Candida albicans exists as a commensal of mucosal surfaces and the gastrointestinal tract without causing pathology. However, this fungus is also a common cause of mucosal and systemic infections when antifungal immune defenses become compromised. The activation of antifungal host defenses depends on the recognition of fungal pathogen-associated molecular patterns (PAMPs), such as β-1,3-glucan. In C. albicans, most β-1,3-glucan is present in the inner cell wall, concealed by the outer mannan layer, but some β-1,3-glucan becomes exposed at the cell surface. In response to host signals, such as lactate, C. albicans induces the Xog1 exoglucanase, which shaves exposed β-1,3-glucan from the cell surface, thereby reducing phagocytic recognition. We show here that β-1,3-glucan is exposed at bud scars and punctate foci on the lateral wall of yeast cells, that this exposed β-1,3-glucan is targeted during phagocytic attack, and that lactate-induced masking reduces β-1,3-glucan exposure at bud scars and at punctate foci. β-1,3-Glucan masking depends upon protein kinase A (PKA) signaling. We reveal that inactivating PKA, or its conserved downstream effectors, Sin3 and Mig1/Mig2, affects the amounts of the Xog1 and Eng1 glucanases in the C. albicans secretome and modulates β-1,3-glucan exposure. Furthermore, perturbing PKA, Sin3, or Mig1/Mig2 attenuates the virulence of lactate-exposed C. albicans cells in Galleria. Taken together, the data are consistent with the idea that β-1,3-glucan masking contributes to Candida pathogenicity. Candida infections alone, over one hundred million women suffer recurrent vaginitis infections, and systemic Candida infections kill more than 200,000 individuals each year    were fouC. albicans. This was achieved by fluorescence imaging of Fc-dectin-1-stained C. albicans SC5314 cells  and quantification of the three-dimensional (3D) images to measure the fluorescence intensity of individual β-1,3-glucan-exposing features. The septal junctions and bud scars were relatively large (>2 μm3), whereas punctate foci were relatively small (<2 μm3)   and b. Ceduction  and c. Gud scars . These bud scars . These cud scars  and in tud scars . Therefoud scars , 48, 49.C. albicans attenuates recognition and cytokine production by innate immune cells  by high-speed time-lapse video microscopy . Significantly, the BMDMs bound most frequently to cell wall regions displaying the most β-1,3-glucan exposure , and the BMDMs bound less frequently to the lateral cell wall  before engulfment. Download Movie S2, AVI file, 11.1 MB.A macrophage binds its Copyright © 2022 de Assis et al.2022de Assis et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans cell after it had bound that cell. C. albicans cells that had been exposed to lactate took significantly longer to phagocytose , washed, and then grown in the absence of CFW for a further 2.5 h (approximately one doubling time) to differentiate older cells  from younger cells (daughters: CFW negative). First, control experiments were performed to confirm that this CFW staining did not interfere significantly with cell growth or β-1,3-glucan staining under our experimental conditions. C. albicans growth was not significantly affected by concentrations of up to 20 μg/mL CFW  and concanavalin A (ConA)-AF647 (for mannan)  . The ren mannan) . Triple  mannan)  to e. Me mannan)  to i.10.1128/mbio.02605-22.7VIDEO S3C. albicans cells. Wild-type (SN250) cells grown in glucose expose β-1,3-glucan at septal junctions/bud scars and at punctate foci on the lateral cell wall  via the protein kinase Stk22   . C. albidundancy . In the pression . In S. cyc8/Ssn6 , which mude Sin3 . BringinC. albicans proteins that coimmunoprecipitate with Sin3-FLAG2. As expected, we reproducibly identified orthologues of other components of the histone deacetylase complex in S. cerevisiae, such as Rpd3   . However10.1128/mbio.02605-22.2TABLE S22 interacting proteins in Candida albicans cells grown on glucose in the presence or absence of lactate. Download Table S2, XLSX file, 0.5 MB.Sin3-FLAGCopyright © 2022 de Assis et al.2022de Assis et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans. Wild-type SC5314 cells were grown in minimal medium containing glucose, exposed to either 0 or 2% lactate for 5 h, and then stained with Fc-dectin-1. The amount of Fc-dectin-1 binding, which reflects β-1,3-glucan exposure levels, was quantified by flow cytometry  reduced XOG1 transcript levels in stationary-phase cells and increased XOG1 and ENG1 levels in exponential-phase cells, whether or not lactate was present  was the parental control for the mig1Δ mig2Δ and sin3Δ strains, and WTt (SN152HLA) was the control for the tpk1Δ tpk2Δ strain. Download Table S1, XLSX file, 0.8 MB.Secretomes of Copyright © 2022 de Assis et al.2022de Assis et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans cells by confocal high-resolution fluorescence microscopy after growth in medium supplemented with glucose or glucose plus lactate  Strains were grown in complete medium for 16 h at 30°C and then washed twice and diluted to 1.0 OD/mL before dropout spot inoculation was done. A serial dilution was applied, namely, 1.0, 0.1, 0.001, 0.001, and 0.0001 OD/mL, and these dilutions were inoculated using a 96-well replica plater. The plates were grown for 3 days at 30°C. The cell wall stress agents used, CFW  and CR (Congo red), are shown in increased concentrations, represented as micrograms per milliliter. The data are the average of three independent experiments, and the replicates are presented as black circles (glucose) and black squares (glucose plus lactate). Standard deviations represent the averages of results from three biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-way ANOVA multiple-comparison test using the WT (isogenic background) as a reference for each condition. Download FIG S1, PDF file, 1.5 MB.Glucose-grown and glucose-lactate-grown cells after 5 h. Strains were inoculated in GYNB medium and grown for 16 h at 30°C. The next day, cells were washed twice with water, inoculated at 0.2 OD/mL, and allowed to grow for 5 h. (a) The glucose and glucose-lactate-grown cells were quantified by ODCopyright © 2022 de Assis et al.2022de Assis et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans . The tpk1Δ tpk2Δ mutant formed fewer pseudohyphae and cell aggregates, as expected  and SN152HLA (WTt). No significant differences in larval survival were observed for fungal cells that were preadapted on glucose plus lactate and their glucose controls. The tpk1Δ tpk2Δ, sin3Δ, and mig1Δ mig2Δ mutants were then compared to their relevant wild-type controls  fibrils  to e  of 0.2. The cells were then grown for 5 h, after which they were fixed with 50 mM thimerosal .ains see  from a s10.1128/mbio.02605-22.3TABLE S3Candida albicans strains used in this study. Download Table S3, PDF file, 0.1 MB.Copyright © 2022 de Assis et al.2022de Assis et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Protein extractions, protein assays, and immunoprecipitations using anti-FLAG M2 magnetic beads and GFP Selector (Nanotag) were performed as described previously .C. albicans cells was quantified by flow cytometry, as described previously (6 cells/mL in fluorescence-activated cell sorting (FACS) buffer . Median fluorescence intensities (MFIs) were determined using FlowJo v.10 software. Each cytometry plot is representative of at least three independent biological experiments.β-Glucan exposure on eviously , 49. BriC. albicans cells were prestained with calcofluor white (CFW) to differentiate mother cells from daughters. To achieve this, C. albicans SN250 was grown overnight in GYNB at 30°C, washed 3 times in deionized water, inoculated into fresh GYNB at an OD600 of 0.2, and grown for 2.5 h at 30°C. These cells were then stained with 20 μg/mL CFW for 5 min and then washed three times with deionized water, and the cells were then grown for additional 2.5 h at 30°C. Control, unstained cells were inoculated into fresh GYNB at an OD600 of 0.2, and these cultures were grown for 5 h at 30°C, with collection of unstained samples at 2.5 and 5 h to check the β-glucan levels. Finally, cells (stained and unstained) were harvested, fixed with 50 mM thimerosal, stained with Fc-dectin-1 (above), and analyzed by flow cytometry using an Attune NxT.For some experiments, ACT1 and TAF14 mRNA controls.RNA was prepared from cells ground under liquid nitrogen using RNeasy plant mini kits , according to the manufacturer’s instructions. cDNA was synthesized from 2 μg RNA using the ImProm-II reverse transcriptase kit , per the manufacturer’s instructions. For qRT-PCR, 1 μL cDNA was used for each SYBR green reaction . Transcript levels were normalized using Bio-Rad CFX96 software against the internal C. albicans cells grown for 5 h in GYNB or GYNB containing 2% lactate were harvested by centrifugation , 100 mL of culture supernatant was concentrated down to 200 μL at 4°C using Amicon Ultra 15- to 10,000-nominal-molecular-weight-limit (NMWL) filters , and the samples were frozen using liquid nitrogen. The samples were then freeze-dried before trypsin digestion.To examine the secretome, 2 interacting proteins, C. albicans Ca1428 cells  was added, and the samples were incubated at 56°C for 