8.3The thermostability of the enzyme was studied at 80 °C. 10 mL of the appropriately diluted enzyme solution was transferred in test tubes and incubated in a water bath at 80 °C at pH 7.0. The residual activity of the enzyme was calculated after 1, 2, 3 and 4 h of incubation at pH 7.0 and 55 °C. Enzyme activity is compared with the activity at time 0 h. as 100 %. The data was fit into the exponential model using Minitab19.0 to calculate half-life.max and Km was determined by incubating the amylase with the final concentration of soluble starch ranging from 0.1 % to 0.7 % in the buffer at optimum pH and temperature. The enzyme activity was measured as explained in section max and Km is determined by nonlinear regression statistical methods using Minitab 19.The kinetic parameter V2.9TLC of the hydrolysis product is performed according to Hansen, . The pro2.102, CaCl2, CoCl2, CuSO4, FeSO4, HgCl2, KCl, MgSO4, MnSo4, NiCl2, Pb(C2H3O2)2, SnCl2 and ZnSO4. The activity of the diluted enzyme was measured at optimum temperature and pH.The stock enzyme solution was added to an equal volume of 2X metal solutions to make a final concentration of 0.1 mM, 1 mM and 10 mM respectively. The salt solution used were BaCl2.11A. pallidus BTPS-2 was tested for the liquefaction efficiency of the algal biomass. The dried biomass of Chlorella vulgaris was provided by Algal Research Laboratory, Department of Biotechnology Kathmandu University. The starch content in algal biomass was quantified by Megazyme starch assay kit K-TSTA-100A. 50 mg/mL of dried algal sample was prepared in 0.01 M phosphate buffer at pH 7.0. The algal sample was sonicated at 20 kHz, maximum intensity in Biologics 150 V T ultrasonic Homogenizer to disrupt algal cells. The sonicated sample was boiled at 100 °C water bath for 10 min to gelatinize the starch. The treated biomass was aliquoted in the Gilmont GV2200 viscometer ensuring no leakage. The sample was allowed to equilibrate to 70 °C in a water bath. The units/mL of the enzyme from A. pallidus BTPS-2 and Bacillus amyloliquefaciens were measured at pH 7.0 and 70 °C. 50 units of the enzyme was added to each algal sample. An algal sample without enzymes was taken as control. The viscosity of the samples was quantified by measuring the time of decent of the glass ball in the interval of 10 min for 1 h. Each sample was run in triplicates. The difference in means was measured by one-way ANOVA at 95 % confidence interval and the means were compared by Tukey’s mean comparison method. The relative viscosity reduction percentage of all samples was measured against the viscosity of the control.Alpha-amylase from 33.1Water samples, algal bio-mats and sediments were collected from Bhurung geothermal springs of Nepal. The maximum temperature of the water sample was 72 °C and pH 7.0. A total of 15 strains were isolated based on colony morphology in nutrient agar plates. Only one isolate (BTPS2) showed significant amylase activity in starch agar plates assay.3.23.2.1A. pallidus DSM 3670. The sequence was published in NCBI gene database with the gene accession no. MH688460. The phylogenetic tree was constructed using the Neighbor-Joining algorithm using NCBI Blast tool  and medium M5 (5.22 ± 0.2 Units/mL) while the enzyme production in medium M1, M2 and M4 were insignificant  was 0.51 ± 0.05 mg/mL. The Vmax value was 1.43 ± 0.02 mM /min .The optimum temperature of the enzyme was 70 °C. However, the enzyme retained 93 % activity at 80 °C and 81 % at 60 °C and 47 % at 100 °C as shown in Figure t pH 7.0 B. The ent pH 7.0 C. The Km3.5+ showed inhibitory effect in the concentration range of 0.1 mM–10 mM. The relative activity of all metal ions investigated were >60 % in 0.1 mM concentration; the maximum inhibition was observed for Hg+ ions with relative activity of 68 %. A stronger inhibitory effect was observed in the presence of Fe2+, Pb2+, Sn2+ and Hg2+ at 10 mM concentration.Alpha-amylase activity at 0.1 mM, 1 mM and 10 mM was studied . All met3.6). The dominant product was maltose and maltotriose seen as distinct spots in the TLC plate. A smear of band corresponding to oligosaccharide with 6–8 degree of polymerization is visible. This suggests that the alpha-amylase of A. pallidus BTPS-2 is a liquefying alpha-amylase.To characterize the mode of action of the alpha-amylase, the hydrolysis products were analyzed by thin-layer chromatography. Soluble starch was converted into glucose maltose and maltotriose . After 1Fig. 73.7A. pallidus BTPS-2 amylase was compared with B.amyloliquefaciens (chlorella vulgaris biomass was estimated to be 27.1 ± 0.38 %(w/w). The sample treated with alpha-amylase from A. pallidus BTPS-2 showed viscosity reduction up to 74.2 % after 60 min incubation. The viscosity reduction ratio of alpha-amylase from B. amyloliquefaciens was 80.5 %.Liquefaction of algal biomass by efaciens . The sta4A. pallidus BTPS-2 were compared with the properties of A. pallidus DSM3670 as reported by Scholz et al. [A. pallidus BTPS-2 was neutrophilic with a pH optimum of 