Saccharomyces cerevisiae Se-enriched biomass using a medium composed of corn bran and soybean bran acid hydrolysates as carbon and nitrogen sources in a stirred-tank reactor. After hydrolysis, hydrolysates presented complex composition and high concentrations of sugars, proteins, and minerals. The use of a stirred-tank bioreactor leads to the production of 9 g/L S. cerevisiae biomass enriched with 236.93 μg/g Se, and 99% cell viability. Likewise, the combination of sugarcane molasses and soybean bran hydrolysate was effective for cell growth of a probiotic strain of S. cerevisiae with a 24.08% β-glucan content. The results demonstrated that starchy acid hydrolysates are low-cost and efficient substrates for the production of yeast biomass and derivate products and may contribute to further studies for a sustainable development of biorefinery technologies.The production of biomolecules using agro-industrial by-products as feedstock is a growing trend worldwide. Selenium (Se) is a trace element essential for health, and the Se-enrichment of yeast biomass can enhance its benefits. This study investigated the feasibility of the production of  The consumption of yeast biomass brings several health benefits. Nutritional yeasts can be divided into probiotics and prebiotics (metabolites isolated from biomass) . ProbiotSaccharomyces cerevisiae cell wall consists of 30–60% of β-glucan, 1–2% chitin, and different proteins linked to glycans [The use of yeasts as prebiotics includes inactivated microorganisms, fragments from the cell wall, autolyzed yeasts, and mineral-enriched biomass . β-gluca glycans . Still, Selenium (Se) has an important role for human and animal health due to its antioxidant and antitumor properties, protection against drug-induced toxicity, regulation of thyroid gland function, prevention of inflammatory diseases, and also acting in animal growth and performance ,9,10,11.−, 2+, 4+, 6+, and “zero”, a biologically inert form) and it has a critical role as the redox center in selenoproteins such as glutathione peroxidase (GPx), thioredoxin reductase (TrxR), and selenoprotein P [In nature, Se is found under different oxidation states  than the acid hydrolysis of soybean bran  . The majXylose, arabinose, and phenolic compounds have been found in both CBAH (corn bran acid hydrolysate) and SBAH (soybean bran acid hydrolysate). The presence of pentose sugars is due to the hemicellulosic fraction in brans  and phenSBAH presented higher protein concentration (13.52 ± 1.06 g/L) than CBAH (7.82 ± 2.33 g/L), which can be related to the high protein content reported for this biomass. Barzegar et al.  reportedRegarding mineral composition, SBAH demonstrates a higher concentration of macrominerals and microminerals than CBAH, as can be observed in S. cerevisiae 193 presented 9.0 ± 0.26 g/L cell biomass  enriched with 236.93 μg/g Se and 99 ± 1% viability at 72 h. Until 24 h cultivation, S. cerevisiae 193 presented rapid cell growth, but the growth remained reasonably stable posteriorly, and cell biomass exhibited a pink-red color starting at 24 h. The color change is probably due to Se biotransformation inside cells since a red color in yeast biomass indicates the reduction of selenite or selenate into elemental Se [The medium presented 44.23 ± 5.48 g/L reducing sugars and 11.42 ± 2.44 g/L total protein before the fermentation assays. At 72 h, cells were recovered by centrifugation and the selenium content was determined. ental Se .C. utilis biomass enriched with 1010 μg/g Se after 18 h fermentation using YPD medium added by 15 mg/L Na2SeO3. Likewise, Kieliszek et al. [C. utilis biomass with 1841 μg/g Se content after 48 h using a YPD medium added by 30 mg/L Se as Na2SeO3 in a stirred tank bioreactor. Yang et al. [C. utilis biomass in medium supplemented with 15 mg/L Na2SeO3 under batch and fed-batch systems, respectively. In another study, Kieliszek et al. [d.w./L of C. utilis biomass and 800 μg/g in 6.7 g d.w./L of S. cerevisiae biomass after 72 h cultivation. Using agro-industrial by-products as substrate, Martiniano et al. [2SeO3, and Egressy-Molnár [Hericium erinaceus mushroom biomass using substrate composed of sawdust, wooden chips, and wheat bran.Most studies of Se enrichment in microorganisms utilize glucose as a carbon source. In the study of Zhang et al. , the autk et al.  producedk et al.  reportedo et al.  obtainedy-Molnár  obtainedX/S) occurred at 48 h (1.77 g/g), but the major volumetric cell productivity (QX) was observed at 24 h (0.25 g/L h) (The maximum cell biomass yield (Y5 g/L h) .S. cerevisiae 193 metabolized 11.46% reducing sugars of medium and the protein concentration did not suffer any significant alteration (11.58 ± 2.85 g/L). The low reducing sugar and protein consumption may be indicative that the yeast utilized other sources for growth, and, likewise, the fermentation time may be decreased since the maximum biomass yield and volumetric cell productivity occurred before 72 h.At 72 h, 4+/L did not inhibit the growth of the evaluated strain. The higher Se concentration in the medium leads to lower cell growth, but different species exhibit different ranges of tolerance towards this compound [Candida utilis biomass yield of 0.45 g/g using medium supplemented with 15 mg/L Se. Santos et al. [Candida utilis biomass using sugarcane vinasse as substrate and obtained 2.25 g/L biomass, 0.13 g/g yield, and 0.08 g/L h productivity. Juszczyk et al. [Yarrowia lipolytica yeast using raw glycerol from flaxseed oil production, resulting in a biomass yield of 0.51 g/g and productivity of 1.33 g/L h.The presence of a high concentration of Se has an inhibitory effect in yeast growth ,47, but compound ,48. Furtcompound . In the compound , the auts et al.  producedk et al.  studied S. cerevisiae strains can colonize oily residues such as wastewater from table olive processing [Some ocessing , and botocessing  reportedocessing  reportedocessing ,42,52,53Neither ethanol nor glycerol production was observed during the fermentation, possibly due to aeration conditions and strain characteristics. Although glycerol and ethanol are common by-products in fermentative processes using yeasts, their production is not always desirable since it is associated with the redox balance in yeasts and can decrease the yeast biomass production due to the deviation of growth metabolism ,54. HoweS. cerevisiae is an important microorganism used for bioethanol production due to its ability to ferment sugars and its high ethanol productivity and tolerance [Even though the Se-enrichment of yeasts has been studied over the years, the use of agro-industrial wastes as feedstock brings a new option especially for developing countries such as Brazil, one of the main producers of protein sources with large area occupied by agriculture. The use of crop by-products represents an important technological advance since most technologies are focused on the development of integrated crop-livestock systems to optimize the yields . Thus, colerance . This yeolerance .X) equaled 0.1 g/L h and occurred at 48 h, with yields (YX/S) of 0.152 and 0.311 g/g in glucose and sucrose, respectively. At 48 h, the yeast consumed 98% of glucose, 68% of fructose, and 59% of sucrose from the medium. After this time, the concentration of sugars remained stable. It is indicative that the time of the fermentation process could be shorted, reducing the costs of the process.The medium presented an initial sugars concentration of 32.01 ± 0.44 g/L glucose, 22.9 ± 2.5 g/L fructose, and 1.1 ± 0.5 g/L sucrose. At 72 h, the production of cell biomass was 6.6 ± 1.1 g/L with 95± 2% viability. Glucose was rapidly depleted by 48 h, followed by the consumption of fructose and sucrose . HoweverSaccharomyces cerevisiae strains are thermotolerant, this species is a mesophilic microorganism [The low biomass production might be related to the temperature chosen for fermentation, 37 °C since the yeast capacity to grow at a temperature tolerated by probiotic yeasts was evaluated . Althougorganism . TherebyX/S of 0.02 g/g, and QX of 0.21 g/L h of a probiotic yeast in an STR bioreactor using YPD medium under microaerobic conditions at 40 °C and 150 rpm. Guluarte et al. [6 of a probiotic Kluyveromyces lactis using YPD medium with pH 5.5 and incubated at 29 °C. Although the use of synthetic and semisynthetic media is a well-established method for cell growth, the agro-industrial by-products are low-cost alternatives for the production of yeast biomass. Paula et al. [S. cerevisiae var. boulardii as brewer’s yeast. These authors observed that glucose was preferably assimilated in the wheat beer wort, followed by maltose, and the strain performance was sensitive over temperature. Singu et al. [Saccharomyces cerevisiae var. boulardii strain coated with hydrocolloids to produce probiotic cornflakes.Cangussu et al.  produceda et al.  producedu et al.  used a tSugarcane molasses is a by-product with a complex composition and rich in sugars, which correspond up to 50% of molasses composition, primarily consisting of glucose, sucrose, and fructose . LikewisS. cerevisiae biomass from date palm waste using an airflow of 1.25 vvm and pH range of 5.0–5.6. Santos et al. [Candida utilis biomass from vinasse, a residue generated after cachaça  and ethanol production. In a study using molasses, Vu et al. [S. cerevisiae using molasses and corn steep liquor under optimized conditions in an STR bioreactor.Putra et al.  produceds et al.  obtainedu et al.  producedS. cerevisiae, whereas Pengkumsri et al. [S. cerevisiae strains.At 72 h, cells were recovery by centrifugation and treated for the extraction of β-glucan from the cell wall. The recovered β-glucan corresponded to 24.08 ± 2.14% from the disrupted yeast biomass. In general, β-glucan comprises 15–30% of the dry weight of the yeast cell . Howeveri et al.  obtainedi et al.  recovereS. cerevisiae is a widely recognized source of fungal β-glucan [The yeast β-glucan . Yeasts β-glucan  and brin2SO4 (w/v), 1:8 solid/liquid ratio (w/v) (dry mass) at 121 °C for 15 min. Soybean bran was hydrolysate with H2SO4 1% (w/v), 1:5 (w/v) (dry mass) in Erlenmeyer flasks in an autoclave at 121 °C, 15 min. After hydrolysis, both hydrolysates were filtered in cotton cloth, had their pH adjusted with 6.5 mol/L NaOH until pH 5.5, were centrifuged , sterilized , and centrifuged aseptically in these same conditions [Corn bran, soybean bran, and sugarcane molasses  were acquired from the local market . The moisture contents of corn bran and soybean bran were determined by infrared radiation at 105 °C in analytical balance. Corn bran hydrolysis was carried out in stainless steel reactor of 80 L operating volume filled with 40 L solution using 1% Hnditions . Sugarca2SO4 solution as eluent, with a flow rate of 0.6 mL/min, refractive index detection (RID6A), and injection of 20 μL of samples. Sugarcane molasses was also characterized regarding pH, reducing sugars (DNS method), and the concentration of sucrose and fructose by HPLC under the conditions of HPX-87H (BIORAD) column (300 × 7.8 