20 min. Next, 2.7 μL of fresh 0.55 M iodoacetamide was added, and the samples were incubated at room temperature for 15 min in the dark. After these reduction and alkylation steps, 1 μL of ProteaseMAX surfactant  and 1 μL of trypsin gold, mass spectrometry grade (Promega V5280), were added, and the samples were mixed and incubated overnight at 37°C. Reactions were terminated by adding 1 μL of trifluoroacetic acid (liquid chromatography-mass spectrometry [LC-MS] grade), and incubated for an additional 5 min. LC-tandem MS (LC-MS/MS) scans and analyses were performed with three independent replicates using the parameters described previously .C. albicans cells were grown in GYNB or GYNB containing 2% lactate for 5 h at 30°C (as above), fixed overnight with 50 mM thimerosal, washed twice with deionized water, and stained with Fc-dectin-1 and IgG-AF488  and ConA-AF647 (mannan). Fixed, unstained cells were used as controls. Slides were prepared by pipetting 300 μL of 2% (wt/vol) agar onto a glass slide, and a second slide was then placed on top to produce a cushion that was approximately 1 mm thick. Once the agar had set (20 min), 3 μL of cell suspension was pipetted onto the agar cushion, and a Zeiss 1.5 thickness high-performance coverslip was place on top . Cells were then observed and imaged using a Zeiss LSM880 confocal system using Airyscan high-resolution modes (Zeiss Zen [blue edition] 2.3). The system was equipped with an alpha Plan-Apochromat 100×/1.46 oil DIC objective. Nyquist sampling in xyz dimensions was used. The sample was excited with a 561-nm (20-mW) and a 488-nm (25-mW) laser, with power set to 5% for both tracks. The main beam splitter was a Zeiss dual pass 488/561 filter, and the other Airyscan path filters were set to plate. All images were then Airyscan processed (Zeiss Zen [blue edition] 2.3) using an automatic Wiener filter setting. Images were subsequently linearly unmixed to adjust for cross talk using automatic two-component extraction (Zeiss Zen [blue edition] 2.3). Contrast and brightness were adjusted, and parameters were set to similar values across all treatments. Scale bars were applied, 2D and 3D projections were generated, and images were exported as 16-bit tiff files or high-resolution Windows media files.C. albicans SN250 cells were grown in GYNB, fixed with 50 mM thimerosal, and stained with 1.5 μg/mL Fc-dectin-1 plus anti-human IgG conjugated to Alexa Fluor 488. Using a Zeiss Axioplan 2 microscope, cells were analyzed by phase DIC and fluorescence microscopy. The images were recorded using Openlab v.4.04  with a Hamamatsu C4742-95 digital camera . The volume and intensity of β-1,3-glucan-exposing features were quantified from cells fixed with thimerosal and stained with Fc-dectin-1. Cells were suspended in ProLong diamond antifade mountant (Invitrogen). 3D images were captured using a Nikon Eclipse Ti UltraVIEW VoX spinning-disk microscope. Volocity software was used to identify objects in the 488 channel and to measure their volume and mean fluorescence intensity. Septal junctions, bud scars, and parts thereof were relatively large (>2 μm3). Punctate foci ranged in volume but were smaller (<2 μm3).For quantitative fluorescence microscopy of β-1,3-glucan-exposing features, C. albicans SC5314 cells (1:3 macrophage-to-yeast cell ratio) that had been grown in the presence or absence of lactate. The BMDM-Candida interactions were imaged in DIC using a Nikon Eclipse Ti UltraVIEW VoX spinning-disk microscope at 5-s intervals for 60 min, and the videos were analyzed using Volocity software. The positions at which BMDMs first attached to Candida cells and the time from this first attachment to the full engulfment of the Candida cargo were monitored. The data represent events from three independent experiments (videos) per condition: n = 48 for glucose; n = 52 for glucose plus lactate.Bone marrow-derived macrophages (BMDMs) were prepared from bone marrow from femurs and tibias of 12-week-old male C57BL/6 mice and differentiated for 7 days, as described previously . BMDMs wGalleria mellonella infections were performed as described previously . C. albicans strains were grown in GYNB with or without lactate for 5 h at 37°C, and the cells were washed and resuspended in PBS. Upon arrival, the larvae were kept in wood shavings at 12°C for up to 2 weeks before the experiments. Initially, to compare killing rates for wild-type and deletion strains, groups of 15 larvae weighing 200 to 300 mg were inoculated with 1 × 108, 5 × 107, 5 × 106, and 4 × 105 cells/mL of the wild-type strain. Based on these comparisons, a dose of 2.5 × 105 cells was used in subsequent experiments. Each larva was injected with 50 μL of fungal cell suspension through the second proleg, using a 0.5-mL BD Micro-Fine insulin syringe, and noninjected and PBS controls were included. The larvae were then incubated at 37°C in the dark, and survival was quantified for 10 days postinfection.eviously . The waxC. albicans mutants were compared to the appropriate isogenic parental strain. Flow cytometry data were analyzed using FlowJo 10.7.2 software for MacBook, and ImageStream analyses were performed using IDEAS 6.3 software. High-resolution confocal microscopy images were analyzed using ZEISS Zen lite, blue and black, version 3.4, and processed images were smoothed using IMARIS software version 9.7. Figures were prepared using Adobe Illustrator 2021 and Adobe Photoshop 2021 for MacBook. Data are expressed as means ± standard deviation of results from at least three independent experiments, and statistical analyses were performed using analysis of variance (ANOVA) with Tukey’s multiple-comparison test or log rank (Mantel-Cox) test for the survival curves in Prism GraphPad 9.0 .


A great number of α-glucosidase inhibitors have been developed based on the molecular skeletons of bioactive natural products. However, efficient fluorescent probes for α-glucosidase detection are still limited, not to mention the probes with additional inhibitory functions. In this work, aiming for the enzyme’s highly specific detection, we designed and synthesized two environmentally sensitive fluorescent probes, namely, LD01 and LD02, respectively, based on conjugates of coumarin and cinnamic acid derivatives. We found a significant responsive emission enhancement upon LD02’s binding to α-glucosidase. These newly designed probes can act as a simple but efficient tool to evaluate the binding affinity of α-glucosidase to their inhibitors. Diabetes mellitus (DM) can be categorized into three types , with type II DM accounting for ninety percent of the total number of patients . PatholoBecause of the biological and pharmacological importance of α-glucosidase, a great number of natural products and synthetic compounds were developed for α-glucosidase-targeting inhibition . HoweverEnvironmentally sensitive fluorescent probes are widely used for protein detection and imaging, whose responsive emissions are highly sensitive to the microenvironmental properties, such as polarity, pH, or viscosity of the surrounding medium where the dyes are located . IntramoCoumarin and cinnamic acid are two types of typical natural products widely distributed in nature, harboring typical useful motifs in medicinal chemistry . RecentlSaccharomyces cerevisiae (EC 3.2.1.20) was purchased from Sigma-Aldrich. p-Nitrophenyl-α-D-galactopyranoside (PNPG) was obtained from Abcam. Nuclear magnetic resonance (NMR) spectra of new compounds were recorded in DMSO-d6 using a Bruker Advance 500 MHz spectrometer. High-resolution mass spectrometry (HRMS) was measured on a Thermo Fisher Scientific Q Exactive Orbitrap mass spectrometer. UV–Vis absorption spectra were measured using an Hitachi 3,900 absorption spectrophotometer . Fluorescence spectra were recorded using an Edinburgh FLS1000 fluorescence spectrophotometer .All chemicals and solvents were obtained from commercial suppliers without further purification. α-Glucosidase from The substituted 3-acetyl-aminocoumarin derivatives (1.0 mmol) were added to a solution of hydroxylamine hydrochloride (3.0 mmol) and pyridine (0.04 mmol) in ethanol (10.0 ml), and the mixture was stirred for 20 h at room temperature. When the reaction ended, the mixture was filtered to obtain precipitate which was then washed with ethanol to generate the substituted coumarin-based oxime product. DCC (1.1 mmol) and DMAP (0.2 mmol) were added to the solution of the substituted cinnamic acid at 0°C, and the mixture was stirred for 15 min; subsequently, the coumarin-based oxime compounds were added into the mixture and thoroughly stirred for another 12 h at room temperature. The mixture was quenched with water and extracted three times with DCM, washed with brine, and dried with magnesium sulfate. The crude product could be obtained under vacuum, and then purified by column chromatography to give the title product.1H NMR  δ 7.86 , 7.64 , 7.60 – 7.57 , 7.49 , 6.69 , 6.56 , 6.52 , 3.42 , 2.05 , 1.11 ;13C NMR  δ 167.89, 160.51, 156.73, 152.69, 151.18, 143.08, 141.91, 133.99, 132.31, 130.61, 130.44, 123.99, 120.58, 117.64, 109.66, 108.03, 96.61, 44.57, 14.10, 12.77; HRMS (+ESI) [M + H]+ calcd. For C24H23BrN2O4: 483.0930; found: 483.0967.Compound LD01. Orange, sold; yield 70%; m.p. 196–198°C; 1H