7.0 in comparison to A. pallidus DSM 3670 with pH optimum of 8.0. Both are obligate aerobes and motile. Both were positive for catalase and oxidase test. Hydrolysis of starch, esculin and tributyrin were positive for both strains. Similarly, hydrolysis of casein and gelatin was negative for both strains. Both strains showed similar results in sugar utilization tests. Both strains were negative for lactose, l-arabinose, d-arabinose, trehalose, sorbitol, xylose and malic acid. Utilization of urea, citrate and nitrate reduction test were negative for both strains. BLAST analysis showed that the 16 s rRNA sequence of A. pallidus BTPS-2 was 99.8 % similar with the type strain. The morphological, physiological, biochemical and molecular data showed that the isolated bacterial culture is a new strain of A. pallidus.The morphological physiological and biochemical properties of z et al. . Both arA. pallidus BTPS-2 showed strong amylolytic activity in the starch agar plate. Preliminary screening was carried out in five different medium formulations to identify the optimum medium for amylase production. Higher amylase activity in the fermentation broth was observed in medium M3 and M4. Medium M3 and M4 both contain complex source of nitrogen as well as trace metal salt supplement (yeast extract and casein hydrolysate in M3 and peptone in M5). The incorporation of complex mediums such as peptone and yeast extract along with the addition of trace metal increased the amylase production by several folds. A. pallidus are fastidious halophilic microbes requiring complex salt-rich medium for their growth. This is also reflected by the result that the strains were negative for casein hydrolysis. Their optimum salt concentration was 3 % which reflects their halophilic property. As alpha-amylase is a primary metabolite, the incorporation of casein hydrolysates or yeast extract along with salt solution may have provided optimum growth condition which resulted in higher amylase production.A. pallidus BTPS-2 alpha-amylase was 100 kDa. This is the first report of the experimental calculation of the molecular mass of A. pallidus. However, amylase from other closely related Geobacillus species has been reported. The molecular mass of Geobacillus sp. IIPTn alpha-amylase was 97 kDa [Geobacillus stearothermophilus [SDS-PAGE result showed that the molecular mass of s 97 kDa . High momophilus .A. pallidus BTPS-2 alpha-amylase at 80 °C was 2.81 h. The half-life of other bacterial alpha-amylases has been reported. The enzyme from Bacillus sp. displayed a half-life of 48 min at 80 °C [B. licheniformis 44MB82 was 10 min at 85 °C which increased to 120 min in 5 mM CaCl2. [Bacillus amyloliquefaciens which retained 100 % activity for 24 h at 65 °C and exhibited a half-life of 9 h at 80 °C [Anoxybacillus rupiensis TS‐4 [A. pallidus BTPS-2 is moderately thermostable at 80 °C.The half-life of at 80 °C . The halM CaCl2. . Alpha-aat 80 °C . The halsis TS‐4 . In compm value of the alpha-amylase on starch was calculated to be 0.51 mg /mL for soluble potato starch as a substrate. This is lower than other reported value for commercially important amylase source (Bacillus licheniformis, Km: 0.9 mg/mL [Bacillus amyloliquefaciens BH072, Km: 4.27 mg/mL [Bacillus subtilis, Km:2.68 mg /mL [m value reflects the higher affinity of alpha-amylase of A. pallidus BTPS-2 for soluble potato starch.The K.9 mg/mL , Bacillu27 mg/mL  and Baci8 mg /mL . Lower K+ ions did not show significant inhibition in 0.1 M concentration. The maximum inhibition in 0.1 mM was found for Hg+ ions at 68 %. Alpha-amylases with no obligate requirements for metal ions have been reported in other bacterial species [A. pallidus BTPS-2 could be an important enzyme for industrial applications.All metal salts investigated except Hg species . Most of species . The proA. pallidus-BTPS2 has more liquefying efficiency for algal biomass compared to alpha-amylase from Bacillus amyloliquefaciens. Microalgae are a good absorber of heavy metals from solution [A. pallidus BTPS-2 could be due to its specificity for algal starch as well as the presence of various metal ions in the algal broth.The study showed that the alpha-amylase from solution . The enz5Aeribacillus pallidus strain. The liquefying maltogenic alpha-amylase was found to be highly thermostable and insensitive to metal inhibition. The enzyme showed higher liquefying efficiency for algal starch than alpha-amylase from B. amyloliquefaciens. The enzyme could be a potential candidate for industrial application.This is the first report of purification and characterization of alpha-amylase from Parash Mani Timilsina: Conceptualization, Methodology, Visualization, Writing - original draft, Software, Validation, Funding acquisition, Data curation, Investigation, Writing - review & editing. Gyanu Raj Pandey: Investigation. Asmita Shrestha: Investigation. Manish Ojha: Investigation. Tika Bahadur Karki: Data curation, Supervision, Writing - review & editing.The authors report no declarations of interest.