mm) at 60 °C, deionized water as eluent, and 0.4 mL/min flow rate.Starchy hydrolysates in natura were characterized in triplicate for reducing sugars by DNS  method , total pSaccharomyces cerevisiae 193  and a commercial probiotic strain S. cerevisiae Sc 47 , were used in this investigation. Yeasts were grown in YPMG agar composed of (g/L) glucose 10.0, peptone 5.0, malt extract 3.0, yeast extract 3.0, and agar 20.0. The incubation of S. cerevisiae 193 and S. cerevisiae Sc 47 agar plates occurred at 30 °C for 48 h.g for 10 min for cell recovery, and the yeast biomass was washed, suspended in sterile distilled water, and adjusted for 1 × 108 cell/mL by counting cells in Agasse Lafont-R chamber (0.0025 mm2 × 0.1 mm3)  under 400× magnification in an optical microscope [The inoculum was prepared by the transference of two loopful of colonies of each strain from YPMG agar to YPG broth containing (g/L) glucose 30.0, peptone 20.0, and yeast extract 10.0. Erlenmeyer flasks were filled with 40% working volume with YPG broth and incubated in an orbital shaker at 30 °C, 200 rpm for 24 h . After 2croscope .v/v) ratio, containing about 40 g/L of reducing sugars and 10 g/L total protein, 10 mg/L of Se as Na2SeO3, and initial pH of 5.5.The medium CSBAH was composed of corn bran acid hydrolysate (CBAH) and soybean bran acid hydrolysate (SBAH) at a 4:1 , washed with distilled water, and stored at 4 °C for the analysis of total Se.Fermentation assays were performed in triplicate in a stirred-tank reactor  with 1.5 L capacity and 1 L working volume, equipped with sensors of pH and temperature, four removable baffles, an agitator shaft with two standard six-bladed impellers, and a glass condenser. Fermentation conditions were 30 °C, 350 rpm, 1.5 vvm  for 72 hS. cerevisiae Sc 47 was performed in triplicate in a stirred-tank bioreactor equipped according to v/v). Sugarcane molasses was diluted until 40 g/L of reducing sugars, and the initial pH was adjusted to 5.5 using 6.5 mol/L NaOH. Fermentation was carried out at 37 °C, 350 rpm, 1.5 vvm for 72 h [The evaluation of cell biomass production and β-glucan content of the probiotic yeast for 72 h ,70. The for 72 h  for prob2 × 0.1 mm3)  at 400× in an optical microscope [v/v).Cell growth was determined in triplicate by counting cells on the Agasse Lafont-R chamber  was calculated by the relation between the concentration of cell biomass (dry weight) produced by the concentration of reducing sugars consumed. The volumetric productivity  was calculated using the biomass production versus time.The cell biomass yield  method [Ethanol, xylitol, glycerol, glucose, xylose, arabinose, and cellobiose were analyzed in triplicate by HPLC in Agilent Technology chromatograph (aminex HPX-87H (Bio-Rad)) column—300 × 7.8 mm, 45 °C, 0.005 mol/L H) method  and tota) method .S. cerevisiae 193 biomass was previously suspended in deionized water and digested with H2SO4  in Erlenmeyer flasks covered with glass funnel, and maintained at 300 °C for 1 h in a heating plate, followed by the addition of HNO3 1:1 (v/v), and heating at 270 °C for 2 h [2SO3 (1000 mg/L) as standard [For Se analysis, standard . All assS. cerevisiae Sc47 biomass aiming to conciliate both probiotic and prebiotic properties in a single product. All assays were performed in triplicate. Yeast was disrupted using autolysis. In this process, a suspension of 45 g/L cell biomass in distilled water was incubated at 50 °C, 120 rpm, for 48 h, and subsequently, at 80 °C for 15 min [g for 10 min and the supernatant was stored at 4 °C.In this first approach, β-glucan content was evaluated only in r 15 min . After tw/v) and incubated in a water bath at 90 °C, for 5 h, centrifuged at 2000× g for 10 min, and neutralized until pH 7.7 with 2 mol/L HCl. Ethanol at −10 °C was added to the cell suspension in a ratio of 3:1 (v/v) and the mixture was left at 4 °C for 24 h. The β-glucan precipitate was oven-dried at 60 °C until constant and determined gravimetrically.The extraction of β-glucan from the disrupted cell biomass was carried out following a method adapted from Kim and Yun . In thisα = 0.05.The data obtained from this study were subjected to analysis of variance using Statistica 13.3 program . The significance of differences between the mean values in each group was tested by Tukey’s test at a significance level of S. cerevisiae 193 biomass enriched with 236.93 μg/g Se after 72 h cultivation in an STR bioreactor. A medium elaborated with sugarcane molasses and soybean bran hydrolysate is efficient for cell growth of a probiotic strain of S. cerevisiae. Yet, further studies of fermentation parameters are necessary to up-scale these processes. Agro-industrial by-products are efficient substrates for the biotechnological production of yeast biomass and derivate products that can be produced altogether with biofuels for a sustainable development of biorefinery technologies.The use of corn bran and soybean bran acid hydrolysates as substrate enabled the production of 9.0 g/L


Staphylococcus aureus (MRSA). The ABC transporter TarGH is indispensable for flipping WTA precursor from cytoplasm to the extracellular space, thus making it a promising drug target for anti-MRSA agents. The 3.9-Å cryo-EM structure of a TarGH homolog helps us to decode the binding site and inhibitory mechanism of a recently reported inhibitor, Targocil, and provides a structural platform for rational design and optimization of potential antibiotics. Moreover, we propose a “crankshaft conrod” mechanism to explain how a big substrate is translocated through subtle conformational changes of type II exporters. These findings advance our understanding of anti-MRSA drug design and ABC transporters.The wall teichoic acid (WTA) is a major component of cell wall and a pathogenic factor in methicillin-resistant  Staphylococcus aureus (MRSA), a common cause of fatal clinical infections in humans. Thus, the indispensable ABC transporter TarGH, which flips WTA from cytoplasm to extracellular space, becomes a promising target of anti-MRSA drugs. Here, we report the 3.9-Å cryo-electron microscopy (cryo-EM) structure of a 50% sequence-identical homolog of TarGH from Alicyclobacillus herbarius at an ATP-free and inward-facing conformation. Structural analysis combined with activity assays enables us to clearly decode the binding site and inhibitory mechanism of the anti-MRSA inhibitor Targocil, which targets TarGH. Moreover, we propose a “crankshaft conrod” mechanism utilized by TarGH, which can be applied to similar ABC transporters that translocate a rather big substrate through relatively subtle conformational changes. These findings provide a structural basis for the rational design and optimization of antibiotics against MRSA.The wall teichoic acid (WTA) is a major cell wall component of Gram-positive bacteria, such as methicillin-resistant  Staphylococcus aureus (MRSA) is a common clinical pathogen leading to difficult-to-treat, in many cases even fatal, infections in humans -modified ribitol-phosphate (RboP) polymer covalently linked to the UND-PP moiety, out of the cell membrane (S. aureus strains has identified a couple of putative Targocil binding residues on TarGH (Undecaprenyl diphosphate (UND-PP) is a key lipid carrier for the biosynthesis of WTAs and a variety of other cell wall polysaccharide components, such as lipopolysaccharides , bacteriiffusion . Therefomembrane , 10  polymer is transported to the outside by TarGH, after which WTA is covalently linked through a phosphodiester bond to the MurNAc sugars of peptidoglycan by an unidentified enzyme. Download FIG S1, TIF file, 2.9 MB.Schematic of the primary Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Campylobacter jejuni, which translocates intracellular UND-PP-GlcGalNAc5Bac out of the membrane for the N-glycosylation of bacterial surface proteins  encoded by the tarG gene.After systematic trials, we failed in overexpressing us TarGH A and B. of 3.9 Å , with th10.1128/mBio.02749-19.2FIG S2Alicyclobacillus herbarius and Staphylococcus aureus. (B) Alignment of NBDs between Alicyclobacillus herbarius and Staphylococcus aureus. (C) Alignment of TMDs from different strains. Download FIG S2, TIF file, 2.8 MB.Sequence alignment of wall teichoic acid transporters. (A) Alignment of TMDs between Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02749-19.3FIG S3E169Q). (A) A representative gel filtration analysis of TarGH (TarHE169Q) in UDM. The second peak fraction is visualized on SDS-PAGE gels by Coomassie blue staining. (B) A representative cryo-EM micrograph. Some particles are circled by red circles. (C) Euler angle distribution of the final 3D refinement of the overall map. (D) Representative reference-free 2D class averages of the TarGH (TarHE169Q) particles. (E) The gold-standard Fourier shell correlation (FSC) curves for the overall map. (F) FSC curves of the refined model versus the overall 3.94-Å map against which it was refined (black), of the model refined in the first of the two independent maps used for the gold-standard FSC versus that same map (green), and of the model refined in the first of the two independent maps versus the second independent map (red). The small difference between the red and green curves indicates that the refinement of the atomic coordinates does not suffer from overfitting. Download FIG S3, TIF file, 2.8 MB.Cryo-EM analysis of TarGH  shift outward, along with the inward shift of TM1 and TM5 (inside TarGH). Consequently, the tunnel becomes narrower, and meanwhile the TMDs are slightly enlarged at the outward face, corresponding to the approach of the NBDs toward each other and a higher ATPase activity. Download Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the https://pymol.org/) by the centers of molecules of our ATP-free TarGH against the Wzm-WztN structures in the ATP-free and ATP-bound forms gave a root mean square deviation (RMSD) of 4.7 Å over 832 Cα atoms and 3.4 Å over 595 Cα atoms, respectively. In addition, superposition of the TMDs yielded an RMSD of 3.9 Å over 375 Cα atoms and 3.4 Å over 341 Cα atoms, respectively. It suggested that our structure resembles the two structures of Wzm-WztN, which undergo subtle conformational changes upon ATP binding. Moreover, compared to the ATP-bound Wzm-WztN, fewer conformational changes could be observed in the overall structure of our ATP-free TarGH, except for a rigid-body rotation of the TMDs at about 15° . From the top view, when TarGH binds to ATP, TMD of TarGH clockwise rotates about 15° as a rigid body. Along with this rotation, the interface helix IF that sticks on the TMD undergoes a horizontal translation of about 5 Å. The corresponding structural segments, including TMD, IF2/IF, and GL/GH, are labeled. Download Movie S2, AVI file, 1.5 MB.Conformational change of TMD in the “crankshaft conrod” mechanism. This video is produced based on the structure alignment of TarGH (cyan) and Wzm-WztN  by using the One Step Cloning kit (Vazyme), with the tarG product containing an N-terminal decahistidine tag. The plasmid was transformed into the