NMR  δ 8.63 , 7.93 , 7.73 , 7.46 , 7.40 , 6.67 , 6.20 , 2.95 , 2.58 ; 13C NMR  δ 195.51, 168.67, 158.90, 155.06, 152.02, 147.52, 145.12, 134.92, 131.24, 130.16, 125.37, 124.81, 121.99, 118.61, 116.56, 113.35, 112.20, 40.14, 30.52; HRMS (+ESI) [M + H]+ calcd. For C22H20N2O4: 377.1509; found: 377.1533.Compound LD02. Yellow, sold; yield 65%; m.p. 167–168°C; All solvents used for spectroscopic studies are spectrum pure grade. The stock solution of each sample was prepared in DMSO (1 mM), and the working solution was obtained by diluting 30 μL of stock solutions into 3 ml of PBS buffer  or other solvents in a quartz cuvette (1 cm × 1 cm). The absorption and emission spectra were measured at room temperature. The widths of the slits for both excitation and emission were 2 nm for all experiments.The emission spectra of the probes in PBS buffer (pH from 3.0 to 8.0) were determined at room temperature. The pKa value was calculated using the Henderson–Hosslbalch equation :pH = pKα-Glucosidase protein was titrated to the solution of the testing probe (LD01 or LD02) (10 μM), and the fluorescence emission spectra were recorded from 380 to 700 nm under excitation of 365 nm. The binding constants of Ka were obtained from a double logarithm regression curve :1g[ to the solution of LD02 (10 μM) in 10 mM PBS buffer (pH = 7.4). The dissociation constant of a competitor to α-glucosidase can be determined by titrating it against binding complexes of α-glucosidase with LD02, and can be calculated according to the Cheng–Prusoff equation .1 and A0 are the absorbance values of testing samples and blank control, respectively. The IC50 value was obtained from a plot of the inhibition rate versus the sample concentration curve. The assays were carried out in triplicate and the mean values were given.The inhibition efficiency of LD01 and LD02 were determined according to the procedures Xu et al. reported previously . Stock sLD01 and LD02, which were well characterized by 1H NMR, 13C NMR, and HRMS  and 290 nm (ε = 9800 M cm−1) assigned to π → π* transitions from diethylamino-coumarin and bromo cinnamic ester moiety, respectively. LD02 has an absorption band at around 365 nm (ε = 4900 M cm−1) resulting from the mixed π → π* transitions of coumarin and cinnamic ester moieties. For the emission spectra, LD01 and LD02 show one emission band with maximum emission at 500 and 457 nm, respectively, while exhibiting very low emission quantum yield, 5.3% for LD01, and 3.2% for LD02 in PBS buffer (pH = 7.4). It has been well documented that dialkyl amine-substituted coumarin or cinnamic acid derivatives are ICT-based dyes, and their emission band or intensity is affected by the surrounding polarity. We first determined the emission spectra of LD01 and LD02 in various solvents of different polarities, such as water, methanol, acetonitrile, N,N-dimethylformamide, and dioxane . LD01 has two absorption bands at 410 nm , were collected in 10 mM PBS buffer (pH = 7.4) at room temperature. As shown in To verify the binding specificity of LD02 toward α-glucosidase, emission spectra of LD02 in the presence of α-glucosidase or various potential competitors such as other enzymes (including glucose oxidase (GOD), polyphenol oxidase (TKY), and pepsin (PEP)), and small molecules of interest  of LD01 to α-glucosidase was calculated, and the value of lgKa was 5.2 for LD01, which means LD01 has a much stronger affinity for α-glucosidase than LD02 does. This result encouraged us to determine the inhibition efficiency of LD01 and LD02 against α-glucosidase.For competitive fluorescence titration, different amounts of LD01 were added to a solution of α-glucosidase/LD02 complex. After the addition of LD01, it was found that the emission of α-glucosidase/LD02 complex decreased as a function of increasing the concentration of LD01, indicating LD01’s competitive binding to the same binding pocket as LD02 . The bin50 values of 4.6 ± 0.5 and 32.5 ± 0.8 μM, respectively, as compared to the standard acarbose (IC50 = 856.45 ± 5.60 μM). In particular, LD01 and LD02 have a relatively stronger inhibition effect against α-glucosidase than the coumarin derivatives that Xu et al. reported previously  as observed from the enzyme inhibitory assays. This research not only lays out a new strategy for quantitative detection of α-glucosidase but also offered a potential tool for binding affinity evaluation between α-glucosidase and its inhibitors, which may provide guidance for developing new α-glucosidase inhibitors treating diabetes.In summary, two environmentally sensitive fluorescence agents, LD01 and LD02, based on the conjugates of substituted coumarin and cinnamic acid, were designed and synthesized. Both LD01 and LD02’s fluorescent emissions show a polarity-dependent enhancement feature, as demonstrated in various solvents of different polarities, and the solvent mixture of dioxane and water. When coming to fluorescence titration, LD02 shows responsive emission changes featured with 2.7 times emission enhancement upon its binding to α-glucosidase. However, LD01 does not have such changes under the same experimental conditions as LD02 does. The competitive fluorescence titration of LD01 to the α-glucosidase/LD02 complex shows a gradually reduced emission as a function of increasing concentration of LD01, which suggests that LD01 can competitively bind to the same pocket of α-glucosidase targeted by LD02. The binding constants of LD01 and LD02 toward α-glucosidase were worked out by the fluorescence titration experiments, from which the values of lg


Root samples of sweet potato varieties originating from South Africa , the USA (‘Beauregard’), and Peru (‘199062.1′) were analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/QTOF/MS) and chemometrics to characterize and compare the locally developed varieties with well-known established overseas varieties. The highest total phenol content was detected in ‘Bophelo’, followed by ‘Beauregard’ and Peruvian variety ‘199062.1’. The Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) model classified the storage roots of six sweet potato varieties into two clusters. In the OPLS-DA scatter plot, one cluster, which included Peruvian variety ‘199062.1’, was separated from the others. L-tryptophan and 3-caffeoylquinic acid (CQA) showed variable importance in projection (VIP) scores greater than 1.5. Based on the OPLS-DA-S-plot, L-tryptophan separated the other varieties from Peruvian variety ‘199062.1’. Peruvian variety ‘199062.1’ contained higher concentrations of CQA  and 5-hydroxy-6-methoxycoumarin 7-glucoside than other varieties. Among all sweet potato varieties analyzed, Peruvian variety ‘199062.1′ showed the highest ferric reducing antioxidant power  free radical scavenging activity, and  scavenging activity. Among the local sweet potato varieties, ‘Bophelo’ has the greatest potential for commercialization as it is the richest source of CQA. Ipomoea batatas (L.) Lam.) is a dicotyledonous plant that belongs to the family Convolvulaceae [−1 dry weight) [Sweet potato  . There a weight) , the amo weight) . Regular weight) . Further weight) . Among the developed sweet potato varieties by the Agricultural Research Council (ARC) in South Africa, the cream-fleshed variety ‘Blesbok’ is by far the most commercially utilized . The ARCHowever, the information is limited on the content of phenolic compounds in the roots of locally used sweet potatoes. The variety ‘Bophelo’ was recommended by Nyati et al.  for its Therefore, the primary objective of our study was to characterize the phenolic compounds in the roots of locally produced sweet potato varieties compared with the well-established varieties from the United States of America (‘Beauregard’) and Peru (‘199062.1’). The second objective was to compare the antioxidant activities of the roots of domestic varieties, USA’s Beauregard, and Peruvian variety 199062.1.N-hexane, acetone, isopropyl alcohol, acetic acid, and acetonitrile), formic acid, 2.2′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′ Azino-bis(3 ethylbenzothiazoline-6-sulphonic acid) ABTS, 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), iron (III) chloride hexahydrate, the analytical standards , sodium hydroxide, hydrochloric acid, ammonium hydroxide, Trolox, and Folin−Ciocalteu reagent. The chemicals used in this study were purchased from Sigma Aldrich, Johannesburg, South Africa.The following chemicals were used in this study: anhydrous sodium sulphate, potassium persulphate, sodium carbonate, butylated hydroxytoluene, solvents  and pith (the inner layer of the cambium). For biochemical analysis, the stem, middle, and bud end of the roots were cut into tiny slices and mixed well. Thereafter, the roots were freeze-dried , ground into a powder, and held at −20°C.The storage roots of orange-fleshed (‘Bophelo’) and three cream-fleshed varieties  developed in South Africa as well as the USA’s ‘Beauregard’ variety (orange flesh) and Peru’s ‘199062.1′ variety (yellow-orange flesh) (average weight 210 g)  were obtv/v) for 1 min; thereafter, the samples were sonicated  for 1 h and centrifuged at 2000× g using a centrifuge . Each sample was extracted three times in a similar way. The supernatants were collected and stored at −5 °C before analysis. Freeze