Zingiber kerrii Craib were investigated using different extraction techniques; including solid-phase microextraction (SPME), hydrodistillation (HD), and organic solvent (OS), and characterized by gas chromatography–mass spectrometry (GC–MS). A total of 37 SPME; 19 HD; and 36 OS compounds were identified from the rhizome extract of Z. kerrii; with the major components being α-pinene; β-pinene; and terpinen-4-ol; respectively. From the flower extract; 16 SPME; 2 HD; and 10 OS compounds were identified; (E)-caryophyllene was found as a major compound by these techniques. The leaf extract exhibited 20 SPME; 13 HD; and 14 OS compounds; with α-pinene; (E)-caryophyllene; and n-hexadecanoic acid being the major compounds; respectively. The rhizome extract showed tyrosinase inhibitory activity of 71.60% and a total phenolic content of 22.4 mg gallic acid/g. The IC50 values of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays were 25.2 µg/mL and 153.6 µg/mL; respectively; and the ferric ion reducing antioxidant power (FRAP) assay value was 318.5 µM ascorbic acid equivalent (AAE)/g extract. The rhizome extract showed weak antibacterial activity. This extract showed no adverse toxicity in human keratinocyte (HaCaT) cell lines at concentrations below 200 µg/mL.The essential oils of the fresh rhizomes; flowers; and leaves of  Zingiberaceae is a large family containing approximately 50 genera and over 1200 species distributed throughout the tropical countries . This faZingiber species -2,5-diphenyl tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich . Folin–Ciocalteu’s phenol reagent was obtained from Merck KGaA . Mueller-Hinton broth was obtained from HiMedia Laboratories . Vancomycin hydrochloride was obtained from the EDQM Council of Europe . Gentamycin sulfate and ampicillin sodium salt were obtained from Bio Basic Canada .Gallic acid, L-ascorbic acid, kojic acid, ferric chloride, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt ABTS, 2,4,6-tris(2-pyridyl)-Z. kerrii. The SPME fiber was purchased from Supelco . Fresh samples (50 g) were transferred to a 250 mL glass septum bottle, then kept in a water bath at 45 °C for 30 min. For each extraction, the SPME fiber was preconditioned for 30 min at 220 °C in the injection port of GC–MS. The fiber was then exposed to the headspace for 30 min. The thermal desorption of constituents was carried out at 250 °C for 5 min.An SPME fiber coated with 50/30 µm divinylbenzene-carboxen-polydimethylsiloxane (DVB/CAR/PDMS) was used to extract the volatile components from all parts of fresh Z. kerrii (100 g) were extracted via hydrodistillation (separately) for 6 h [All fresh parts of  for 6 h  using a Z. kerrii parts were extracted using n-hexane as a solvent. A total of 50 g of each sample was weighed and suspended in 300 mL of solvent, then shaken for 6 h in an electronic shaker at room temperature. Whatman No. 1 filter paper was used for filtering. The solvent was removed via reduced pressure with rotary evaporator at 40 °C, then stored at 4 °C for further studies.Soluble compounds from all of the fresh Dried rhizomes (1 kg) were extracted three times with ethyl acetate (EtOAc) for 72 h at room temperature. The resulting mixtures were filtrated, then the solvent was removed at 40 °C using a vacuum to produce the EtOAc extract (7.67 g), which was stored at 4 °C for further studies.m/z 29–300. The electron multiplier voltage was 1150 V. The ion source and the quadrupole temperatures were set to 230 °C and 150 °C, respectively. The volatile components were identified by comparing their Kovát retention indices relative to the C8–C20n-alkanes standard and comparing the mass spectra of individual components with the reference mass spectra via Wiley and National Institute of Standards and Technology (NIST) database matching. The relative concentrations of the volatile compounds were investigated using a percent relative peak area, as shown in Table 1.The volatile components of essential oils were performed by GC–MS using the Hewlett Packard model HP6890 gas chromatograph  with an HP model 5973 mass-selective detector. The analyses were carried out using a capillary column  of HP-5ms (5% phenylpolymethylsiloxane). The temperature program was set at 60 °C and increased to 220 °C at a rate of 3 °C/min. The temperatures of the injector and the detector were set at 250 °C and 280 °C, respectively. The carrier gas was purified helium (99.99%) at a flow rate of 1 mL/min in split mode 1:70 with an injection volume of 1 μL, which was injected 3 times. For electron ionization, mass spectra were used with an ionization energy of 70 eV and ionization voltages over the range of w/v) sodium carbonate was added to the mixture and incubated for 90 min at the same conditions. The absorbance of the mixture was monitored at 750 nm using a UV-Vis Genesys 30 Visible spectrophotometer . Gallic acid  was used as a positive control to generate a standard calibration curve. The total phenolic content was expressed as gallic acid equivalents in grams per 100 g extract (GAE/100 g).The total phenolic content was determined with the Folin–Ciocalteu assay. The Folin–Ciocalteu reagent was diluted 10-fold with Milli-Q water prior to use . The ext−5 M) of DPPH methanolic solution, mixed well, and incubated in the dark at room temperature for 30 min. The absorbance of the reaction solution was measured at 517 nm using the microplate reader . Ascorbic acid at serially diluted concentrations  was used as the positive control. The DPPH radical scavenging activity was expressed as the inhibitory concentration at 50% (IC50), which was calculated in comparison with the ascorbic acid [One hundred microliters of sample at serially diluted concentrations  was added to 100 µL (6 × 10●+) was prepared by reacting 7 mM of ABTS with 2.45 mM of potassium persulfate and allowing the mixture to stand in the dark at room temperature for 16 h before use. Prior to the assay, the working solution of ABTS●+ was diluted with ethanol to an absorbance of 0.70 ± 0.05 at 734 nm to give the ABTS●+ solution. Twenty microliters of serially diluted sample  was mixed with 180 µL of ABTS●+ solution. The reaction of mixture was allowed to stand in the dark at room temperature for 5 min, then the absorbance of the reaction solution was measured at 734 nm. Serially diluted concentrations of ascorbic acid  were used as the positive controls. The ABTS radical cation scavenging activity was expressed as the inhibitory concentration at 50% (IC50), which was calculated in comparison with ascorbic acid [The working solution of ABTS radical cation (ABTSbic acid .v/v). The FRAP reagent was prepared fresh daily and warmed at 37 °C in a water bath for 15 min prior to use. Fifty microliters of sample was added to 1.5 mL of FRAP reagent and incubated in the dark at room temperature