Escherichia coli BL21(DE3) strain (Invitrogen), growing at 37°C in lysogeny broth (LB) culture medium, supplemented with 50 μg ml−1 ampicillin. Overexpression of TarGH was induced by adding 0.2 mM IPTG  (Biofroxx) when the cell density reached an optical density at 600 nm (OD600) of 0.6 to 0.8. At 4 h after induction at 37°C, cells were collected by centrifugation; homogenized in buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 7 mM β-mercaptoethanol (β-ME) plus 10% glycerol; and stored at −80°C before use.The codon-optimized g for 1 h. The membrane fraction was collected and incubated for 1.5 h with buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 7 mM β-ME, 10% glycerol, 1% (wt/vol) dodecyl-β-d-maltopyranoside , and 1% (wt/vol) octaethylene glycol monododecyl ether  at 4°C. After another round of ultracentrifugation at 200,000 × g for 30 min, the supernatant was collected and loaded onto nickel affinity resin . The column was then washed with 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 7 mM β-ME, 10% glycerol, and 0.04% DDM. Contaminants were removed by washing with 50 mM Tris (pH 7.5), 500 mM NaCl, 7 mM β-ME, 10% glycerol, and 0.04% DDM plus 20 mM imidazole and 40 mM imidazole successively. Protein was eluted from the affinity resin with buffer containing 50 mM Tris (pH 7.5), 500 mM NaCl, 7 mM β-ME, 10% glycerol, 0.04% DDM, and 400 mM imidazole. Afterward, the eluent was concentrated to 2 ml before further purification by gel filtration  in the buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 7 mM β-ME, 10% glycerol, and 0.03% n-undecyl-β-d-maltopyranoside . Peak fractions were pooled, and desalting was implemented with buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 7 mM β-ME, 10% glycerol, and 0.03% UDM. Then, it was concentrated for EM analysis.For purification, cells were thawed followed by disruption with a Constant cell disruption system (Constant Systems) with two passes at 30 kpsi. Cell debris was removed by low-speed centrifugation for 15 min. The supernatant was collected and ultracentrifuged at 200,000 × tarGH (E169QtarH) was also constructed by using the One Step Cloning kit (Vazyme). The mutated protein purification was the same as the wild type.The mutated plasmid pET19b-E169Q was concentrated to 8 mg ml−1 for cryo-EM sample preparation implemented with FEI Vitrobot Mark IV. A 3.5-μl amount of protein was applied to a glow-discharged Quantifoil holey carbon grid . Grids were blotted for 4 s with 100% humidity and plunge-frozen in liquid ethane.Purified TarGH−/Å2 for each stack.Cryo-EM data were collected at liquid nitrogen temperature on a Titan Krios transmission electron microscope (FEI), operating at 300 kV and equipped with a K2 Summit direct electron detector (Gatan) and a Gatan GIF quantum energy filter . A total of 4,985 micrograph stacks were automatically collected with SerialEM  at a nomA simplified flow chart for the image processing procedure is presented in 10.1128/mBio.02749-19.4FIG S4FIG S4, TIF file, 2.7 MB.Cryo-EM data processing. A flow chart for cryo-EM data processing. Details can be found in under “Image processing” in Materials and Methods. Download Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02749-19.5FIG S5E169Q). (A) EM maps for IF helices, Latch Loop and each TM helix in the TMD. (B) EM maps for the overall NBD. The gate loop (GL) is shown inside a red rectangle. (C) EM maps for representative segments in the NBD. Download FIG S5, TIF file, 1.8 MB.EM maps for representative segments of TarGH , the main TMs were built by homology modeling. The side chains were assigned mainly by bulky residues such as Arg, Phe, Tyr, and Trp. High homology existed between WztN and TarH, so the structure of the WztN of Wzm/WztN was docked into the map and manually adjusted in Coot.The overall TarGH map at 3.9 Å was used for  in Coot . Based oThe model was further improved by iterative refinement in real space with secondary structure and geometry restraints using Phenix , rebuilt10.1128/mBio.02749-19.7TABLE S1Table S1, DOCX file, 0.02 MB.Cryo-EM data collection, refinement, and validation statistics. Download Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli polar lipids (Avanti) were resuspended in 20 mM HEPES-KOH (pH 7.0) to the final concentration of 20 mg ml−1. A 0.45% final concentration of Triton X-100 was added to destabilize the liposomes for 0.5 h at room temperature. The proteins were added to the destabilized liposomes and incubated for 1 h at 4°C. The ratio of lipids and proteins was kept at 100:1 (wt/wt). Triton X-100 was removed by SM-2 adsorbent biobeads (Bio-Rad) by incubation at 4°C overnight and repeated for another 2 h. Proteoliposomes were diluted and resuspended twice with the ice-cold buffer at 250,000 × g for 1 h at 4°C. After the final centrifugation, the pellets were resuspended in 20 mM HEPES-KOH (pH 7.0), 50 mM KCl, and 2 mM MgCl2 to 100 μl as one reaction sample. For each reaction sample, 0.1 μM TarGH was added.All ATP hydrolysis assays were performed in the proteoliposomes. 2O). For each 100-μl reaction sample, 1 μM Targocil was added, indicating that 0.01% DMSO was included. Thus, we added equal amounts of DMSO as supplements for control groups. Afterward, ATP was added to each sample at a final concentration of 2 mM by three rounds of freezing in liquid nitrogen followed by thawing in a bath sonicator. Reactions were performed at 37°C for 1 h, and the amount of released phosphate ion was quantitatively measured using the ATPase colorimetric assay kit (Innova Biosciences) in 96-well plates at OD650.Targocil was first solubilized in dimethyl sulfoxide (DMSO) to 10 mM and then diluted to 100 μM with double-distilled water , and the size of the space for the docking was restrained as a 16.0- by 28.0- by 18.0-Å10.1128/mBio.02749-19.6FIG S6S. aureus pLI50-tarG (lane 1). (D) Targocil docking models. All nine models calculated by AutoDock Vina are displayed in different colors for one symmetric unit. Download FIG S6, TIF file, 2.3 MB.(A) Nuclear magnetic resonance data for Targocil. (B) Structural formula of undecaprenyl diphosphate. (C) Western blot assay for verifying the expression of Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the tarG and its native promoter were amplified from S. aureus NCTC8325 genomic DNA. The fragment was cloned into the shuttle plasmid pLI50, and a Flag tag was added at the N-terminal part of the gene product to derive the plasmid pLI50-tarG. This plasmid pLI50 with the N-terminal Flag tag was constructed by using the One Step Cloning kit (Vazyme). The mutated pLI50-tarG mentioned in this paper was constructed by using the One Step Cloning kit (Vazyme). The wild-type tarG and tarG mutant plasmids were transformed by electroporation into S. aureus strain RN4220 for modification and subsequently into S. aureus NCTC8325.The gene fragment encompassing tarG-transformed S. aureus NCTC8325 was cultured at 37°C in 400 ml tryptic soy broth (TSB) culture medium overnight, supplemented with 17 μg ml−1 chloramphenicol. Then, the cells were collected by centrifugation; homogenized in buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 2 mM dithiothreitol plus 10% glycerol; and stored at −80°C if not for immediate use.The pLI50-g for 30 min. The supernatant was collected and incubated with the anti-Flag M2 affinity gel (Sigma) on ice for 1 h. Then, the resin was loaded onto the column and washed with 5 ml of wash buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% (wt/wt) glycerol, and 0.04% (wt/wt) DDM. Protein was eluted with 6 ml of wash buffer plus 200 μg ml−1 Flag peptide. Subsequently, the protein was used for Western blotting assay. Flag tag antibody  was purchased from Proteintech.The cells were disrupted with a Constant cell disruption system (Constant Systems) with one pass at 30 kpsi. Cell debris was removed by centrifugation at 12,000 × S. aureus NCTC8325 strain cultured overnight. The extraction method strictly followed the protocol of the work of Covas et al. (650.Each portion of WTA was extracted from 10 ml of the s et al. . The quaThe accession number of our structure in PDB is 6JBH and in EMDB is EMD-9790.


Scientific Reportshttps://doi.org/10.1038/s41598-019-47112-9, published online 22 July 2019Correction to: This Article contains errors.https://doi.org/10.5281/zenodo.39072891, referenced in the figure legend.The original source of Fig. 1A, B, G, and H could not be identified. A revised version of Figure Figure 3A (control start point) was a duplication of Figure 3B (control start point). The correct Figure 3 appears below as Figure The cell depicted as a representative PV-CG4 cell, shown in Fig. 6A, was selected erroneously from a movie frame of GFP-CG4 cells, shown in Fig. 7A. A correct version of Figure 6 appears below as Figure A) and differentiated (B) CG4 cells were incorrect. A corrected version of Figure 7 appears below as Figure In Figure 7, the specified time points  of representative IncuCyte images for undifferentiated ”“The datasets generated and analyzed during the current study are available in the ZENODO repository, , https://doi.org/10.5281/zenodo.3754905 , and https://doi.org/10.5281/zenodo.3335842 .”“The datasets generated and analyzed during the current study are available in the ZENODO repositories


DSTMS emits the broadest spectrum, covering the entire bandwidth of our detector from ca. 0.5 to ~7 THz, also at a laser wavelength of 2100 nm. Future improvements will require handling the photothermal damage of these delicate organic crystals, and increasing the THz frequency.Water is the most prominent solvent. The unique properties of water are rooted in the dynamical hydrogen-bonded network. While TeraHertz (THz) radiation can probe directly the collective molecular network, several open issues remain about the interpretation of these highly anharmonic, coupled bands. In order to address this problem, we need intense THz radiation able to drive the liquid into the nonlinear response regime. Firstly, in this study, we summarize the available brilliant THz sources and compare their emission properties. Secondly, we characterize the THz emission by Gallium Phosphide (GaP), 2–{3–(4–hydroxystyryl)–5,5–dimethylcyclohex–2–enylidene}malononitrile (OH1), and 4–N,N–dimethylamino–4′–N′–methyl–stilbazolium 2,4,6–trimethylbenzenesulfonate (DSTMS) crystals pumped by an amplified near-infrared (NIR) laser with tunable wavelength. We found that both OH1 as well as DSTMS could convert NIR laser radiation between 1200 and 2500 nm into THz radiation with high efficiency (> 2 × 10 Terahertz (THz) radiation probes the collective intermolecular modes of hydrogen-bonded water molecules from ~0.2 to ~20 THz . These mWhile infrared spectroscopy (IR) typically probes intramolecular modes, the absorption features in the THz range can be broad . These bAt equilibrium, the absorption by liquid water in the THz spectrum is continuous and characterized by two prominent bands  that havIn order to explore the complex energy landscape of liquid water and address its unique