dried roots of each variety (1 g) were separately homogenized using 10 mL of 80:20 methanol/water . As a reference standard, chlorogenic acid was made at concentrations varying from 0 to 100 µg mL−1. Total phenolic content was expressed in mg of chlorogenic equivalent (CAE) per kg based on a dry weight (DW) basis.The Folin−Ciocalteu method ,18,19 wa−1. Data were acquired by scanning from m/z 150 to 1500 m/z in resolution mode as well as in MSE mode. In MSE mode, two channels of MS data were acquired: one at a low collision energy (4 V) and the second using a collision energy ramp (40–100 V) to obtain fragmentation data as well. Leucine enkephalin was used as reference mass for accurate mass determination and the instrument was calibrated with sodium formate. In addition, a Waters HSS T3, 2.1 × 100 mm, 1.7 μm column was used, and the injection volume was 2 μL with the mobile phase consisting of 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid as solvent B. The gradient started at 100% solvent A for 1 min and changed to 28% B over 22 min in a linear way. It then went to 40% B over 50 s and a wash step of 1.5 min at 100% B, followed by re-equilibration to initial conditions for 4 min. The flow rate was 0.3 mL min−1, and the column temperature was maintained at 55 °C. Using the technique of Mashitoa et al. , phenoli−1 phenolic compounds, were quantified in mg kg−1 of dried plant material.Compounds were tentatively identified based on comparison of mass fragmentation data with that of known compounds in mass bank libraries. Quantification of different phenolic compounds was conducted based on peak area relative to the reference standards; chlorogenic acid, catechin, and rutin, which were injected at a concentration range of 0 to 100 µg mL−1 of TPTZ in 40 mM of HCl, and 20 mM FeCl3·6H2O in 20 mM of acetate buffer (pH 3.6) mixed in a 1:1:10 ratio, respectively. Afterward, the absorbance was read at 593 nm using a spectrophotometer. Trolox solution ranging from 0–30 mM was prepared to construct the calibration curves, and the antioxidant power was expressed in mM TEAC g−1. Ferric Reducing Antioxidant Power (FRAP) assay was conducted in accordance with Seke et al. . FRAP reg for 5 min at 4 °C using a centrifuge . Different sample concentrations (100 µL) were made by serial dilution (0–10 mg mL−1), and 200 µL DPPH solution (13 µL DPPH mL−1 methanol) was pipetted into each well. Thereafter, the set up was held for 20 min at 25 °C, and the absorbance was read at 517 nm. Based on the following equation, we determined the % inhibition:Following the procedure described by Seke et al. , the 2,20 − A1/A0) × 100, where A0 is the absorbance of the DPPH radical solution and A1 is the absorbance of the sample.DPPH% Inhibition =  radical scavenging was conducted according to Seke et al. [+) was produced by allowing the reaction between 7 mM ABTS stock solution with 4.9 mM potassium persulphate at a 1:1 ratio and incubating the mixture at 25 °C for 12–16 h prior to use. An aliquot of 40 µL of the sample  was pipetted into 200 µL of ABTS+. The mixture was incubated at 37 °C for 10 min in the dark; a decrease in absorbance at 734 nm was measured.2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)  was calculated from the graph of the inhibition percentage versus the concentration. The % inhibition was determined according to the following equation:p < 0.05. A set of three replicate samples of sweet potato roots per variety were analyzed by UPLC-Q-TOF/MS and imported into MetaAnalyst 5.0 for Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) variable importance in projection (VIP) and heat maps.The experiments were laid out in a completely randomized design with 10 replicates per variety, and the roots were harvested twice, namely in December 2019 and January 2020. The Genstat  for Windows 13th Edition (2010 version) analyzed the differences between the roots of different sweet potato varieties using a one-way ANOVA. In order to compare the means of the different biochemical components from the roots, ANOVA with Tukey’s honestly significant difference (HSD) post hoc test was used with −1 CAE ± 0.56), followed by ‘Beauregard’ (3462.22 mg kg−1 CAE ± 0.84) and cream-fleshed Monate (2925.18 mg kg−1 CAE ± 0.78) compared to ‘Ndou’, ‘Blesbok’, and Peruvian variety ‘199062.1’ (−1) [−1, the USA’s purple-fleshed line) and ‘Quarter Million’  showed slightly higher total phenolic content than that of ‘Bophelo’ on a dry weight basis.Other authors reported that the total phenolic content, the components of phenolic compounds, and the type of phenols vary widely, and sweet potato varieties differ in their antioxidant properties . The tot99062.1’ . The lev.1’ (−1) . Padda av/v) solvent used in the extraction of derivatives of caffeoylquinic acids. However, Usuki et al. [Twelve compounds were tentatively identified in the roots of six sweet potato varieties using UPLC-QTOF/MS, which were similar to the compounds characterized in the leaves of these same varieties in our previous investigation   technique allowed the separation of sweet potato varieties based on their phenolic compounds A. Princi2 = 0.70), and the predictability of the model (Q2 = 0.60) predicted the changes of phenolic compounds from the data. Score scatter plots resulting from the OPLS-DA model are shown in l-tryptophan (1.67) and 5-CQA (1.78). Based on the OPLS-DA-S-plot, the amino acid L-tryptophan separated the other varieties from Peruvian variety ‘199062.1’ , 1,4-diCQA (21.40 ± 0.93 mg kg−1), 3,5-diCQA (36.77 ± 0.63 mg kg−1), 4,5-diCQA (0.86 ± 0.08 mg kg−1), 3-CQA (35.14 ± 0.45 mg kg−1), and 5-CQA (9.60 ± 0.23 mg kg−1) and 5-hydroxy-6-methoxycoumarin 7-glucoside (1.04 ± 0.05 mg kg−1) were detected at the highest concentrations in Peruvian variety ‘199062.1’ compared to the other varieties. Moreover, the roots of the local variety ‘Bophelo’ 0.70 ± 0.11 (mg kg−1) also showed a similar concentration of 4,5-diCQA as the Peruvian variety ‘199062.1’ (0.86 ± 0.08 mg kg−1). Among the local sweet potato varieties, ‘Bophelo’ is the richest source of CQA and other identified phenolic compounds. Furthermore, the concentrations of derivatives of CQA were higher in the storage roots of ‘Bophelo’ than in the USA’s ‘Beauregard’ variety. In contrast, CQA derivatives were detected at higher concentrations in the roots of ‘Blesbok’ than in those of ‘Ndou’ and ‘Monate.’ Individual phenolic components differed significantly among sweet potato varieties, as reported by Padda and Picha [−1 ± 7.87) had the highest antioxidant power (FRAP) compared to all local varieties (184.20 to 525.63 µM TEAC g−1) and the USA’s ‘Beauregard’ (459.53 µM TEAC g−1 ± 1.84) variety. Moreover, the storage roots of the local variety ‘Bophelo’ (4.27 IC50 mg mL−1 ± 0.10), the USA’s ‘Beauregard’ variety 4.42 (IC50 mg mL−1 ± 0.03), and Peruvian variety ‘199062.1’ (4.60 IC50 mg mL−1) exhibited the maximum DPPH scavenging activity. Similarly, the ‘Bophelo’ (4.32 IC50 mg mL−1 ± 0.20), Beauregard (4.32 IC50 mg mL−1 ± 0.20), and ‘199062.1′ varieties (4.73 IC50 mg mL−1 ± 0.5) showed higher ABTS activities, while the local varieties ‘Monate’, ‘Ndou’, and ‘Blesbok’ showed the lowest DPPH and ABTS scavenging activities. Therefore, of all the analyzed storage roots of sweet potato varieties for antioxidant properties, Peruvian variety ‘199062.1′ showed the maximum FRAP, DPPH, and ABTS activities. In comparison to DPPH, which normally takes time to complete the reaction, ABTS is soluble in all solvents, and it acts swiftly [ swiftly . However swiftly . Further swiftly . 2) between antioxidant activity and phenolic content of roots. Furthermore, Padda et al. [2 = 0.76, p < 0.05) in the present study. The 5-CQA correlated to the FRAP activity , DPPH scavenging activity , and ABTS activity . Similarly, 3,5-diCQA correlated to the FRAP activity , DPPH scavenging activity , and ABTS activity . 1,3-diCQA showed a moderate correlation with FRAP activity , DPPH scavenging activity , and ABTS activity . Likewise, 3-CQA also showed a moderate correlation with FRAP activity , DPPH scavenging activity , and ABTS activity .Prada and Picha  establisa et al.  demonstra et al. . Strong a et al. . In the a et al. . Lebot ea et al.  showed ta et al.  also shoTherefore, 5-CQA, 3,5-diCQA, 1,3-diCQA, and 3-CQA were responsible for the observed antioxidant activity in theses varieties. According to Islam et al. , the preFurthermore, according to Cho et al. ,32, highWhen comparing four local sweet potato varieties, ‘Beauregard’ from the USA, and ‘199062.1′ from Peru for antioxidant properties, Peruvian variety ‘199062.1′ showed the maximum antioxidant properties . Among the local sweet potato varieties, ‘Bophelo’ showed the highest antioxidant activity, the richest source of CQA, and other identified phenolic compounds, and these concentrations were higher than in the USA’s ‘Beauregard’ while being in the range of Peruvian ‘199062.1’. Thus, the local sweet potato variety ‘Bophelo’ can be recommended to produce functional products. In addition, the 5-CQA showed a positive correlation between antioxidant activities. Tryptophan and 5-CQA can also be used as markers to distinguish the roots of Peruvian variety ‘199062.1’ from the other five varieties included in this study.