for 5 min, then the absorbance of the reaction solution at 593 nm was recorded. Ascorbic acid at serially diluted concentrations  was used to generate a calibration curve. The results were expressed as µM ascorbic acid equivalents per gram of extract [The working solution of FRAP was prepared by mixing 300 mM of sodium acetate buffer at a pH of 3.6 with 10 mM of TPTZ (solution in 40 mM of HCl) and 20 mM of ferric chloride solution in the proportion 10:1:1  for 24 h at 37 °C in 5% CO2. MTT solution (5 mg/mL) was added to each well and incubated for another 4 h at 37 °C before dissolving the formazan product in 10% SDS–0.01M HCl. Finally, absorbance was measured at 595 nm after 24 h using a microplate reader . The viability of the cells was reported as the mean ± SD of three independent experiments performed in quadruplicate (Table 3) [Human keratinocyte cells (HaCaT) were seeded at a density of 2 × 10Table 3) ,29,30.Bacillus cereus TISTR 687, Staphylococcus epidermidis TISTR 2141, Bacillus subtilis TISTR 1248, and Staphylococcus aureus TISTR 746, and four Gram-negative bacteria, Salmonella typhimurium TISTR 1470, Pseudomonas aeruginosa TISTR 1287, Escherichia coli TISTR 527, and Serratia marcescens TISTR 1354, were obtained from the Microbiological Resources Centre of the Thailand Institute of Scientific and Technological Research. The minimum inhibitory concentrations (MICs) were determined using Mueller-Hinton broth microdilution with serially diluted (two-fold) plant extracts using 96-well microtiter plates. Vancomycin, gentamycin, and ampicillin were used as the positive controls, while DMSO was used as the negative control (Table 4) [Four Gram-positive bacteria, Table 4) .The tyrosinase inhibition activity was determined using a slightly modified dopachrome method with L-DOPA as the substrate ,33. BrieThe statistical analyses were performed using IBM SPSS Statistics, version 23 (IBM Crop.). The principal component analysis (PCA) was conducted to reveal interrelationships between plant parts and extraction methods based on measured characteristics. A two-way analysis of variance (ANOVA) was used to compare the volatile constituents exhibited by plant parts after various extraction methods.v/w) from HD and OS extracts of fresh rhizomes, flowers, and leaves of Z. kerrii were , , and , respectively. Rhizomes represented the highest percentage of essential oil yields using the OS technique. The chemical compositions of rhizomes, flowers, and leaves essential oils are shown in , β-pinene , sabinene , and (E)-β-ocimene  using the SPME and the HD techniques, respectively, whereas the major components of the OS technique included β-pinene (7.7% ± 1.1%), (E)-β-ocimene (4.1% ± 0.8%), terpinen-4-ol (11.5% ± 0.6%), germacrene B (4.2% ± 0.6%), caryophyllene oxide (4.4% ± 0.7%), and n-hexadecanoic acid (8.7% ± 0.9%). The flower essential oils showed 16, two, and nine compounds, of which 99.1%, 97.2%, and 97.1% were identified. The main compound was E-caryophyllene , which was identified via the SPME, the HD, and the OS techniques, respectively. The SPME technique showed α-pinene (22.2% ± 1.8%) and β-pinene (8.6% ± 1.1%) as dominant compounds, but n-hexadecanoic acid (14.9% ± 0.4%) was as a major compound identified by the OS technique. The isolated essential oils of leaves contained 20, 13, and 14 components, which represented 95.4%, 91.8%, and 93.1% of the total oil compositions, respectively. Most of the compounds in the leaves belonged to five chemical classes, which are listed in (α-pinene (39.7% ± 0.7%), β-pinene (6.3% ± 0.5%), and (E)-caryophyllene (21.2% ± 0.8%) using the SPME technique, but the HD technique identified β-elemene (12.2% ± 1.3%), (E)-caryophyllene (24.2% ± 1.6%), and valencene (21.2% ± 1.3%) as the major components. (E)-caryophyllene (6.0% ± 0.8%), isodaucene (4.2% ± 0.9%), and n-hexadecanoic acid (55.7% ± 1.3%) were identified as the main compounds when the OS technique was used.The yields of essential oils (shown in . GC–MS aisted in . The majZ. kerrii (E)-Caryophyllene was the main compound seen in the flowers as identified by several techniques, with the exception of the SPME technique in the rhizomes. In addition, α-pinene and β-pinene were found in several parts, except the flowers when the HD and the OS techniques were used. The results revealed that the HD and OS techniques lost some highly volatile compounds during the process of extraction [Z. officinale include zingiberene, geranial, and other sesquiterpene hydrocarbons [Z. zerumbet leaves reported (E)-nerolidol, α-pinene, and β-pinene as the main compounds [α-pinene, β-pinene, and (E)-caryophyllene were found to be the major constituents of Z. kerrii, which corroborated previous literature reports of Z. zerumbet leaves [Z. kerrii using different techniques.The essential oil compositions of . kerrii  were diftraction ,8,9. Theocarbons ,35, wherompounds . In our t leaves . This woZ. kerrii according to the plant part and the extraction technique -caryophyllene, β-elemene, α-zingiberene, valencene, (E)-β-ocimene, terpinen-4-ol, α-humulene, linalool, and myrcene, all of which were present in large amounts. This was considered to be the strongest determinant of chemical composition identification.PCA was used to determine the differences in the obtained volatile components of echnique . The priechnique . The PCAp < 0.05) interaction between the plant part and the extraction method with the volatile components.Two-way ANOVA analysis was performed to define the variables of the chemical composition according to plant part and extraction method, revealing a significant (Z. kerrii rhizome extract were evaluated using DPPH, ABTS, and FRAP assays. The total phenolic content of the extract was low, at 2.2 ± 0.1 mg GAE/100 g extract. Regarding antioxidant activity, the extract showed IC50 values of 143.5 and 169.7 μg/mL using the DPPH and ABTS assays ascorbic acid was used as a positive control, with IC50 values of 1.6 ± 0.8 and 5.2 ± 0.8 μg/mL, respectively. The FRAP were also low, at 0.3 ± 0.01 mM AAE/g extract, as shown in The total phenolic content and antioxidant activity of the Z. kerrii rhizome contained a small amount of phenolic compounds. The chemical compositions of the Z. kerrii essential oils showed that terpenoid was a main compound. Similarly, previous studies reported that the phenolic content in leaves was higher than in rhizomes [Phenolic compounds are secondary metabolites in plants that show strong antioxidant activities . They plrhizomes , providiZ. kerri extract was non-toxic to the cells in a concentration range of 12.5–100 μg/mL.To assess cytotoxicity, HaCaT cells were treated with increasing doses of extract, after which cell viability was estimated using the MTT assay. Percentage of cell viability is reported as the mean ± SD of three independent