solvation properties, highly brilliant THz sources are required. Unfortunately, it is challenging to generate intense radiation in the so-called “THz gap”, which nowadays spans between about 7 and 19 THz and largely overlaps with water librations. We report in There are two ways to generate intense THz pulses . The firFurthermore, THz generation is reported on using novel spintronic devices ,56 as we3 [2 [Another way to generate THz is based on non-linear optical methods, by which a strong laser (often in the NIR) induces a non-linear polarization in a birefringent medium. In turn, the non-linear polarization can be the source of pulsed radiation . Emissio3 ,75,76,773 ,79,80, Z3 ,82, LiGa3 [2 , GaP [843 [2 ,87,88,893 [2 ,91,92, O3 [2 ,95,96,973 [2 ,99, DPFO3 [2 , HMQ-TMS3 [2 , OHQ-N2S3 [2 ) crystal3 [2 ,64 and o3 [2 ,65 crystThe detection of THz radiation is based on similar concepts. The highest dynamic range (DR) reported to date is above 100 dbm when antennas are used in conjunction with multi-MHz laser oscillators ,104. ForIn the quest to find intense sources operating in the THz gap, here, we report the experimental characterization of the OR in GaP, OH1, and DSTMS over the full range of the wavelengths emitted by an optical parametric amplifier (OPA), i.e., between 1200 and 2500 nm.An amplified Ti:Sa laser  emits 90 fs long pulses centered at 790 nm with a repetition rate of 1 kHz. Part of the fundamental laser output (ca 2 mJ/pulse) seeds a commercial and automated OPA . The non-linear process of parametric amplification generates simultaneously both the signal (s) as well as the idler (i) beams . The totP = 1200 (s), 1400 (s), 1600 (s), 2100 (i), 2300 (i), 2500 nm (i). We chose the wavelengths to avoid photodamage. OH1 [Here we investigate the THz emission by ~0.5 mm thick crystals  triggered by pump pulses centered at λage. OH1   strage. OH1 ,120,121 age. OH1 ). We encIn the following, we report the THz emission by both the inorganic GaP as well as the organic DSTMS and OH1 crystals  at the OPA wavelengths. The results are summarized in 2. The transient THz pulses are detected by delaying the arrival time of the electro-optical sampling beam (tEOS). We obtain almost single-cycle fields with a duration of ca. 0.35 ps (time difference between the minimum at tEOS ~ −0.15 ps and the next minimum at tEOS ~ +0.2 ps). This duration increases for longer pump wavelengths. A THz peak field of ~30 kV/cm is obtained at tEOS = 0 ps and for λP = 1200, 1400, and 1600 nm. The THz field amplitude emitted by the non-linear optical process of OR should scale linearly with the intensity of the driving pump field [P = 1600 nm and varied the fluence from 3.2 mJ/cm2 to 19.1 mJ/cm2. We found a linear relationship between the THz field and the pump intensity, as indicated by the black line fit with an R2 value of 1.02.mp field , which i2 was obtained from the pump spot size at the generation crystal (~1 mm FWHM) and the input pump power, measured with a power meter . We calculated the THz fluence from the THz peak field ) and the THz spot size at the detection crystal (estimated to ~0.4 mm FWHM with an iris). For the 0.5 mm thick GaP, we found that η decreases for increasing pump wavelength, η~3 × 10−5 at λP = 1200 nm and η~5 × 10−6 at λP = 2500 nm . A THz peak field of ~100 kV/cm is obtained at tEOS = 0 ps and for λP = 1200, 1400, and 1600 nm. In P = 1600 nm and varied the fluence from 0.4 mJ/cm2 to 1.9 mJ/cm2. As proved by the black line fit (R2 = 1.07), a linear relationship held. P = 2100 nm. In −4 for λP = 1200 nm, 1400 nm, 1600 nm. The efficiency dropped at longer wavelengths.2). For most pump wavelengths, we obtained single-cycle fields with a duration of ca. 0.27 ps (the first minimum is at tEOS ~ −0.15 ps and the second at tEOS ~ +0.12 ps). A THz peak field of ~177 kV/cm was obtained at tEOS = 0 ps for λP = 1200 nm, 1400 nm, 1600 nm, and 2100 nm. We chose the pump wavelength of λP = 1600 nm and varied the fluence from 0.6 mJ/cm2 to 6.4 mJ/cm2. The result is plotted in 2 = 1.01). −4 for λP = 1200, 1400, 1600, and 2100 nm. The efficiency dropped for the larger wavelengths and amounts to η~6 × 10−5 at λP = 2500 nm.P = 900 nm, and that the frequency range of the emitted spectrum decreases for longer pump wavelengths. The results reported in P = 1600 nm are η~2 × 10−5, η~3 × 10−4, and η~2 × 10−4, respectively. Previously, we investigated the THz emission by GaP pumped by a mode-locked oscillator tunable between 700 and 1000 nm . We foun2, 5 TW/cm2, 1 GW/cm2, and 5 TW/cm2, the peak power required to induce a pump-probe signal of roughly 1% at the frequency of ~1 MHz [In order to perform non-linear THz experiments on liquid water , broad af ~1 MHz ,128, ~1 f ~1 MHz , ~10 THzf ~1 MHz , and ~20f ~1 MHz ,139,140,2. Both organic crystals also emit some radiation over the whole detection range and up to 7 THz [P ≥ 2100 nm. As shown in P = 2100 nm with an unchanged high efficiency (η~2 × 10−4). Thus, this crystal could generate intense THz radiation from special laser sources operating in the NIR.With the aim of generating such intense pump fields, here, we investigated the THz emission by organic crystals with high electro-optic coefficients and pumped by laser pulses spanning the entire OPA spectrum. Peak fields above 0.1 MV/cm were emitted by both OH1  as well to 7 THz , with DSFuture experimental developments should aim both at scaling up the pump fluence without damaging these fragile organic crystals, as well as extending the frequency bandwidth of the detection apparatus without losing dynamical range. Pulse-shaping techniques ,141 cont


Lamprocapnos spectabilis (L.) Fukuhara is a perennial plant species valued in the horticultural, cosmetic, and pharmaceutical markets. To date, however, there were no studies on tissue culture systems in this species when adjusted from non-meristematic explants. The aim of this study is to induce callogenesis, organogenesis, and somatic embryogenesis in non-meristematic explants of Lamprocapnos spectabilis ‘Alba’ cultured in various media and to analyze the chemical diversity of the produced callus. Leaf, petiole, and internode explants were cultured on the modified Murashige and Skoog (MS) medium fortified with various combinations and concentrations of 6-benzyladenine (BA), indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorphenoxyacetic acid , and picloram (PIC). After 10 weeks of culturing, the morphogenetic response of explants was evaluated and the concentration of chlorophylls, carotenoids, anthocyanins, and polyphenols in callus was analyzed. There was no influence of explant type on the callogenesis efficiency (62.1–65.3%). The highest fresh weight of callus was produced on leaf explants in the presence of 2,4-D or PIC. In contrast, the highest share of dry weight was found in internode-derived calli and cultured on IAA-supplemented medium (up to 30.8%). Only 2.5% of all explants regenerated adventitious shoots, while rhizogenesis was reported in 4.5% of explants. Somatic embryos were produced indirectly by 0% to 100% of explants, depending on the culture medium and explant type. The highest mean number of embryos (11.4 per explant) was found on petioles cultured in the MS medium with 0.5 mg·L−1 BA and 1.0 mg·L−1 PIC. Calli cultured in media with NAA usually contained a higher content of primary and secondary metabolites. There was also a significant impact of explant type on the content of anthocyanins, polyphenols, and carotenoids in callus. Further studies should focus on the elicitation of metabolites production in callus culture systems of the bleeding heart. Lamprocapnos spectabilis (L.) Fukuhara (syn. Dicentra spectabilis (L.) Lem.) or bleeding heart is a perennial plant species, originating from Asia. It is a member of a small botanical family known as Fumariaceae, which is closely related to Papaveraceae. Due to its spectacular flowers, the species is commonly used as both an indoor and outdoor/landscape plant [L. spectabilis extracts, were observed even at a low (0.1% w/v) concentration [Staphylococcus aureus F. J. Rosenbach [pe plant . There ipe plant . Strong ntration . Studiesntration  revealedntration  and seventration . The speosenbach . TherefoL. spectabilis and achieved up to 3.3 new shoots from a single-node explant. On the other hand, there are no reports on adventitious organogenesis induced from non-meristematic explants in this species.Similar to other plant species, the bleeding heart was introduced to in vitro culture conditions for large scale reproduction. Micropropagation techniques used with horticultural crops include shoot culture  and regeneration via adventitious organogenesis, somatic embryogenesis (SE), and protocorm-like-bodies. Regeneration may occur either directly, from explant cuttings to shoots, or indirectly, with passage through a callus phase. Lee and Lee  reportedDue to the high accessibility and totipotency of plant cells, non-meristematic organs such as leaf blades, leaf petioles, and internodes are a good explant source for micropropagation, which is useful in breeding . By placPGRs were first described in the mid-20th century by Skoog, Miller, and Tsui (reviewed by Thorpe ). Among Centella asiatica L., Stevia rebaudiana Bertoni, Vaccinium myrtillus L., Hypericum perforatum L. var. angustifolium, Maackia amurensis Rupr., Sophora flavescens Aiton, Ononis arvensis L., and Maclura pomifera Raf. [L. spectabilis.Besides biomass formation, PGRs affect the plant metabolism and synthesis of chemical compounds in which some are important for the industry. Plant tissue culture systems are used in the acquisition of secondary metabolites used as pharmaceuticals, agrochemicals, flavors, fragrances, coloring agents, biopesticides, and food additives . Callus era Raf. . Productera Raf. . UnfortuLamprocapnos spectabilis ‘Alba’ cultured in various media in order to analyze the chemical diversity of the produced callus.The aim of this study was to induce callogenesis, organogenesis, and somatic embryogenesis in non-meristematic explants of −1 BA + 1.0 mg·L−1 IAA) and M4 (1.0 mg·L−1 BA + 2.0 mg·L−1 IAA) were less effective in stimulating callus development (59.3–66.7% of responding explants) than other PGR combinations (70.4–100%). On the other hand, there was no influence of explant type on this parameter. The callogenesis is reported in 62.1%, 62.4%, and 65.3% of internode, petiole, and whole-leaf explants, respectively  medium (642.8 mg). In contrast, calli produced on internodes had a higher share of dry weight  compared to leaf-derived biomass  medium (30.8%). There was no impact of AXs and CKs ratio on the general callus regeneration frequency, callus FW, and DW  than NAA (134.4–246.9 mg), while calli regenerated in the presence of IAA were of the lowest FW  regenerated indirectly a total of 36 adventitious shoots, usually in the presence of IAA  and at the cutting site A. SimilaBoth factors tested (explant type and medium composition) affected the embryogenic potential of the bleeding heart . Leaves –1 BA + 0.5 mg·L–1 2,4-D) and M14 (0.5 mg·L–1 BA + 1.0 mg·L–1 PIC) as well as petioles in the M14 medium  than 2,4-D or PIC  than other explant types . Also, IAA and NAA auxins favored non-embryogenic callus development , chlorophyll b (91.09–95.72 µg·g−1 FW), and chlorophyll ct (154.16–155.04 µg·g−1 FW) were found in calli produced on leaf explants in media M5 (0.5 mg·L−1 BA + 0.5 mg·L−1 NAA) and M8 (1.0 mg·L−1 BA + 1.0 mg·L−1 NAA) (–1 BA + 0.5 mg·L–1 PIC) medium produced the lowest chlorophyll content (6.91 µg ct per gram FW). A balanced ratio between CKs and AXs promoted chlorophyll biosynthesis  A–C. In c−1 BA + 0.5 mg·L−1 2,4-D), M15 (1.0 mg·L−1 BA + 0.5 mg·L−1 PIC), and M16 (1.0 mg·L−1 BA + 1.0 mg·L−1 PIC) had a chlorophyll a/b ratio above 1.0 (−1 BA + 0.5 mg·L−1 PIC) medium (2.44), while the lowest in callus produced on leaves in M14 (0.5 mg·L−1 BA + 1.0 mg·L−1 PIC) medium (0.47) and internodes in M9  medium (0.50)  D. No impm (0.50) .