Vitis vinifera seed extract using an excision wound model with focus on wound healing molecular targets including TGFBR1, VEGF, TNF-α, and IL-1β. The wound healing results revealed that V. vinifera seed extract enhanced wound closure rates (p < 0.001), elevated TGF-β and VEGF levels, and significantly downregulated TNF-α and IL-1β levels in comparison to the Mebo®-treated group. The phenotypical results were supported by biochemical and histopathological findings. Phytochemical investigation yielded a total of 36 compounds including twenty-seven compounds (1–27) identified from seed oil using GC-MS analysis, along with nine isolated compounds. Among the isolated compounds, one new benzofuran dimer (28) along with eight known ones (29–36) were identified. The structure of new compound was elucidated utilizing 1D/2D NMR, with HRESIMS analyses. Moreover, molecular docking experiments were performed to elucidate the molecular targets  of the observed wound healing activity. Additionally, the in vitro antioxidant activity of V. vinifera seed extract along with two isolated compounds  were explored. Our study highlights the potential of V. vinifera seed extract in wound repair uncovering the most probable mechanisms of action using in silico analysis.This study explored the in vivo wound healing potential of  Wounds are a significant global health issue that has serious commercial and social impacts on health institutes, patients, and caregivers . Wounds Vitis vinifera Linn. (F. Vitaceae) is a climber which is woody in nature and containing coiled climbing tendrils. It bears small, pale, tender flowers, which are modified to berry fruits that vary from green to various degrees of purple black  ethyl ester (g 71.16%  of the t2:1 (13) . It was yl ester . While hyl ester .1H), with DEPT-Q NMR, along with the relation to the biography and some authoritative references, the crude extract of V. vinifera seeds provided the new benzofuran dimer (28) along with eight known compounds , suggesting 12 degrees of unsaturation. The 1H and DEPT-Q 13C NMR data  δC 145.9, δH 7.09, dd  δC 104.0, δH 7.40, d (2.5) δC 108.8, δH 6.37, dd  δC 114.0, and δH 7.30, d (8.0) δC 123.8, δH 7.79, d (3.0) δC 144.5, δH 7.40, d (2.5) δC 108.8, δH 6.37, dd  δC 114.0, δH 7.30, d (8.0) δC 123.8, and seven quaternary carbons at δC 113.5, 116.8, 148.4, 148.4, 157.3, 157.3, and 160.8, indicating the characteristic core structure for dimeric benzofuran derivatives [28 , 1D, and 2D NMR analysis data of compound scaffold . The HR-NMR data , as wellNMR data , predictivatives , where Hives [28  confirme1H and DEPT-Q 13C NMR data , and C3‵  δC 144.5, CH) with C2  δC 145.9, CH), and C3  δC 104.0, CH) suggested the attachment of one of the benzofuran units at C2‵ by converting the methine of C2‵ to quaternary carbon (13C NMR data (δC 148.4) and C3‵ (δC 144.5) compared with C2 (δC 145.9) and C3 (δC 104.0), suggesting O attachment at C2‵. Accordingly, compound 28 was identified as 2-(benzofuran-5-yloxy) benzofuran-5-ol.Comparing the NMR data , along wNMR data , for C2‵y carbon . The DEPNMR data  showed dV. vinifera seed extract, TNF-α, PDB ID: 2AZ5, TGFBR1, PDB ID: 6B8Y, and IL-1β, PDB ID: 6Y8M, which were downloaded from the protein data bank (PDB). The binding free energy represented by  and the Root Mean Square Deviation  in the Molecular Operating Environment (MOE) program were utilized in ranking different isolated compounds in comparison to the co-crystallized ligand. Besides, the different interactions within the active sites of amino acid residues along with their energies were listed. Firstly, the docking studies within the TNF-α active site showed that all the test compounds attained binding energies of −3.7887 to −6.3236 kcal/mol), close to that of the co-crystallized ligand , with an accuracy of less than 2 Å RMSD. Interestingly, the binding accuracy of these test compounds was better than the co-crystallized ligand and one of them has higher binding energy than the co-crystallized ligand  showed higher affinity to the active site over the co-crystallized ligand, this was presented by their better binding energy value  compared to the co-crystallized ligand  with an accuracy of less than 2 Å RMSD  than co-crystallized ligand but its accuracy was as not high as its test congeners (>2 Å RMSD).All the isolated compounds showed binding free energy comparable to that of the co-crystallized ligand within the interleukin 1 beta active site with accuracy in the same way below 2 Å RMSD . Interes28 with various amino acid residues of three active sites comparable to the co-crystallized ligands.Moreover, the 2D-interaction diagram 28 with various amino acid residues of three active sites comparable to the co-crystallized ligands; results were listed in Moreover, the 2D-interaction diagram 34 and β-sitosterol-3-O-glucopyranoside 36 as a scavenger potential against H2O2 were investigated in this study. The maximal H2O2 radical scavenging activities of compounds 34 and 36 were 44.44% and 46.42% at 1000 µg/mL concentration, respectively, according to the data. Compounds 34 and 36 suppressed the formation of H2O2 radicals in a dose-dependent manner, demonstrating a consistent antioxidant potential with IC50 of 197.1 and 222 μg/mL concentration, respectively (50 = 181.2 μg/mL).The antioxidant activities of ursolic acid ectively , in comp34, and β-sitosterol-3-O-glucopyranoside 36 was evaluated. The results showed that both compounds had 55.66%, and 63.63% SOD at 1000 μg/mL concentration, respectively. The concentration of compounds 34, and 36 needed for 50% inhibition (IC50) was 221.4, and 205 µg/mL, respectively, whereas 156.6 μg/mL was demanded for ascorbic acid  diminishing in wound area compared to the untreated wounds  is required to improve neutrophils, and remove bacteria and pollution from the wound section and identifies dynamic inducer MMPs  regeneration in inflammatory and fibroblast cells. In the wound healing process, the MMP diminishes and excludes broken extracellular matrixes (ECM) to aid wound reconstruction [α is one of the growth factors excreted from macrophages, which incorporates IL-1β to enhance and overcome respective collagen manufacture and fibroblast proliferation [α prompts NF-κB, which in time encourages gene interpretation of an overabundance of pro-inflammatory cytokines including TNF-α as well as proteases, as MMP, to clear dispersed TNF-α and potentiate the effects of such inflammatory cytokines [α; and IL-1β) by V. vinifera seed extract can inhibit continued inflammation and hence avoid impaired wound repair.Expression of IL-1truction . Howeverferation . The TNFytokines . So, supV. vinifera seed extract enhanced VEGF expression and hence improved wound healing. The relative protein expression of VEGF was developed in V. vinifera seed extract-treated wound tissues related to untreated wound tissues, which might indicate that V. vinifera seed extract increased VEGF expression in wound tissues.Additionally, healing the wounds is resolved by various growth factors which are excreted in feedback to injury, such as VEGF, which exerts a significant role in the regenerationof new blood vessels . VEGF stV. vinifera seed extract can thus prevent extended inflammation and hence promote wound healing.Moreover, wound improvement is interfered in by type I collagen, which is the primary protein inside skin tissue  and exer2O2 radical scavenging activity of V. vinifera seed extract was 48.1% at 1000 µg/mL concentration. According to the data. V. vinifera seed extract suppressed the formation of H2O2 radicals in a dose-dependent manner, demonstrating a consistent antioxidant activity with IC50 of 175.8 μg/mL concentration (50 = 178.1 μg/mL) [Antioxidants are thought to help manage wound oxidative stress and hence speed up the healing process. They usually play a critical role in controlling the damage that biological components such as DNA, protein, lipids, and body tissue may sustain in the presence of reactive species. The maximal Hntration  compared1 μg/mL) . High le1 μg/mL) ,46. More1 μg/mL) .V. vinifera seed extract was evaluated. The results showed that the scavenging impact of the standard and extract rises with concentration  identified from seed oil using GC/MS analysis, along with nine isolated compounds, including one new benzofuran dimer (28), and eight known ones (29–36). The structure of the new compound was elucidated using 1D and 2D NMR and HRESIMS analyses  play a vital role in the wound healing process. The high scores and