experiments. The cytotoxicity of the extract at a concentration of 200 μg/mL demonstrated a cell viability of 23.1% ± 10.4%, whereas lower concentrations of extract did not inhibit the growth of the cell lines . The resS. aureus and S. typhimurium in concentrations of 640 μg/mL with less activity toward other bacterial strains in concentrations 1280 μg/mL or more compared with the standard antibiotics vancomycin, gentamycin, and ampicillin, respectively (The antibacterial activity of the extract was assayed in vitro using the Mueller-Hinton broth microdilution method against eight types of resistant bacteria. The extract showed the smallest MIC value only to ectively .Z. kerrii extract was different from other Zingiber species, such as the n-hexane solvent extract of the Z. officinale rhizome, which exhibited sensitivity to Gram-positive and Gram-negative bacteria, including S. epidermidis, S. aureus, and E. coli. It was clear from the results that its extract contained fewer phenolic compounds and oxygenated sesquiterpenes than indicated by previous studies, as these compounds showed antimicrobial potency [We found that the antimicrobial activity of  potency .The extract exhibited moderate tyrosinase inhibitory activity at 22.7 ± 0.8 mg kojic acid equivalence per gram extract . In thisZ. kerrii rhizomes, flowers, and leaves, as well as their biological activities. Z. kerrii showed moderate activity against bacterial strains and tyrosinase activity. The extract was also non-toxic to human keratinocyte cells at lower than 100 μg/mL concentration; therefore, this is a promising candidate for the development of cosmetic products. Further investigation is required to identify the phenolic compounds of crude rhizome extract.This is the first report of the chemical profiles of fresh


The aim of this study is to preliminary evaluate the antiparkinsonian activity of furanocoumarin—xanthotoxin, in two behavioral animal models, zebrafish larvae treated with 6-hydroxydopamine and mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in order to compare both models.Pastinaca sativa L. (Apiaceae) fruits. Then, the compound was administered by immersion to zebrafish 5 days after fertilization (dpf) larvae or intraperitoneally to male Swiss mice, as a potential therapeutic agent against locomotor impairments.Xanthotoxin was isolated from Acute xanthotoxin administration at the concentration of 7.5 µM reversed locomotor activity impairments in 5-dpf zebrafish larvae. In mice model, acute xanthotoxin administration alleviated movement impairments at the concentration of 25 mg/kg.The similar activity of the same substance in two different animal models indicates their compatibility and proves the potential of in vivo bioassays based on zebrafish models. Results of our study indicate that xanthotoxin may be considered as a potential lead compound in the discovery of antiparkinsonian drugs. Main manifestations of the disease cover motor function decline, e.g., tremor, bradykinesia, postural instability; non-motor symptoms, e.g. nausea, depression, dementia, sialorrhea; as well as cognitive impairment , 2. Sincagonists .Apiaceae family. It was found in 1911 in an alcoholic extract of Fagara zanthoxyloides Lam. The structure of the compound was determined in 1936 and in the same year, it was reproduced synthetically [8-Methoxypsoralen known also as methoxsalen or xanthotoxin (XT) is a natural furanocoumarin occurring in many plants belonging to the etically . Today, etically . In receetically . After setically . XT showThe most commonly used animal model of PD developing syndromes of the disease is based on the administration of neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is metabolized to 1-methyl-4-phenylpyridinium (MPP +), which is a radical interfering with the mitochondrial respiratory chain of neural cells. As a consequence of mitochondrial dysfunction, this neurotoxin induces the loss of dopaminergic neurons in the substantia nigra , 13.Another model of PD in rodents, as well as zebrafish, involves the induction of symptoms of the disease using 6-hydroxydopamine (6-OHDA), a neurotoxin selectively destroying catecholaminergic neurons including the nigrostriatal system. Pathophysiological changes in nigrostriatal neurons are similar to those caused by MPTP and, in both models, the participation of oxidative stress is firmly established –15.The aim of the study was to compare effects of XT on motor functions in zebrafish and mice, applying different models of PD, but with similar molecular mechanism. For this reason, two models of neurodegeneration in the substantia nigra were used: treatment with 6-hydroxydopamine (6-OHDA) in zebrafish and with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. This is the first and preliminary study of potential antiparkinsonian activity of XT in PD models.Danio rerio stocks of the AB strain were maintained at 28.5 °C, on a 14-/10-h light/dark cycle under standard aquaculture conditions, and fertilized eggs were collected via natural spawning. Embryos were reared under standard light/dark cycle in embryo medium: pH 7.1–7.3, 17.4-µM NaCl, 0.21-µM KCl, 0.12-µM MgSO4 and 0.18-µM Ca(NO3)2 in an incubator at 28.5 °C. To the procedure, 5 days post fertilization (dpf) larvae  were used, and all experiments were completed before 120-h post fertilization, before larvae start feeding independently. According to EU Directive, 2010/63/EU, there is no need of local ethics committee approval for larvae on this stage of development. Immediately after the experiment, larvae were killed by immersion in 15-μM tricaine solution.2.Naive male Swiss mice , 6 weeks old (25–30 g), delivered from the Centre of Experimental Medicine, Medical University of Lublin, Poland and kept under standard laboratory conditions  were used in experiments. Animals had free access to tap water and laboratory chow . Animals were housed 4 per cage, in Individually Ventilated Cages (IVC)—Techniplast UK. Each experimental group consisted of 8 animals. All experiments were conducted in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and to the European Community Council Directive for the Care and Use of Laboratory Animals of 22 September 2010 (2010/63/EU) and were approved by the local ethics committee (63/2015). The experimenter was unaware of the animal’s group during experimentation. To minimize animal suffering, qualified and experienced experimenters handled the animals with utmost care. All animal experiments were performed between 9 a.m. and 2 p.m. Mice were subsequently euthanized with COPastinaca sativa L. (Apiaceae) fruits, collected in the Medical Plant Garden of the Department of Pharmacognosy, Medical University of Lublin (Poland). The location of the garden is as follows: Situation (position) 700 m N-W near Center of town