−1 FW) than from whole-leaf explants  medium (22.71 µg·g−1 FW), for internode-derived in M5 and M13 (0.5 mg·L−1 BA + 0.5 mg·L−1 PIC) media (22.58–22.69 µg·g−1 FW), and for petiole-derived callus in medium M8 (1.0 mg·L−1 BA + 1.0 mg·L−1 NAA)  medium was the least abundant in those pigments (4.5 µg·g−1 FW). Balanced low concentrations of cytokinin and auxin were preferable for the biosynthesis of carotenoids  to carotenoids ratio (−1 BA + 1.0 mg·L−1 NAA) (9.34) and callus formed on internodes in medium M15 (1.0 mg·L−1 BA + 0.5 mg·L−1 PIC) (0.53), respectively  than those from whole-leaf explants  (−1 BA + 0.5 mg·L−1 NAA) and M8 (1.0 mg·L−1 BA + 1.0 mg·L−1 NAA)  medium contained the lowest concentration of those pigments (3.40 µg·g−1 FW).Calli produced from internodes and petioles generally contained more anthocyanins (mean 32.25–35.86 µg·gL−1 NAA) . The hig−1 FW) compared to those formed on whole leaves  or moderate (0.3 < |r| ≤ 0.5) but always negative correlations between the content of metabolites tested and the share of explants forming embryogenic callus/number of somatic embryos per inoculated explant were observed (r = 0.20–0.93).Low  and cytokinins  ranged from 1 to 7 per explant [Chelidonium majus L., Eschscholtzia californica Cham., and Papaver somniferum L., have proven to be difficult to culture [Sesamum indicum L. [Non-meristematic explants, such as whole leaves, leaf petioles, and internodes, were used in the present study since they are the easiest and most abundant to obtain. It was found that most of the indirectly regenerating adventitious shoots were produced on the cutting site of the explant. This could be a result of mechanical stimuli, which is known to promote the regenerability of the plant tissue. A similar phenomenon was observed by Tymoszuk et al. , with trn et al. . Those o explant . Similar culture . Problem culture , but the culture . The sca culture . Another culture  medium, dicum L. , which mL. spectabilis was much more effective. The produced embryos had a typical morphology as those described in several other plant species [−1 BA and 1.0 mg·L−1 PIC) is quite high. As for chrysanthemum, a maximal of 5.7 embryos per explant (with 85% regeneration frequency) were produced in the MS medium with 4.0 mg·L−1 2,4-D and 2.0 mg·L−1 KIN [Glaucium flavum Crantz.), the efficiency of somatic embryogenesis reached 17.2 embryos per explants in the MS medium supplemented with 1.0 mg·L−1 2,4-D, 0.5 mg·L−1 TDZ and 0.2 mg·L−1 BA [L. spectabilis, but an additional subculture on an embryo-germination medium is often a necessity [On the other hand, SE in  species ,16,28. T·L−1 KIN . In yellg·L−1 BA . Somaticecessity . PerhapsBryophyllum subgenus. Auxin NAA enhanced root formation (data not shown), which coincides with the previous findings with a bleeding heart [Auxins and cytokinins are considered the principal PGRs involved in modulating the occurrence of signaling events during morphogenesis. The results obtained in this case allowed us to decipher that BA had a pleiotropic effect in the bleeding heart, as it caused different morphogenetic responses depending on its concentration and presence of other PGRs, including callus formation, indirect shoot organogenesis, and/or SE. On the contrary, IAA showed an inhibiting role in callus and somatic embryo formation, which stimulates adventitious shoot regeneration. Similar results were reported by García-Pérez et al.  with theng heart . As for ng heart .The balance between AXs and CKs determines the morphogenetic response in explants. Normally, the dominance of CKs over AXs promotes cell division and shoot elongation. A balanced ratio between CKs and AXs favors callus formation and embryogenesis, and AXs predominance results in root formation and elongation . In the Oncidium Gower Ramsey and subsequent plant regeneration [Anthurium andreanum Linden [It is well known that somatic embryos develop mostly from a single totipotent somatic cell in the explant (the pre-embryogenic determined cells in the direct path) or in pro-embryogenic masses re-differentiated from the parenchyma-like callus cells in the indirect path . Nonetheneration . In contm Linden . The higArtemisia annua L., tissue cultures are used for the synthesis of artemisinin [Plants are a tremendous source for the discovery of new products with medicinal and industrial importance. Approximately 25% of all drugs approved by the Food and Drug Administration proceed from plant sources . For exaemisinin . The preChlorophylls and carotenoids are involved in photosynthesis, but their derivatives consistent with cancer prevention include antioxidant and antimutagenic activity. They are also used as a food coloring agent ,39. AnthL. spectabilis can be controlled by medium variables and explant type, which is in agreement with the reports of other authors [The present study showed that metabolite biosynthesis in  authors . Seconda authors , which cRosa spp. were obtained in vegetative calluses, especially in the leaf and stem, compared with flower calluses. In the present study, calli derived from petioles and internodes contained more anthocyanins, carotenoids, and polyphenols than those produced on whole leaves, but did not differ in terms of chlorophyll content. This could be the effect of mechanical injury during explant excision, which stimulated the production of stress-related metabolites [When an explant is inoculated in vitro, diverse responses are expected, depending on its origin. According to Tarrahi and Rezanejad , the higabolites .Gerbera jamesonii Bolus cultured in vitro [a/b ratio or anthocyanins and polyphenols content. In contrast, NAA elevated the levels of all metabolites analyzed in this study, which suggests strong antioxidant activities in calli. Similarly, NAA proved to stimulate isoflavone production in Genista tinctoria L. calli at 5.0 mg·L−1 when applied jointly with 0.5 mg·L−1 cytokinin [Cytokinins play an important role in the development and structural differentiation of chloroplasts . Consequin vitro . Callus ytokinin . Perhapsytokinin .r = 0.20–0.93), the in vitro production of those metabolites can be performed simultaneously.In the present study, a negative correlation between the content of chemical compounds in callus and SE efficiency was found . To the Further studies should focus on the use of exogenous melatonine ,47, elic2·6H2O), iron sulfate (FeSO4), and Na2EDTA·2H2O, supplemented with 3% (w/v) sucrose, and solidified with 0.8% (w/v) agar . A cytokinin (BA) and auxin  were added into the culture medium in combination, at the concentration of 0.5; 1.0, or 2.0 mg·L−1 each. Based on the qualitative and quantitative composition of PGRs, the media were assigned the following symbols: M0 (a PGR-free control medium), M1 (0.5 mg·L−1 BA + 1.0 mg·L−1 IAA), M2 (0.5 mg·L−1 BA + 2.0 mg·L−1 IAA), M3 (1.0 mg·L−1 BA + 1.0 mg·L−1 IAA), M4 (1.0 mg·L−1 BA + 2.0 mg·L−1 IAA), M5 (0.5 mg·L−1 BA + 0.5 mg·L−1 NAA), M6 (0.5 mg·L−1 BA + 1.0 mg·L−1 NAA), M7 (1.0 mg·L−1 BA + 0.5 mg·L−1 NAA), M8 (1.0 mg·L−1 BA + 1.0 mg·L−1 NAA), M9 , M10 , M11 , M12 , M13 (0.5 mg·L−1 BA + 0.5 mg·L−1 PIC), M14 (0.5 mg·L−1 BA + 1.0 mg·L−1 PIC), M15 (1.0 mg·L−1 BA + 0.5 mg·L−1 PIC), and M16 (1.0 mg·L−1 BA + 1.0 mg·L−1 PIC) , prior to sterilization at 105 kPa and 121 °C for 20 min. The medium (40 mL) was poured into 350-mL glass jars and sealed with plastic caps.The Murashige and Skoog  medium (L−1 PIC) . All PGRLamprocapnos spectabilis (L.) Fukuhara ‘Alba’ were used as the donor plant material. Whole leaves (7–10 mm-long), leaf petioles (3–5 mm), and internodes (1–2 mm) were excised from the central part of shoots and inoculated polarly (leaves) or vertically (petioles and internodes) in the modified MS medium with nine explants per jar. Each jar was considered a single repetition. The experiment was repeated thrice.The 10-week-old in vitro-derived shoots of −2·s−1 provided by standard cool daylight TLD 54/36W fluorescent tubes with a color temperature of 6200 K .The cultures were kept in the growth chamber at 24 ± 1 °C, under 16-h photoperiod conditions and photosynthetic photon flux density of approximately 30 µmol·mAfter 10 weeks of culturing, the share of explants forming callus, adventitious shoots, and roots was counted. The fresh weight and share of dry weight of calli (dried in a laboratory oven at 105 °C for 3 h) were also evaluated. The share of inoculated explants forming somatic embryos, and the number of embryos per one inoculated explant was included.The spectral analysis of metabolites was performed for in vitro regenerated calli in the 10th week of the culture in three repetitions. Twelve experimental combinations (M5–M16) were included in the array. Due to an insufficient amount of callus produced in combinations M0–M5 (PGR-free control and media with BA and IAA), those experimental objects were not included in the study.v/v), according to the Harborne [Chlorophylls and carotenoids were extracted from fresh calli, as described by Lichtenthaler  using 10Harborne  method. Harborne . The totmax): for carotenoids at 470 nm, for anthocyanins at 530 nm, for chlorophyll a and b at 645 and 662 nm, and for phenolics at 765 nm, respectively. The content of pigments and phenolics was calculated per gram of fresh matter.The spectrophotometric analysis of extracts was performed in a spectrophotometer SmartSpec PlusTM  at specific wavelengths  with the alphabet indicating the homogeneous groups. The experiment was set up in a completely randomized design. To obtain the normal distribution of the data expressed as a percentage, the Freeman-Tukey transformation was used. Data were statistically verified by applying Statistica 12.0  software. The analysis of variance (ANOVA) was performed and means were evaluated with the Newman-Keuls test at the significance level of r, p ≤ 0.05).Associations between the content of chemical compounds in callus and the share of explants forming (non-)embryogenic callus and number of somatic embryos per explant were also analyzed, based on the Pearson correlation coefficient (Lamprocapnos spectabilis. Due to the multifactorial behavior, the design of a universal protocol for plant regeneration is a challenging task. The induction of adventitious organogenesis seems difficult and not efficient in the bleeding heart. Somatic embryogenesis is more promising, especially when using whole-leaf or petiole explants and 2,4-D or PIC-fortified media. Medium composition affects not only the production of biomass but also the biosynthesis of metabolites. Calli cultured on media with NAA usually contain a higher content of primary and secondary metabolites, but produce less somatic embryos. There is also a significant impact of the explant type on the content of anthocyanins, polyphenols, and carotenoids in callus. Further studies should focus on the application of elicitor stimulation in the overproduction of economically important compounds.This is the maiden research on the in vitro culture systems adjusted from non-meristematic explants in