extremely comparable interaction patterns of multiple ligands in V. vinifera seed extract with the stated wound healing targets provided some molecular explanation for the extract’s wound healing effect. Additionally, the binding modes and free energies obtained for the isolated compounds during the molecular docking studies within active sites of TGFBR1, TNF-α, and IL-1β (β (increased) and the inflammatory markers, TNF-α and IL-1β (decreased). Our data suggested that V. vinifera seed extract could accelerate the switching process from inflammatory to anti-inflammatory responses, which afterwards promotes healing.Molecular docking was carried out to explore the molecular targets that might contribute to the wound healing potential. The three examined targets . Nine compounds were isolated and identified from V. vinifera seeds. Molecular docking analysis on three molecular targets predicted the possible mode of action in the wound healing activity. Additionally, the potent in vitro antioxidant activity of Vitis vinifera seed extract, and two isolated compounds  that are attributed by its SOD, and H2O2 scavenging activity can eliminate ROS and hence enhance its wound-healing activity. This antioxidant potential may be endorsed to the phenolic content of the extract. Finally, this study recommended the application of V. vinifera seed extract in wound care as a promising therapy to accelerate the wound healing process; however, future detailed mechanistic studies are still required to confirm those results.In this study,


Acinetobacter baumannii (A. baumannii) is one of the members of ESKAPE bacteria which is considered multidrug resistant globally. The objective of this study is to determine the protein docking of different antibiotic resistance gene (ARGs) in A. baumannii. In silico analysis of antibiotic resistance genes against carbapenem are the blaOXA-51, blaOXA-23, blaOXA-58, blaOXA-24, blaOXA-143, NMD-1 and IMP-1 in A. baumannii. The doripenem, imipenem and meropenem were docked to blaOXA-51 and blaOXA-23 using PyRx. The top docking energy was -5.5 kcal/mol by imipenem and doripenem and meropenem showed a binding score of -5. 2 kcal/mol each and blaOXA-23 energy was -4.3 kcal/mol by imipenem and meropenem showed a binding score of -2.3 kcal/mol, while doripenem showed the binding score of -3.4 kcal/mol. Similarly, doripenem imipenem and meropenem were docked to blaOXA-58, IMP-1, Rec A and blaOXA-143, with docking energy was -8.8 kcal/mol by doripenem and meropenem each while imipenem showed a binding score of -4.2 kcal/mol and with IMP-1 demonstrated their binding energies. was -5.7 kcal/mol by meropenem and doripenem showed a binding score of -5.3 kcal/mol, while imipenem showed a binding score of -4.5 kcal/mol. And docking energy was -4.9 kcal/mol by imipenem and meropenem showed binding energy of -3.6 kcal/mol each while doripenem showed a binding score of -3.9 kcal/mol in RecA and with blaOXA-143 docking energy was -3.0 kcal/mol by imipenem and meropenem showed a binding score of -1.9 kcal/mol, while doripenem showed the binding score of -2.5 kcal/mol respectively. Doripenem, imipenem, and meropenem docking findings with blaOXA-24 confirmed their binding energies. Doripenem had the highest docking energy of -5.5 kcal/mol, meropenem had a binding score of -4.0 kcal/mol, and imipenem had a binding score of -3.9 kcal/mol. PyRx was used to dock the doripenem, imipenem, and meropenem to NMD-1. Docking energies for doripenem were all – 4.0 kcal/mol, whereas meropenem had docking energy of -3.3 kcal/mol and imipenem was -1.50 kcal/mol. To the best of our knowledge the underlying mechanism of phenotypic with genotypic resistance molecular docking regarding carbapenem resistance A. baumannii is unclear. Our molecular docking finds the possible protein targeting mechanism for carbapenem-resistant A.baumannii.  A. baumannii is one of the most important nosocomial pathogens and a great resistance model to antimicrobials [According to the World Health Organization, (WHO), crobials . Antibiocrobials . The carcrobials –5.Molecular docking provides successful insights into the structure–activity relationships, mode of activity, and further analysis from protein–ligand interaction. Such studies would culminate in the development of novel drug molecules at a faster pace against infectious pathogens. Additionally, the physicochemical properties of the molecule would provide vital information on the initial phase of drug development –8.A. baumannii has a high level of resistance against available antibiotics carbapenem and colistin which are the last line of treatment [A. baumannii (CRAB) is the major mechanism of carbapenem resistance in A. baumannii as well as other Gram-negative bacteria. The carbapenemase enzyme-encoded genes are located on mobile genetic elements like plasmid, transposon and integron [A. baumannii are important to implement the new drug for overcoming the resistance in such types of pathogens [Pathogen like reatment . Carbapeintegron , 11. Dueintegron . We haveintegron . The comathogens .All lists of electronic tools were briefly elaborated in this work as swiss prot and uniprot model.https://www.rcsb.org/) while IMP-1 and rec A against carbapenem  were accessed from UniProtKB/Swiss-Prot database (https://swissmodel.expasy.org/interactive) because protein structures of IMP-1 and rec A were not present in PDB.The three-dimensional (3D) structure of resistant beta-lactam oxacillinase-51, beta-lactam oxacillinase, beta-lactam oxacillinase-58, beta lactam oxacillinase-24, beta lactam oxacillinase-143, and New Delhi metallo-ß-lactamase-1) were accessed from the protein data bank (PDB) (https://www.rcsb.org/) with sequence entries 5KZH (https://www.rcsb.org/structure/5KZH), 4JF5 (https://www.rcsb.org/structure/4jf5), 4OH0 (https://www.rcsb.org/structure/4oh0), 3G4P (https://www.rcsb.org/structure/3g4p), 6NZ8 (https://www.rcsb.org/structure/6nz8) 6O3R (https://www.rcsb.org/structure/6o3r) respectively and IMP-1 and rec A were retrieved from the swiss model (https://swissmodel.expasy.org/interactive) with sequence entries Q6ZXZ6 (https://www.uniprot.org/uniprotkb/Q6ZXZ6) and PS50163 (https://prosite.expasy.org/PS50163) respectively. The UCSF chimera 1.6 was used to minimise the selected protein models [http://redpoll.pharmacy.ualberta.ca/vadar) and WinCoot was used to assess the Ramachandran plot value [These proteins are involved in different activities such as antibiotic-resistant gene blaOXA-51, blaOXA-23, blaOXA-58, blaOXA-24, blaOXA-143, and NMD-1, with target antibiotics retrieved from the protein data bank  were used, respectively. GalaxyRefine employs the CASP10 assessment to refine the query structure, improving the structural and global quality of the three-dimensional model. This method initially rebuilds side chains and performs side-chain repacking and subsequently uses MD simulation to achieve overall structure relaxation.To perform structure refinement and energy minimization of the three-dimensional modelled protein structures, the online server GalaxyRefine (https://pubchem.ncbi.nlm.nih.gov/) having accession No 73303, 104,838, 441,130 respectively and downloaded in SDF format. The retrieved antibiotics were designed in ACD/ChemSketch (https://www.acdlabs.com/) and accessed in PDB format for further docking process.A comprehensive literature survey was employed and checked all Doripenem, imipenem and meropenem target of a particular resistance protein. Doripenem, imipenem and meropenem were retrieved from the PubChem database (http://cospi.iiserpune.ac.in/depth/), an online source which explores the probability of amino acids involved in the formation of active binding sites.The active sites gene blaOXA-51, bla23, blaOXA-58, blaOXA-24, blaOXA-143, NMD-1, IMP-1 and rec A provide significant information regarding the functionality of mediated signalling pathways. The active binding sites of blaOXA-51, blaOXA-23, blaOXA-58, blaOXA-24, blaOXA-143, NMD-1, IMP-1 and rec A were predicted by using Depth Residue  and 250 amino acids having a molecular mass of 113.28 kDa. The VADAR 1.8 structure analysis of blaOXA-51 consists of f 40% α-helices, 25% β-sheets, 34% coils and 5% turns, respectively. The Ramachandran plots and values of blaOXA-51 indicated that 98.94% of blaOXA-51 amino acids were present in the preferred region and 1.06% residues were in the allowed region. blaOXA-23 is another target protein consisting of a single chain (A) having 243 amino acids with a molecular weight of 27.90 