Lublin, Latitude 51º 15′ 22″; Longitude 22º 33′ 51″; Altitude ca 185 m above sea level. Fruits were collected in the summer of 2014. The species was identified by specialists in botany—Mrs Krystyna Dąbrowska from Botanical Garden of Marie Curie University, Lublin and a voucher specimen (17/20) is kept in the Department of Pharmacognosy with the Medicinal Plant Unit.XT was purified from dichloromethane extract of Isolation was carried out by high-performance counter-current chromatography (HPCCC) with a two-phase solvent system composed of n-heptane, ethyl acetate, methanol and water with the ratio of 1:1:1:1 (v/v/v/v) according to a previously published method . The purMPTP hydrochloride (M103) and 6-OHDA hydrobromide  were delivered by Sigma-Aldrich . MPTP was dissolved in a saline solution (0.9% NaCl) and 6-OHDA was dissolved in embryo medium. For mice experiments, XT was suspended in a 50 µL of 1% solution of Tween 80 and dissolved in a saline solution to achieve a concentration of 5 mg/kg. Tween 80 can be employed safely as a vehicle for neuropsychopharmacological experiments in doses not exceeding 1 ml/kg . For zebThe doses of 6-OHDA (250 µM) was chosen based on the literature data . The dosLocomotor activity in zebrafish larvae was measured using the dark chamber of an automated tracking device . Locomotor activity was calculated using ZebraLab software . The total distance moved was defined as the distance (in cm) that a larva moved during one 10-min session.To evaluate the influence of XT on MPTP-induced hypokinesia, the Opto-Varimex-4 Auto-Track apparatus  was used. The locomotor cages are built from a transparent material (43 × 43 × 32 cm) with a lid. The cages are equipped with two rows of infrared emitters (each emitter has 16 laser beams), with detectors monitoring animal movements. Each mouse was placed individually into the cage for 30 min.5 dpf larvae were allocated into groups (24 larvae per group) as follows:(1) Group I: control (1% DMSO solution in embryo medium); (2) Group II: 6-OHDA control (250 µM); (3) Group III: XT (1.5 µM); (4) Group IV: XT (3.0 µM); (5) Group V: XT (5.0 µM); (6) Group VI: XT (7.5 µM); (7) Group VII: 6-OHDA (250 µM) + XT (1.5 µM); (8) Group VIII: 6-OHDA (250 µM) + XT (3.0 µM); (9) Group IX: 6-OHDA (250 µM) + XT (5.0 µM); (10) Group X: 6-OHDA (250 µM) + XT (7.5 µM).The neurodegeneration in larvae was induced by maintaining zebrafish from 2 to 5 dpf in medium containing 6-OHDA in the concentration of 250 µM. The larvae were maintained in groups of 30 individuals. Three times a day, medium with neurotoxin was exchanged and a new concentration of 6-OHDA was prepared directly before administration. 5-dpf larvae were transferred to 96-well plates, 1 larva per each well, and different concentrations  of XT were administrated by immersion method. The incubation with drug lasted 30 min  Group I: control ; (2) Group II: MPTP control ; (3) Group III: XT ; (4) Group IV: XT ; (5) Group V: MPTP  + XT ; (6) Group VI: MPTP  + XT .On the day of the experiment, mice received four ip injections of MPTP 20 mg/kg) in saline at 2-h intervals, 4 doses a day; control mice received saline only. 14 days after MPTP treatment, XT was administered acutely, each animal received a single XT injection, and then the locomotor activity test was performed  ± SEM were used. GraphPad Prism version 7.0  was used for the data analysis and for graphics.Two-way ANOVA was used to perform the statistical analyses. Bonferroni’s post hoc test was calculated when appropriate. When the confidence limit was calculated as F4,144 = 15.81, p = 0.0001; treatment  F5,144 = 25.45, p < 0.001; and interaction F5,144 = 21.28, p < 0.001. Treatment with 6-OHDA significantly decreased distance traveled by larvae as compared to 1% DMSO control group, p < 0.05. Acute administration of XT at the concentration of 7.5 µM reversed locomotor activity impairment induced by 6-OHDA, p < 0.05  F2,48 = 3,94, p = 0.0261; and interaction F2,48 = 3.83, p = 0.0273. MPTP treatment (4 × 20 mg/kg) significantly decreased distance traveled by mice (p < 0.001) as compared to normal control group. Acute administration of XT (25 mg/kg) alleviates movement impairment induced by MPTP (p < 0.05); whereas, coumarin did not influence the locomotor activity in healthy mice in comparison with saline-treated control group  did not influence locomotor activity in mice; whereas, an acute dose of 25 mg/kg induced an increase in motor activity observed in the MPTP-treated mice. Many mechanisms may be responsible for observed effect. XT, as well as auraptene and daphnetin, may change the dopamine levels as MAOs or COMT inhibitor. Auraptene shows selective inhibitory effects against MAO-B at the concentration of 0.6 µM . DaphnetFerulago angulata (Schltdl.) Boiss, known for the content of furanocoumarins, e.g., XT, isopimpinellin, oxypeucedanin and oxypeucedanin hydrate [In the previous studies, activity of two simple coumarins: umbelliferone and esculetin, was evaluated in MPTP-induced PD symptoms in mice, and both compounds showed neuroprotective activity. Umbelliferone and esculetin were administrated in the diet for 7 days at the dose of 0.75 mg/kg/day. The neuroprotective effect was measured as attenuation of the MPTP-induced decrease in TH neuronal staining in the substantia nigra pars compacta and both coumarins restored it to 75% of the control unexposed to the neurotoxin. Moreover, protection against MPTP-induced tyrosine nitrosylation, MPTP-induced decrease of glutathione level in the brain and MPTP-induced apoptosis were observed. The authors hypothesized that both coumarins were acting as antioxidants and, in consequence, reduced the oxidative and nitrosative damage caused by MPTP exposure . Another hydrate , was exa hydrate , 38.Since characteristic features of PD are high levels of basal oxidative stress markers in the substantia nigra, the important factors to prevent the disease progression are antioxidants . NaturalIn this study, the therapeutic potential against PD-like motor symptoms was evaluated. For this purpose, two different animal and pharmacological models were used. The studies confirmed that the zebrafish PD model and mice PD model are comparable. In both the models, we observed the locomotor activity impairments after administration of 6-OHDA and MPTP. Regardless of the pharmacological model, XT reversed the locomotor impairment induced by neurotoxins damaging nigrostriatal cells. Considering that XT belongs to coumarins, this effect may be a result of combined properties like antioxidative, inhibition of MAOs and COMT or agonistic effect on dopamine receptors. Although we are aware of the limitation of this study, it shed new light on neuropharmacological properties of XT and placed it as a potential lead compound in the discovery of antiparkinsonian drugs.