The outbreak of Zika virus (ZIKV) in 2016 created worldwide health emergency which demand urgent research efforts on understanding the virus biology and developing therapeutic strategies. Here, we present a time-resolved chemical proteomic strategy to track the early-stage entry of ZIKV into host cells. ZIKV was labeled on its surface with a chemical probe, which carries a photocrosslinker to covalently link virus-interacting proteins in living cells on UV exposure at different time points, and a biotin tag for subsequent enrichment and mass spectrometric identification of the receptor or other host proteins critical for virus internalization. We identified Neural Cell Adhesion Molecule (NCAM1) as a potential ZIKV receptor and further validated it through overexpression, knockout, and inhibition of NCAM1 in Vero cells and human glioblastoma cells U-251 MG. Collectively, the strategy can serve as a universal tool to map virus entry pathways and uncover key interacting proteins. The mechanism underlying the cellular entry of Zika virus is not fully understood. Here, the authors use a chemically modified virus and time-resolved proteomics to capture interacting host proteins during virus entry and identify NCAM1 as a ZIKV receptor. Flaviviridae, some notable differences such as the Asn154 glycosylation site present on each of the E proteins may affect receptor interactions, antibody response, and downstream biology of the virus2. For the past several years, efforts have been made to understand fundamental biology of ZIKV infection, including the use of animal models to evaluate their immune response to the virus invasion3, as well as potential therapeutics against ZIKV infection6. However, unanswered questions remain regarding molecular mechanisms of host restriction and immune evasion7. Identification of direct interactors of ZIKV during its entry into host cells could not only suggest the molecular pathways manipulated by the virus, but also provide immense opportunity to develop antivirals by offering new potential drug targets. Owing to the transient nature of these interactions and the extreme rapidness in the flavivirus entry in general, identification of dynamic interactors of virus is a formidable task.Zika virus (ZIKV) has been the focus of immense investigation since the recent epidemic. While studies on ZIKV structure revealed its structural similarity to other virus relatives within the family 9. The movement of single virus demonstrated an intriguing and dynamic process. The molecular information, in particular protein machinery involved in the process, is typically limited to labeled molecules in the amazing technique. On the other hand, affinity and chemical proteomics studies identified virus-interacting proteins12. The molecular mechanisms and dynamic virus–host interactions responsible for the internalization of ZIKV, however, have remained unresolved. A systematic quantitative measurement of temporal changes in virus–protein interactions may prove extremely valuable for the identification of host molecules as potential therapeutic targets. We have previously used chemical proteomics strategies and modified a nanoparticle, polyamidoamine generation 3 dendrimer, to understand the endocytic pathways of a nanoparticle13 followed by a recent study to investigate the entry of Salmonella into host cells14. Here, we expand the concept and hypothesize that chemical modification of ZIKV would not significantly affect its infectivity and would allow us to track the virus entry into living cells and identify virus-interacting proteins by mass spectrometry (MS), revealing the spatiotemporal distribution of the key proteins involved in the pathways for ZIKV entry and trafficking.Our understanding of ZIKV internalization and cellular trafficking would greatly benefit from a systematic, temporal characterization of major proteins involved in the dynamic virus entry. Real-time fluorescence microscopy has been used to study the transport, acidification, and fusion of single virus2, there are 13 cysteines in ZIKV E  and no cysteine in M protein -like linker to improve water solubility, while offering the flexibility for efficient crosslinking and enrichment.We devised and synthesized a multifunctional chemical probe Fig. –3 bearinThe labeling was first examined with a standard protein and then with intact ZIKV. Bovine serum albumin (BSA) was incubated with 1 mM of reagent in phosphate buffer pH 7 at 4 °C and the labeled protein was enriched on streptavidin beads and analyzed by SDS-PAGE. The labeling efficiency was estimated to be 25–30%  and plays a key role in cellular attachment and entry of neHEV21. Receptor of activated protein C kinase 1 (RACK1) is important for lymphocystis disease virus entry and infection22. 4F2 cell-surface antigen heavy chain (SLC3A2) was reported interacting with ZIKV NS4B protein and was indicated as a candidate host factor for ZIKV infection12. In this study, co-immunoprecipitation followed by western blotting of transduced cells verified that SLC3A2 specifically associates with ZIKV ENV  complex that recognizes the tyrosine-rich sorting signals on the cytoplasmic tail of receptor proteins24. Our crosslinking experiment validates the complex’s structural assembly on the membrane as AP2M1 was selectively crosslinked at 0 min of infection, while no other AP-2 subunits were observed. AP2M1 has further been shown to modulate early-stage infectious entry of HCV, in its phosphorylated form25. CALML5 was discovered as a potential host factor for ZIKV infection12.Notably, we identified 121 crosslinked proteins at 0 min, 24 of which were membrane proteins, such as C1QBP, CD9, ITGA3, LETM1, NCAM1, RACK1, and SLC3A2. Complement C1q binding protein is a key host factor for efficient respiratory syncytial virus (RSV) production26. Previous studies also have suggested the mechanism of blocking immune response by ZIKV as either the degradation of signal transducer and activator of transcription (STAT2)27, or antagonism of STAT1 and STAT2 phosphorylation28. RACK1, which mediates the interactions between IFN receptor and STAT1, was also identified as a ZIKV-interacting protein at that time point. Previously, a different class of virus was shown to interact with RACK1, thus initiating the dissociation of RACK1–STAT1 complex and inhibition of interferon signaling by the virus29. Our study emphasizes the involvement of both STAT1 and RACK1 in the immune response elicited by the ZIKV infection, suggesting ZIKV might employ a similar approach to suppress the host immune response.We further demonstrated that the strategy allowed us to identify clusters of proteins representing a temporal shift of ZIKV subcellular localizations and the functions of crosslinked proteins are highly correlate to the temporal information in many cases Fig. . For exa30. However, some viruses may also utilize the recycling endosomal pathway to evade lysosomal degradation. Our study suggests the use of the recycling pathway by ZIKV after entry. Besides RAB11A, we also observed RAB5C at 8 min. Previous studies have established the importance of RAB5C in the entry of ZIKV16. The identification of RAB5C and RAB11A in our chemical proteomics experiment at a late time point of entry is consistent with the published data on Japanese Encephalitis Virus (JEV)31, a neurovirulent pathogen from the flavivirus genus structurally similar to the ZIKV. Other proteins identified of infection include HSPA8, also known as heat shock cognate 70 (Hsc70), known for its role in vesicle uncoating in the later stage of endocytosis32. Overall, more than 50 direct interactors such as AP2B1 and ITGB1 were also identified as ZIKV interactors in previous work33, further indicating the confidence of the identified interactors in our study.We also identified an isoform of a key component of recycling endosomes, RAB11A. The role of RAB11A in transport of viral ribonucleoprotein or core proteins to plasma membrane for the generation of new virus particles of influenza and Hepatitis C Virus (HCV), respectively, is well documented35, indicating the requirement of clathrin-mediated endocytosis and low pH for ZIKV infection. In our study, ITGB1, HSPA8, RAB5C, RACK1, and RAB11A were crosslinked, suggesting the clathrin-mediated pathway employed by the virus to infect Vero cells. Furthermore, identification of ARCN1, ITGA3, FLNA, and FLNC also suggests the utilization of a caveolar-mediated pathway by ZIKV for endocytosis.In addition, we examined temporally ZIKV-crosslinked proteins against protein components involved in major endocytic mechanisms for ZIKV internalization. Prior studies showed ZIKV infection could be prevented by lysosomotropic agents which neutralize the normally acidic pH of endosomal compartments and was also blocked by chlorpromazine32, also bound to the ZIKV ENV protein directly  and dengue virus (DENV-2). NCAM1 ECD and anti-NCAM1 antibody had minimal effects on IAV binding and entry . 