kDa. The structural analysis revealed that blaOXA-23 is composed of 39% α-helices, 23% β-sheets, 36% coils and 23% turns. The Ramachandran plots and values of blaOXA-23 indicated that 98.33% of protein amino acids were present in the preferred region and 1.67% of residues lie in the allowed region. blaOXa A-58 has a single chain (A) having 280 amino acids with a molecular weight of 98.01 kDa. TRPV4 consists of 37% α-helices, 25% β-sheets, 37% coils and 21% turns, respectively. The Ramachandran plots and values of blaOXA-58 indicated that 97.90% of blaOXA-58 residues were present in the preferred region and 2.10% of residues lie in the allowed region.The blaOXA-24 from the hydrolase family, comprises a single chain (A) having a length of 250 amino acids having a molecular mass of 27.59 kDa. The VADAR 1.8 structure analysis of blaOXA-24 consists of f 39% α-helices, 26% β-sheets, 34% coils and 5% turns, respectively. The Ramachandran plots and values of blaOXA-24 indicated that 93.80% of blaOXA-24 amino acids were present in preferred region and 4.55% residues were in allowed region. blaOXA-143 is another target protein that consists of a single chain (A) having 258 amino acids with molecular weight of 29.60 kDa. The structural analysis revealed that blaOXA-143 is composed of 38% α-helices, 25% β-sheets, 35% coils and 18% turns. The Ramachandran plots and values of blaOXA-143 indicated that 97.30% of protein amino acids were present in preferred region and 2.70% residues were lie in allowed region. NMD-1 has a single chain (A) having 233 amino acids with molecular weight of 25.02 kDa. NMD-1 consists of 25% α-helice, 31% β-sheets, 42% coils and 31% turns, respectively. The Ramachandran plots and values of NMD-1 indicated that 97.36% of NMD-1 residues were present in preferred region and 1.76% residues were lie in allowed region.IMP-1 consists of 246 amino acids. IMP-1 consists of 26% α-helices, 34% β-sheets, 38% coils and 21% turns, respectively. The Ramachandran plots and values of IMP-1 indicated that 94.67% of IMP-1 residues were present in preferred region and 3.56% residues were lying in the allowed region. Rec A is having 349 amino acids. The structural analysis revealed that rec-A is composed of 38% α-helices, 29% β-sheets, 31% coils and 12% turns. The Ramachandran plots and values of blaOXA-143 indicated that 94.15% of protein amino acids were present in preferred region and 4.62% residues were lie in allowed region   has a molecular weight of 420.5 g/mol. Doripenem is a broad-spectrum carbapenem antibiotic used primarily for the treatment of aerobic gram-negative bacterial infections (https://www.ncbi.nlm.nih.gov/books/n/livertox/Doripenem/). Imipenem (C12H17N3O4S) , ’ is a broad-spectrum, intravenous beta-lactam antibiotic of the carbapenem subgroup. It has a role as an antibacterial drug. It is a beta-lactam antibiotic allergen and a member of carbapenems (http://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI:471744). Meropenem (C12H17N3O4S)  is the anhydrous form of meropenem, a broad-spectrum carbapenem with antibacterial properties, synthetic Meropenem inhibits cell wall synthesis in gram-positive and gram-negative bacteria .The Doripenem C15H4N4O6S2 . These binding pocket residues have good probability values in the range from 0 to 0.4 value as shown in Fig. The binding pocket of blaOXA-23 consists of three amino acids . All the amino acids selected for the active site have a probability greater than 0 to 1 as shown in Fig. Figure The binding pocket of rec A consists of seven amino acids . All the amino acids selected for the active site have probability greater than 0 to 1 as shown in Fig. The binding pocket of blaOXA-143 consists of seven amino acids . All the amino acids selected for active site have probability greater than 0 to 1 as shown in Fig. The binding pocket of blaOXA-24 consists of seven amino acids . All the amino acids selected for active site have probability greater than 0 to 0.8 as shown in Fig. The binding pocket of NMD-1 consists of seven amino acids . All the amino acids selected for active site have probability greater than 0 to 0.3 as shown in Fig. The docking results showed that all ligands were confined in the active binding region of target proteins . The superimposition results of all three docking complexes showed that ligands bind in similar conformational behavior and have similar binding interactions pattern as shown in the results in Fig. The docking results of doripenem, imipenem and meropenem with blaOXA-23 demonstrated their binding energies Table . The dorThe doripenem, imipenem and meropenem were docked to Rec A using PyRx. The top docking energy was -4.9 kcal/mol by imipenem and meropenem showed binding energy of -3.6 kcal/mol each while doripenem showed a binding score of -3.9 kcal/mol. The docking results of doripenem, imipenem and meropenem with blaOXA-143 demonstrated their binding energies. The top docking energy was -3.0 kcal/mol by imipenem and meropenem showed a binding score of -1.9 kcal/mol, while doripenem showed a binding score of -2.5 kcal/mol.Doripenem, imipenem, and meropenem docking findings with blaOXA-24 confirmed their binding energies. Doripenem had the highest docking energy of -5.5 kcal/mol, meropenem had a binding score of -4.0 kcal/mol, and imipenem had a binding score of -3.9 kcal/mol.PyRx was used to dock the doripenem, imipenem, and meropenem to NMD-1. Docking energies for doripenem were all – 4.0 kcal/mol, whereas meropenem had a docking energy of -3.3 kcal/mol and imipenem was -1.50 kcal/mol. Selim et al. 2022  reportedHydrogen and hydrophobic interactions are used to evaluate the bonding interactions of docking complexes. In the doripenem blaOXA-51 docking complex, five Hydrogen bonds have been observed at different residual positions within the active region of the target protein, Ala-209, Asn-213 Ser-232. The doripenem shares the two hydrogen bonds with Ala-209 having a distandistance08Å and2.13 Å, two hydrogen bonds with Asn-213 2.17 Å and1.92 Å respectively as shown in Table The target protein is indicated with the color orchid and grey in ribbon line format. The binding active amino acids all around ligands are outlined in yellow as shown in Fig. Doripenem- blaOXA-23 docking complex shows three hydrogen bonds in the active region of the target protein, at Ser-48, Lys-51, and Lys-185 having a bond distance of 1.69 Å, 2.98 Å and 2.11 Å respectively (Table The target protein is indicated with the color dark khaki and light grey in ribbon line format. The binding active amino acids all around ligands are outlined in sienna color Fig. . HydrogeThe doripenem- blaOXA-58 docking complex reveals four hydrogen bonds in the active region of the target protein, at Ala-209, Ala-211, and Ser-229, with bond distances of 3.06 Å, 2.26 Å, and 2.76 Å,2.38 Å correspondingly Table . MeropenThe colours gold and sienna in ribbon line format identify the target protein. All around ligands, cornflower blue highlights delineate the binding active amino acids. blaOXA-58 amino acids and ligands are demonstrated to form hydrogen bonds at various distances. Red dots show the binding distance in angstroms Fig. .Fig. 14TDoripenem- IMP-1 docking complex shows three hydrogen bonds in the active region of the target protein, namely Gly-168, Ser-202and Glu-203 having bond distances 2.53 Å, 2.45 Å, 2.41 Å (Table The target protein is indicated with the color grey and sky blue in ribbon line format. The binding active amino acids all around ligands are outlined in sandy brown color Fig. . HydrogeThe doripenem- rec A docking complex exhibits three hydrogen bonds in the active region of the target protein, at Glu-232, Asn-268, and Arg-306 with a bond distance of 2.18 Å, 2.04 Å and 2.95 Å respectively. Imipenem bonded with rec A at Lys-265 and Asn-268. Bond distances were 2.38 Å and 2.93 Å respectively (Table Gold and salmon ribbons identify the target protein. Skyblue binding amino acids surround ligands. IMP-1 amino acids and ligands have hydrogen bonding at varying distances. Red dots represent binding distance in angstrom Å Fig. In the doripenem blaOXA-143 docking complex, two hydrogen behave have been observed at different residual positions within the active region of the target protein. Couple of bonds at Glu-140 involved in hydrogen bond having a bond distance of 2.15 Å and 2.40 Å Table . In imipThe target protein is indicated with the color dark cyan and cyan in ribbon line format. The binding active amino acids all around ligands are outlined in violet red color Fig. . HydrogeDoripenem blaOXA-24 docking complex shows four hydrogen bonds in the active region of the target protein, namely Glu-39, Asn-62, Thr-65 and Gl-67 having bond distances 2.80 Å, 2.41 Å,2.20 Å and 2.58 Å respectively as shown in Table The target protein is indicated with the color and gold in ribbon line format. The binding active amino acids all around ligands are outlined in brown Fig. . HydrogeThe Doripenem- NMD-1docking complex reveals two hydrogen bonds in the active region of the target protein, at Leu-139 and Ala-198, with bond distances of 2.25 Å, 2.36 Å correspondingly (Table The target protein is indicated with the color khaki and sky blue in ribbon line format. The binding active amino acids all around ligands are outlined in yellow color as shown in Fig. A. baumannii.Our result may give a clear spot on carbapenem molecular docking data required for protein targeting the resistant gene need more advanced design of drugs and synthesis of new candidates to minimise the resistant crisis against