The situation of renal impairment among HIV-infected patients treated with TDF-based antiretroviral (ARV) regimen greater than 3 years is little known when TDF use has been promptly increasing in Vietnam. 2. Multivariate regression analyses were used to explore the risk factors associated with renal impairment.  We analyse demographic and clinical data from a cross-sectional survey of 400 HIV-infected patients aged ≥18 years, who were treatment-naive or switched TDF regimen within over 3 years between November 2018 and March 2019. Serological tests for serum creatinine, ALT, and AST were performed. Renal impairment was defined as an estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2 and 0.8% had eGFR of 15–29 mL/min/1.73 m2, out of 34 (8.5%) of participants who had renal impairment. Multivariate analysis showed that participants who had preexposure to isoniazid (adjusted PR [aPR] = 0.35 Cl: 0.14–0.91) compared with nonexposure to isoniazid who had a BMI from 18.5 up to 25 kg/m2 (aPR = 0.31 Cl: 0.15–0.62) compared with BMI below 18.5 kg/m2 were less likely to suffer from renal impairment. Patients aged greater than 60 years  compared with those aged 20–29 years were more likely to have increased risk of renal impairment.  At the baseline, 7.8% of respondents had estimated glomerular filtration rate (eGFR) of 30–59 mL/min/1.73 m Our findings underscore the need for longitudinal studies to assess the influence of TDF on maintaining the low prevalence of renal impairment among HIV-infected patients in Vietnam. From the first HIV case detected in 1990 to June 30, 2016, there were 227,225 reported cases of HIV infection in Vietnam, of which the number of patients turned to AIDS was 85,753, and 89,210 HIV/AIDS infected cases died . Recentl3, long duration of HIV infection, WHO stage, and coinfection with hepatitis B and hepatitis C were risk factors for renal dysfunction [Until December 31, 2018, Soc Trang had 1,204 patients on antiretroviral drugs (ARV), of which 88.2% used TDF-containing regimens and 98% of new patients received ARV in 2018 with TDF + 3TC + EFV regimen . Howeverfunction . A studyThis is a cross-sectional study conducted in the department of infectious diseases, general hospital, Soc Trang Province from November 2018 to March 2019. The study protocol was reviewed and approved by the review board of the general hospital, Soc Trang Province on October 25, 2018. All participants provided written informed consent forms.2, and were treatment-naive or switched TDF regimen within over 3 years. The study was powered to obtain 80% power with a two-sided 5% significant level for estimating the overall prevalence of 50% with the desired precision of 5%, which required 384 HIV-infected patients; this was rounded up 400 to allow approximately 5% participant refusal and specimen damage rate , we filtered all patients using TDF regimen over 36 months since July first, 2018 (approximately 435 participants). Second, we recruited participants admitted to the hospital for reexamination to collect data and take blood samples based on the treatment-naive regime until we recruited enough 400 participants.The information sheet describing the purpose and method was shown to participants, and after giving informed consent, we collected baseline demographic and clinical data from medical records including sex, age, weight, height, HIV transmission, WHO stage, tested time for HIV positive, treatment time, cotrimoxazole, isoniazid, viral load, baseline CD4 count, baseline serum creatinine, ALT, AST, hepatitis B, hepatitis C, WHO stage in which treatment started, and baseline hemoglobin. Neither names nor detecting information like phone numbers or addresses were collected.3, measured by the FacsCount, Becton Dickinson), and viral load (COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test) testing were performed at general hospital Soc Trang, Center of HIV/AIDS prevention, Can Tho Province, and Pasteur Institute Ho Chi Minh City, Vietnam, respectively, between October 2018 and March 2019.After the completed physical examination, 03 mL blood specimens were collected and stored at −20°C and transported to the Center of Testing Calibration, University of Medicine and Pharmacy at Ho Chi Minh City for serum creatinine, ALT, AST analysis by chemistry analyzer . Hemoglobin , CD4 cell count (cell/mm2. According to the guideline of CKD EPI equation [−0.329 × (0.993)Age (in year); female with serum creatinine >0.7 mg/dl: GFR = 166× (Scr (in mg/dl)/0.7)−1.209 × (0.993)Age (in year)For female with serum creatinine ≤0.7 mg/dl: GFR =166 × (Scr (in mg/dl)/0.7)−0.411 × (0.993)Age (in year); female with serum creatinine >0.9 mg/dl: GFR = 163× (Scr (in mg/dl)/0.9)−1.209 × (0.993)Age (in year)For male with serum creatinine ≤0.9 mg/dl: GFR =163 × (Scr (in mg/dl)/0.9)Measurement of weight and height: we used Health-scale weight scales and height ruler  to measure weight and height. Body mass index (BMI) was calculated as BMI = weight (in kg) ÷ (height (in m))equation  to calcu2) group including 6 stages (stage 1 (>90), stage 2 (60–89), stage 3a (45–59), stage 3b (30–44), stage 4 (15–29), and stage 5 (<15)).In this study, we