293T and Vero cell lines were purchased from the American Type Culture Collection (ATCC). Low passage cells were used for the virus propagation and other infection experiments. The NCAM1 ECD protein (Cat. No. 10673-H08H) for inhibition experiment and the rabbit anti-NCAM1 antibodies (PE) (Cat. no. 10673-MM05-P) for staining of surface expression of NCAM1 were purchased from Sino Biological. The anti-NCAM1 monoclonal antibody for inhibition experiment and immunofluorescence assay was purchased from BD Biosciences (Cat. no. 559043), while the antibody for western blot was obtained from Cell Signaling Technology (Cat. no. 3576). All reagents for synthesis were obtained from Sigma-Aldrich, ChemPep Inc, Peptides International Inc, Novabiochem (EMD Millipore), and Alfa Aesar. pCMV-Zika-ENV (Cat. No. VG40543-UT) and pCMV-Zika-NS1 (Cat. No. VG40544-UT) natural native open reading frame mammalian expression plasmids were purchased from Sino Biological. pcDNA3.1+/C-(K)DYK-NCAM1 (Cat. no. OHu00262), pCMV-GFP-Flag (Cat: AG13105-CF), pCMV-EGFR (Cat: HG10001-UT) and pcDNA3.1+/C-(K)DYK-HSPA8 (Cat. no. OHu27538) mammalian expression plasmids were purchased from GenScript.U-251 MG , Vero , and HEK293T  were maintained in DMEM media (Gibco) supplemented with heat-inactivated 10% FBS (Gibco), 100 IU/mL of penicillin and 100 µg/mL of streptomycin, at 37 °C and under 5% CO9 Vero-Furin cells36 were infected at a multiplicity of infection (MOI) of 0.1 with ZIKV (strain H/PF/2013) at 37 °C2. The ZIKV MR766 stock was purchased from ATCC (ATCC® VR-1838™). Virus particles were purified from media collected at 60 and 72 h post infection (hpi) according to Sirohi et al.2. Briefly, virus particles were precipitated from the media with 8% polyethylene glycol (PEG) 8000 overnight at 4 °C, pelleted at 10,000 × g for 50 min at 4 °C. Resuspended particles were pelleted through a 24% sucrose cushion, resuspended in 0.5 mL NTE buffer , and purified with a discontinuous gradient in 5% intervals from 35% to 10% K-tartrate, 20 mM Tris pH 8.0, 1 mM EDTA. Mature virus was extracted from the gradient, concentrated, and buffer exchanged into NTE buffer.Approximately 1 × 10The plaque assay was performed as described below. Briefly, virus was diluted serially in the order of ten folds and incubated with monolayers of Vero cells for 1 h at room temperature. Cells were layered with agarose and incubated at 37 °C for 3 days. Plaques were counted following cell staining using neutral red.tert-Butoxycarbonyl) group deprotection. HCTU (O-(1H-6-Chlorobenzotriazole-1-yl)−1,1,3,3-tetramethyluronium hexafluorophosphate) was utilized as an activating agent for the carboxyl group on the incoming reactant, in presence of the base NMM (4-methylmorpholine). The synthesis was performed on the 30 µmol scale and using 2.5-fold excess of the reagents compared with the resin. Each step involved the deprotection of amine group, activation of carboxyl group followed by coupling reaction. The excess reagents were removed by thorough washing of beads by DMF. Ninhydrin test was performed after each deprotection and coupling reaction.The virus-labeling chemical probe was synthesized on the Rink-Amide-AM-Resin (200–400 mesh) 1% DVB manually, using standard solid phase peptide synthesis approach  of Rink-Amide-AM-Resin was added to the fritted reaction vessel. Beads were conditioned with DMF for 15 min. The solution was removed by filtration, and 20% piperidine in DMF was added to the beads for fmoc deprotection. The mixture was end-to-end rotated for 30 min, and solution was removed followed by washing of beads with DMF. A reaction mixture of Fmoc-Lys-Biotin-OH , HCTU , and NMM  in DMF was added to the resin, and rotated for 4 h at room temperature. The resin was washed with DMF, and fmoc deprotection steps were repeated. A solution of N-Fmoc-Nʺ-succinyl-4,7,10-trioxa-1,13-tridecanediamine , HCTU , and NMM  in DMF was rotated with the resin for 4 h. Excess reagents were removed, and the resin was washed with DMF. Similarly, Fmoc-Lys(Boc)-OH , N-Fmoc-Nʺ-succinyl-4,7,10-trioxa-1,13-tridecanediamine , and 6-Maleimidohexanoic acid  were coupled to the resin after fmoc deprotection steps. The resin was washed with DMF and dichloromethane, and the molecule was cleaved from the resin using 95:5 mixture of TFA:TIS (Triisopropylsilane) for 1.5 h. The cleavage step also deprotects the boc group, making available a free amine in the product. The crude product was concentrated and HPLC (Agilent 1100) purified using a gradient of 5–85% B  over 30 min on Waters XBridge Prep BEH130 C18 column  to yield 1 . MALDI-TOF and NMR were used to characterize the product  δ 7.91 , 7.86–7.71 , 7.66 , 7.54 , 7.26–7.18 , 6.91 , 6.89–6.83 , 6.42–6.20 , 4.28–4.16 , 4.10–3.94 , 3.49–3.18 , 3.04–2.85 , 2.70 , 2.71–2.61 , 2.49 , 2.33–2.16 , 1.94 , 1.68–1.47 , 1.46–1.32 , 1.31–0.99 .13C NMR  δ 174.1, 172.0, 171.8, 171.6, 171.6, 171.5, 171.1, 162.8, 158.2, 158.0, 134.5, 117.4, 115.1, 112.7, 69.8, 69.6, 68.1, 68.1, 61.1, 59.3, 55.5, 52.4, 37.0, 36.0, 35.9, 35.8, 35.3, 35.2, 31.4, 31.2, 30.8, 30.7, 30.6, 29.4, 29.4, 29.3, 29.0, 28.3, 28.1, 27.8, 26.7, 25.8, 25.4, 24.9, 23.0, 22.5.+.A 1297.358 peak in MALDI was observed corresponding to the expected M + H1  was dissolved in DMF and reacted with excess NHS-LC-Diazirine hexanoate) in phosphate buffer pH 8, for 2 h at room temperature. The product was purified by directly injecting into the HPLC and using similar conditions as described above. The virus-labeling chemical probe 2 was obtained as a white powder  and characterized by MALDI-TOF, 1H and 13C NMR  δ 8.00–7.88 , 7.88–7.77 , 7.72 , 7.28 , 6.99 , 6.94 , 6.45–6.31 , 4.32–4.26 , 4.15–4.03 , 3.54–3.41 , 3.40–3.24 , 3.14–2.92 , 2.81 , 2.56 , 2.40 – 2.23 , 2.06–1.96 , 1.96–1.88 , 1.72–1.52 , 1.52–1.40 , 1.40–1.08 , 0.96 .13C NMR  δ 174.0, 171.8, 171.6, 171.5, 171.5, 171.4, 171.1, 170.5, 162.7, 134.5, 69.8, 69.5, 68.1, 61.1, 59.2, 55.4, 52.6, 52.4, 37.0, 35.8, 35.7, 35.4, 35.2, 35.2, 31.5, 31.4, 30.7, 29.9, 29.8, 29.4, 29.3, 29.2, 28.9, 28.9, 28.2, 28.0, 27.8, 26.1, 25.8, 25.3, 25.1, 24.8, 23.0, 19.3.m/z, corresponding to the M–N2 + H+. This is due to the loss of N2 from diazirine under MALDI conditions.MALDI showed a peak at 1492.232 Fifty micrograms BSA or Purified ZIKV was diluted to 500 µL with PBS pH 7, and mixed with the labeling chemical probe in the final concentration of 1 mM . Cell lysates were cleared by centrifugation at 14,000 rpm to pellet down cell debris, and supernatant were used for the biotin-Neutravidin affinity purification. Bicinchoninic acid assay (Thermo Fisher Scientific) was performed for protein quantitation, and the lysates equivalent to 1 mg protein for each sample were reduced and alkylated by boiling at 95 °C, in 10 mM TCEP (Tris(2-carboxyethyl)phosphine) and 40 mM CAA (chloroacetamide) respectively. The lysates were then diluted to 0.1% SDS and rotated with 50 µL preconditioned Neutravidin beads slurry in 4 °C overnight. The beads were washed with 0.1% SDS in 50 mM Tris (pH 8.0), 5 M NaCl, 300 mM glycerol in 50 mM Tris (pH 8.0), and then transferred to the low protein binding Eppendorf tubes, where they were further washed three times with 25 mM ammonium bicarbonate (ABC) buffer pH 8. Two hundred microliters ABC buffer was added to the beads, and proteins were digested on-bead at 37 °C using 2 µg Lys-C for 3 h and 200 ng trypsin for 12 h. The supernatant containing peptides was collected and beads were washed twice with 50 µL ABC buffer, further pooled with the supernatant. Peptides were acidified and desalted using in-house StageTips with SDB-XC (3M). The peptides were dried in SpeedVac before subjecting to LC–MS/MS analysis.m/z 350–1800 with a resolution of 30,000 at m/z 400) was followed by MS/MS scans of top 10 intense ions  with a dynamic exclusion for 60 s and dynamic list of 500.The peptides were dissolved in 0.1% formic acid and injected into Easy-nLC 1000 (Thermo Fisher Scientific). The peptides were separated on a 45 cm in-house column (360 µm OD × 75 µm ID), packed with C18 resin  and heated to 60 °C with a column heater . The mobile phase was comprised of 0.1% formic acid in ultra-pure water (solvent A) and 0.1% formic acid in 80% acetonitrile (solvent B), and the gradient used for separation was 10–30% B over a linear 60 min at a flow rate of 250 nL/min. The EASY-nLC 1000 was connected online to the LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific) by a nanospray source. Data acquisition was performed in the data-dependent mode, in which a full scan (range from Rhesus macaque) FASTA database  with Andromeda search engine, using default parameters. The first peptide precursor mass tolerance was set at 20 ppm, and MS/MS tolerance at 0.6 Da. Carbamidomethylation was set as a fixed modification for cysteines, while oxidation of methionine and acetylation at N-terminus were selected as variable modifications. Enzyme specificity was set to trypsin with maximum two missed cleavages. The search was performed with 1% false discovery rate at both peptide and protein levels. The identifications were transferred from the sequenced peaks to the unidentified peaks of the same m/z within a time window of 0.7 min (match between runs) across samples. For LFQ, initially, the LFQ intensities were extracted from the MaxQuant output file. Proteins with valid values in minimum two out of three replicates in at least one group were only considered, and values were imputed for all missing values based on normal distribution. Significant proteins at different time points vs. control  was performed. The raw data were searched against UniProtKB  of total quantified proteins were clustered based on Euclidean distances with average linkage using modified function Clustergram in MATLAB (Version R2019b), and heatmap was also visualized by MATLAB. The color shows that there are great changes from sample to sample, where red is upregulation, green is downregulation, black represents no change, and gray represents non available. Each column in the graph represents an experiment condition, and each row corresponds to a gene. Row of protein were normalized by maximum value of corresponding row. The rows and columns are displayed in the order given by the clustering output trees in the two dimensions.p). p value was calculated using the hypergeometric formula as below:N is the number of all identified proteins that can be connected with GO analysis information.GO enrichment analysis of the differentially expressed proteins were conducted according to the information from GO databases, each bar in the figure denotes the enrichment score from different sample, and enrichment score was defined as −log10 were preincubated with 20 µg NCAM1 ECD  or control protein for 1 h at 37 °C. The viruses (MOI = 100) were then added to U-251 MG cells in twelve-well plate and incubated on ice for 1 h. The supernatant was then removed and the cells were washed three times with cold PBS. Cellular RNA was extracted and purified for test the viral attachment. Otherwise, prewarmed medium was added to the cells to initiate ZIKV internalization. The cells were incubated at 37 °C for additional 40 min and then cellular RNA was extracted and purified. Then RT-qPCR was performed to measure viral entry.For antibody inhibition experiment, U-251 MG cells in twelve-well plate were preincubated with 20 µg/mL anti-NCAM1 antibody  or control isotype IgG antibody  in DMEM supplemented with 2% FBS for 1 h at 37 °C. Cells were then incubated with purified ZIKV (MOI = 100) on ice for 1 h in the presence of antibody. The supernatant was then removed and the cells were washed three times with cold PBS. Cellular RNA was extracted and purified for test the viral attachment. Otherwise, prewarmed medium was added to the cells to initiate ZIKV internalization. The cells were incubated at 37 °C for additional 40 min and then cellular RNA was extracted and purified. Then RT-qPCR was performed to measure viral entry. For NCAM1 protein inhibition experiment, purified ZIKV  on ice for 1 h after 36 h transfection. The supernatant was then removed and the cells were washed three times with cold PBS. The increase in viral attachment was measured with RT-qPCR. On the other hand, prewarmed medium was added to the cells to initiate ZIKV internalization. The cells were