Streptomyces cattleya cultured on an isoflavone-containing agar plate. We further identified a cytochrome P450 monooxygenase, CYP158C1, which is able to catalyze the dimerization of isoflavones. CYP158C1 can also dimerize plant-derived polyketides, such as flavonoids and stilbenes. Our work represents a unique bacterial P450 that can dimerize plant polyphenols, which extends the insights into P450-mediated biaryl coupling reactions in biosynthesis.Dimerization is a widespread natural strategy that enables rapid structural diversification of natural products. However, our understanding of the dimerization enzymes involved in this biotransformation is still limited compared to the numerous reported dimeric natural products. Here, we report the characterization of three new isoflavone dimers from  Dimerization is a ubiquitous biotransformation in nature. Almost all forms of life, including bacteria, fungi, and plants, have the ability to produce homo- or heterodimeric natural products, which enables rapid structural diversification from simple monomers . Often, With the advance of biosynthetic studies on natural products, a number of enzyme classes that are responsible for the dimer formation have been identified ,10–14. IPhenol coupling in plant polyphenol biosynthesis is one of the earliest documented biocatalytic dimerization reactions . ContrarStreptomyces cytochrome P450 enzyme that catalyzes dimerization of plant isoflavones. By untargeted metabolomics, we isolated three new isoflavone dimers, namely cattleyaisoflavones A–C (1–3), from Streptomyces cattleya cultured in soy flour-containing media. We then identified a P450 enzyme, CYP158C1, which is able to dimerize the isoflavones as well as other plant-derived phenolic compounds. This work extends the insight on the substrate scope of P450-mediated dimerization and provides a potential biocatalytic tool for the synthesis of isoflavone dimers in future.In this study, we report the discovery of a Streptomyces species, we identified several unknown isoflavone-like metabolites from S. cattleya NRRL8057 cultured on mannitol soya flour (MS) agar plates , B (2), and C (3)  . HRMS an1–3 were biosynthesized de novo by the strain or derived from the media that contain isoflavones, we cultured S. cattleya on International Streptomyces Project-2 (ISP-2) liquid medium that does not contain isoflavones. These cultures did not produce 1–3, but daidzein (4) supplementation in ISP-2 liquid medium restored the production of 1–3, suggesting that S. cattleya utilizes exogenous isoflavone to form the dimer products 1–3  and H-6’’  revealed C-8’’ as one of the coupling sites. The methoxy group  was assigned to the C-7 site because of the 1H,1H-NOESY correlation with H-6 . The HMBC correlations of H-6 to C-8 (δC 136.3), C-10 (δC 118.9), C-7 (δC 151.7), C-5 (δC 123.6), and C-8’’ (δC 119.1) suggested that C-5 is the coupling site on this unit, and C-8 was substituted by a hydroxy group. Finally, the structure of 1 was determined as a dimer with a rare 5,8’’ biaryl coupling skeleton. Likewise, C-8’’ was assigned as one of the coupling sites of 2. Three B-ring protons, H-2’ , H-5’ , and H-6’ , suggested that C-3’ was another coupling site. Thus, the structure of 2 was established as a 3’,8’’-coupled dimer  did not encode any laccase homologs but encoded 41 P450 enzymes in total. To investigate whether P450 enzymes mediate this dimerization reaction, we first employed a chemical genetics screening using three P450 inhibitors, CTA (strong inhibitor), FCA (moderate inhibitor), and RVT (polyphenol inhibitor) + calcd for C31H20O9, 537.1180; found, 537.1206.Cattleyaisoflavone B (2): yellowish powder; UV (MeOH) λmax 255, 310 nm; for NMR spectral data, see Table S5, m/z): [M + H]+ calcd for C30H18O8, 507.1074; found, 507.1091.Cattleyaisoflavone C (3): white powder; UV (MeOH) λmax 254, 300 nm; for NMR spectral data, see Table S6, m/z): [M + NH+ calcd for C30H18O8, 507.1074; found, 507.1091.4, as 1 mL DMSO solution added after autoclave) by 1% inoculation from the above seed culture. On the 5th day, 2 mL of medium from each sample was taken and freeze-dried. Then, 1 mL MeOH was added and the mixture was treated with ultrasound for 15 min. After centrifugation, the supernatant was directly analyzed by HPLC following analytical method A.Feeding experiments were performed in 500 mL flasks each containing 100 mL of ISP-2 liquid medium and 10 mg daidzein (4), and the P450 inhibitor, by 1% inoculation from the above seed culture. On the 9th day, 0.5 mL of medium from each sample was taken and freeze-dried. Then, 0.2 mL MeOH was added, and the mixture was treated with ultrasound for 15 min. After centrifugation, the supernatant was detected following analytical method B.All samples involved in inhibition experiments were set up using 100 mL flasks containing 20 mL ISP-2 medium, 0.02 mg/mL daidzein (https://www.geneious.com).Multiple sequence alignment was constructed by MAFFT 7.450 using the E-INS-I method , and theStreptomyces genome was extracted by TIANamp Bacteria DNA Kit (TIANGEN BIOTECH), and the P450 genes were amplified by PCR  for protein expression. Cells harboring expression plasmid were inoculated into LB medium with 50 µg/mL kanamycin and incubated at 37 °C until the culture reached an OD600 of 0.4–0.6. The cultures were then cooled to 16 °C and induced with 0.1 mM isopropyl β-ᴅ-1-thiogalactopyranoside (IPTG) for 16 h. Cells were harvested by centrifugation .The 2PO4, 400 mM NaCl, 1 mM TCEP, pH 7.5) and lysed by sonication. After removing cell debris by centrifugation, the lysate supernatant was purified by Ni-NTA resin with washing buffer  and elution buffer  and then dialyzed into the storage buffer  and stored at −80 °C for further use. The P450 partner proteins, FdR and Fdx, were constructed according to the reported method [For protein purification, cell pellets were resuspended in lysis buffer  contained 0.4 mM substrate, 3 µM P450 enzyme, 5 µM FdR, 5 µM Fdx, 1 mM NADPH, 0.2 mM DTT, and 1 mM EDTA in 50 mM PBS buffer (pH 7.4). The reaction mixture was incubated at 37 °C for 1 h and quenched by adding 50 µL MeOH. After centrifugation , the mixture was subjected to HPLC–UV or UHPLC–HRMS analysis.The antioxidant activity was assayed using a Total Antioxidant Capacity Assay Kit with ABTS method (Beyotime Biotechnology). Briefly, the fresh ABTS working solution was prepared by mixing ABTS stock solution with oxidant solution for an overnight reaction, and then this mixture was diluted 50 times by 80% ethanol. To initiate the assay reaction, 10 µL of each sample with a concentration of 1.0 mM in 80% ethanol was mixed with 200 µL of fresh ABTS working solution. After being incubated at 30 °C for 5 min, the 734 nm absorbance was measured on a Thermo Varioskan LUX Microplate reader. Trolox was used as the reference compound for calculation of ABTS-reducing activity (in %).G) at 298.15 K. The two conformers were chosen for ECD calculations in methanol at the B3LYP/6-311+G  level. Solvent effects were taken into consideration using the self-consistent reaction field (SCRF) method with the polarizable continuum model (PCM). The ECD spectrum was generated by the SpecDis program [Merck molecular force field (MMFF) and DFT as well as TDDFT calculations were carried out with the Spartan 14 software (Wavefunction Inc.) and the Gaussian 16 program , respect program  using a File 1Detailed descriptions of the experimental procedures and comprehensive analytical data.