used the Kidney Disease Outcomes Quality Initiative (KDOQI) of the National Kidney Foundation to clarify the eGFR , and all statistical analysis was implemented using Stata version 13.0 , and The patient's characteristics were described using frequency and proportion for categorical variables and mean and standard deviation or median and interquartile ranges for continuous variables.2 [P less than 0.25 or were previously known to be an important risk factor in existing literature were included in multivariate analysis. In multivariate analysis, we used Poisson multivariate regression with backward elimination to detect the final model that describes contributing variables.In this study, renal impairment was defined as eGFR less than 60 ml/min/1.73 m2 . In the A total of 400 participants were recruited in the present study. The mean (±SD) age was 39.8 (±8.8); 51.5% of respondents were male. The mean (±SD) age was 21.2 (±3.4).The majority of study participants suffered from HIV through sexuality (89.5%); up to 98.3% of respondents were found in the World Health Organization (WHO) stage 1, and fifty percent recorded WHO stage 3 for starting treatment.µmol/L, and hemoglobin (g/L) were 517 (±250), 75.7 (±18.2), and 13.5 (±1.6), respectively.The mean (±SD) time for HIV positive testing and treatment was 7.0 (±7.0); 6.5 (±2.5), respectively. Up to 93.5% and 94.7% of respondents were preexposed to isoniazid and cotrimoxazole preventive therapy. The majority of study participants had a viral load less than 20 copies per mL (93.5%), and the mean (±SD) CD4 count, serum creatinine 9.7% of study participants were with comorbidity of hepatitis B and hepatitis C (7.2%). Among study participants, approximately one-third had an AST > 37 U/L and ALT > 40 U/L, and 26.2% had an AST > 37 U/L and ALT > 40 U/L .2) was 96.0 (±24.5), 57.3% of respondents had baseline eGFR greater than 90 ml/min/1.73 m2, and none of them had had baseline eGFR less than 15 ml/min/1.73 m2, out of 34 (8.5%) of participants who had renal impairments  eGFR (ml/min/1.73 mairments .P < 0.001). Compared with KDIGO stage 1, patients who were diagnosed with KDIGO stages 2, 3, and 4 had significantly higher mean (±SD) baseline creatinine (70.1 (±12.6) vs 77.5 (±13.7), 97.2 (±18.8), 151.6 (±51.5), 167.9 (±16.9)), respectively. By contrast, compared with KDIGO stage 1, those with KDIGO stages 2, 3, and 4 had significantly lower mean (±SD) baseline eGFR, (111.9 (±18.1) vs 80.3 (±6.6), 56.4 (±3.7), 40.7 (±3.9), 21.8 (±5.5)), and mean (±SD) CD4 count (531.5 (±233.8) vs 530.7 (±404.9), 470.2 (±251.2), 526.5 (±345.8), 130.7 (±24.4)), respectively (There were significant differences between the mean (±SD) baseline creatinine, eGFR, baseline CD4 count, and KDIGO stage among study participants  compared to BMI less than 18.5 kg/m2 and who were preexposed to isoniazid (aPR = 0.35 Cl: 0.14–0.91) were less likely to suffer from renal impairment  compared with those aged 20–29 years had significantly increased risk of renal impairment. Patients who had BMI ranging from 18.5 to 25 kg/mpairment .In this study, we present findings from the renal impairment prevalence survey of semiurban HIV/AIDS patients conducted in southern Vietnam. A striking finding in this study is that participants who were preexposed to isoniazid and TDF were less likely to report renal impairment compared with those who were not preexposed to isoniazid. According to Decision No. 2495/QD-BYT dated July 18, 2012 , on issuWe also found a low prevalence of renal impairment among study participants. This finding was consistent with findings in South Africa (7.6%)  and MalaIn the current study, we also found that patients who had low BMI were significantly associated with renal impairment compared with those who had normal BMI; this result was similar to studies conducted in Vietnam and Japan , 28, 29,As shown in our study, we did not find the relationship between cotrimoxazole, hepatitis B, C virus infection, and renal impairment which was consistent with the study conducted in Vietnam ; howeverCompared with patients aged 20–29 years, patients with ages equal to and greater than 60 years were more likely to report renal impairment. This finding was in agreement with studies conducted in Ethiopia  and Mala3 was significantly associated with renal impairment, which was in line with the previous studies [In the univariate analysis, we found that baseline CD4 count less than 200 cell/mm studies , 33, 34. studies , 35.The current study was the first study to assess the risk factors among participants who were treated with ARV based TDF regimen over 3 years in Vietnam. However, there are several limitations. In this present study, we did not collect urine data for analysis so that we could not assess chronic kidney disease among participants . In a ruThis survey has important implications for the prioritized strategies for the prevention of renal impairment as TDF is still an important drug for HIV-infected patients . This stOur findings underscore the need for longitudinal studies to assess the influence of TDF to maintain the low prevalence of renal impairment among HIV-infected patients in Vietnam.