incubated at 37 °C for additional 40 min and then cellular RNA was extracted and purified. Then RT-qPCR was performed to measure internalized ZIKV RNA.6 cells per 10 cm dish) were transiently transfected with 14 µg of pCDNA3.1-NCAM1/ pCDNA3.1-HSPA8 and pCMV-NS1/pCMV-E separately for 48 h using TurboFect Transfection Reagent (Thermo Fisher Scientific). Cells were rinsed twice with cold PBS and were then transferred to clean tubes and lysed in cell lysis buffer for immunoprecipitation supplemented with 1% protease inhibitor cocktail . Cell lysates were incubated with PierceTM Protein A/G Agarose  for 4 h at 4 °C and were then subjected to centrifugation at 10,000 × g for 10 min at 4 °C. The supernatant was transferred to a new tube and incubated with 30 µL anti-Flag M2 affinity gel  overnight at 4 °C. The Sepharose samples were centrifuged, washed five times with cell lysis buffer and eluted using 3 × Flag peptide . Then all samples were boiled with SDS loading buffer for 10 min.For co-immunoprecipitation experiments, 293T cells   at room temperature for 20 min. Cells were washed three times with PBS supplemented with 2% FBS. All flow cytometry experiments were carried out using an LSRFortessa cell analyser (BD Bioscience). Samples were analysed using FlowJo software version 10 (TreeStar).17. The oligo sequences of the sgRNAs targeting NCAM1 and HSPA8 are listed as follows. NCAM1-sgRNA1: AACGCCAACATCGACGACGC; NCAM1-sgRNA2: ACACCACTGAGATCCGCTGC; HSPA8-sgRNA1: ACAGATGCCAAACGTCTGAT; HSPA8-sgRNA2: CTAGACTGTTACCAATGCTG. LentiCRISPRv2 clones containing the guide sequences were sequenced, purified, and used for lentiviral production. To generate heterogeneous knockout cell populations, U-251 MG cells were infected with the lentiCRISPRv2-derived lentivirus for 36 h and were then reseeded into complete DMEM containing 2 µg/mL puromycin for 14 days to select for transduced cells. Surviving populations derived in this manner were propagated and expanded for using.Oligos encoding sgRNAs for generating knockout cells using CRISPR-Cas9 were cloned into the lentiCRISPRv2 plasmid  as previously describedRNA was isolated from mammalian cells  using RNeasy mini kit  and normalized based on total RNA amount as determined by NanoDrop™ 2000/2000c Spectrophotometer (Thermo Fisher Scientific). RT-qPCR was performed using SuperScript III Platinum SYBR Green One-Step qPCR Kit w/ROX (Thermo Fisher Scientific) and analyzed on Applied Biosystems 7300 real-time PCR system. The samples were subjected to thermal cycling for 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C, at which point data were collected, and this was followed by dissociation curve analysis. The Ct values obtained were converted to the number of ZIKV RNA molecules using a standard curve generated from in vitro-transcribed viral RNA. ZIKV cDNA clone, used for in vitro transcription, was kindly provided by Shi. For the standard curve, the plasmid containing ZIKV cDNA, was linearized by Cla1 and viral RNA was transcribed using T7 RNA polymerase (New England Biolabs). The DNA template was digested by RNAase-free-DNAase enzymatic treatment for 30 min at 37 °C and the viral RNA was subsequently purified by RNeasy mini kit (Qiagen). RNA concentration and quality were determined by NanoDrop and 10E10 copies of RNA were serially diluted ten-fold and subjected to thermal cycling as described above to obtain the standard curve and PCR efficiency. All PCR primers are listed as follows: ZIKV-F1 TGGGAGGTTTGAAGAGGCTG; ZIKV-R1 TCTCAACATGGCAGCAAGATCT; GAPDH-FCTGGGCTACACTGAGCACC; GAPDH-RAAGTGGTCGTTGAGGGCAATG; DENV-F1AAGGACTAGAGGTTAGAGGAGAC; DENV-R1 GGCGTTCTGTGCCTGGAATGAT; IAV-F1 CGCACAGAGACTTGAGGATG; IAV-R1TGGGTCTCCATTCCCATTTA.HEK293T cells (for transfection with NCAM1) were seeded on cover slips in 24-well plate. Cells were washed with PBS and fixed with 3.7% paraformaldehyde (PFA) for 10 min at room temperature. Cells were again washed with PBS three times and blocked with 2% BSA in PBS for 1 h. Anti-NCAM1 antibody in blocking solution was incubated with the cells for 1 h at room temperature. Cells were washed three times and incubated with anti-mouse FITC or anti-mouse Alexa Fluor 488 for 1 h at room temperature. DAPI staining was performed for 10 min, followed by final three PBS washes. Cover slips were mounted on glass slide and images were captured using Olympus IX81 fluorescence microscope with a 60X oil immersion objective.For detecting ZIKV, cells on slides were fixed with 4% PFA for 20 min at room temperature, permeabilized with 0.1% Triton-X 100 in PBS for 5 min, and blocked with blocking buffer (1% BSA and 2% donkey serum diluted in PBS) for 30 min. Immunofluorescence analyses of ZIKV-infected cells were performed using a mouse anti-flavivirus envelop protein antibody , with a Alexa Fluor 568 donkey anti-mouse IgG (H + L)  as the secondary antibody. All cells were mounted with ProLongTM Gold Antifade with DAPI  and imaged with a TissueFAXS 200 flow-type tissue cytometer . All statistical analyses of immunofluorescence staining present the results from at least 3000 cells per replicate, and data are shown as the mean ± s.e.m.Following overexpression of NCAM1 in HEK293T, cells were lysed at 48 h post transfection. The samples were boiled at 95 °C in gel loading buffer and 1,4-Dithiothreitol (DTT) for 5 min. The cell lysates were separated on the precast NuPAGE 4-12% Bis-Tris polyacrylamide gels (Invitrogen) for 90 min at constant voltage of 150 V. A MOPS solution  was used as a running buffer. The proteins were transferred onto polyvinylidene fluoride membranes in Bicine-Bis-Tris transfer buffer containing 12% methanol, for 75 min at a constant current of 275 mA. The membrane was blocked with 2% BSA in TBST, and probed with anti-human NCAM1  for 1 h at room temperature. Following washings, anti-mouse IgG HRP-conjugated secondary antibody  was utilized for visualization (Supplementary Fig. Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


Sosiho-tang (SSHT), a traditional herbal formula consisting of a mixture of seven herbs, has been used to treat allergic reactions and chronic hepatitis disease in East Asia. In this study, we determined whether SSHT has an inhibitory effect against lipopolysaccharide- (LPS-) induced acute lung injury (ALI) in mice. 0.05, 0.55, and 5.55 mg/kg of SSHT were orally administered to C57BL/6J mice for 7 days prior to the administration of LPS. After 2 h of LPS sensitization, lung tissues were collected to confirm the lung histology and ALI-related inflammatory factors. SSHT ameliorated the LPS-induced alveolar hemorrhage, alveolar wall thickening, and the shrinkage of the alveolar spaces in the ALI mice model. Proinflammatory cytokines including IL-6, TNF-α, and IFN-γ in the lung tissue were significantly regulated in the SSHT-treated groups compared to the LPS only-treated group. Also, increases of IL-6 and TNF-α and decrease of IFN-γ expressions were dose-dependently modulated by SSHT treatment in LPS-induced raw 264.7 cells. Additionally, the translocation of NF-κB into nucleus and phosphorylation of mitogen-activated protein (MAP) kinase were significantly attenuated by the treatment of SSHT in LPS-sensitized ALI mice. SSHT showed anti-inflammatory activities by inhibiting proinflammatory cytokines and NF-κB signaling in LPS-induced ALI. This study demonstrates that SSHT has preventive effects on LPS-induced ALI by regulating inflammatory responses as an alternative for treating lung diseases.Acute lung injury (ALI) is a series of syndromes with persistent inflammation and abnormally increased vascular permeability.  ALI is hrinkage .Clinically, treatment of ALI focuses on counterbalance of inflammatory response by regulating inflammatory/anti-inflammatory cytokines production . Some stSosiho-tang, known as Sho-saiko-to in Japanese and Xiao-Chai-Hu-Tang in Chinese, is an herbal medicine in Korea for medical insurance. Sosiho-tang consists of seven herbs: Bupleurum falcatum Linne 12 g, Scutellaria baicalensis Georgi 8 g, Pinellia ternata Breitenbach 4 g, Panax ginseng C. A. Meyer 4 g, Glycyrrhiza glabra Linn var. glandulifera Regel & Herder 2 g, 3 of Zingiber officinale Roscoe, and 2 of Ziziphus vulgaris Lamarck var. inermis Bunge. It is widely used for treating allergic diseases, cold-related symptoms, and chronic liver disease [Sosiho-tang include the dyshepatia and hepatosis of chronic liver disorders [Sosiho-tang improved liver inflammation and fibrosis by regulating liver toxicity levels, demonstrating that research of Sosiho-tang is focused on liver disease [Sosiho-tang is reported to decrease the production of T helper 2-type cytokines and chemokine [Sosiho-tang has a therapeutic effect on ALI via its anti-inflammatory effects. In the present study, inflammatory effects of Sosiho-tang against ALI were determined in LPS-sensitized mice and raw 264.7 cells. disease –19. In pisorders , 21. In  disease , 23. Bechemokine , we postSosiho-tang used in this study was Jungwoo Pharmacy Sosiho-tang Soft Ext. (SSHT), a prescription-based medicine in Korea. SSHT, a Korean Medicine for national health insurance, was obtained from Jungwoo Pharmacy Co., Ltd. (Lot. no. 702). Briefly, SSHT consists of 750 mg of Bupleurum falcatum Linne, 1.115 g of Scutellaria baicalensis Georgi, 588 mg of Pinellia ternate Breitenbach, 514 mg of Panax ginseng C. A. Meyer, 246 mg of Glycyrrhiza glabra Linn var. glandulifera Regel & Herder, 75 mg of Zingiber officinale Roscoe, and 518 mg of Ziziphus vulgaris Lamarck var. inermis Bunge soft extracts based on the general requirements in the Korean Pharmacopoeia. A total of 9 g of SSHT was dissolved in distilled water with appropriate doses for mice.The sample of Thirty-six males C57BL/6 mice at 5 weeks of age were purchased from Raon Bio Inc. . Mice were housed under controlled conditions . After 1-week acclimation, mice were randomly divided into six groups with 6 per group: CTR , LPS (LPS-sensitized group as a negative control), DEX (LPS-sensitized and DEX-treated group as a positive control), S0.05 (LPS-sensitized and SSHT 0.05 g/kg-treated group), S0.55 (LPS-sensitized and SSHT 0.55 g/kg-treated group), and S5.55 (LPS-sensitized and SSHT 5.55 g/kg-treated group). 7 mg/kg of dexamethasone (DEX) and SSHT were orally preadministrated for 1 week, followed by intraperitoneal injection of 10 mg/kg LPS. The SSHT that was used for the human being 9 g/60 kg/day was converted by using the human equivalent dose (HED) equation used for mice into 5.55 g/kg. There was no toxicity to mice during sample administration of SSHT 5.55 g/kg. Following LPS sensitization for 2 h, blood samples were collected by cardiac puncture. Lung tissues were collected at 10% neutralized formalin solution. All experiments were approved by the Committee on Care and Use of Laboratory Animals of Kyung Hee University (KHUASP(SE)-18-078; Seoul, Korea).μm thickness and mounted on slides. These slides were stained with hematoxylin and eosin solution. The stained slides were photographed by digital microscope at a magnification of 200× and 400×. The blind test for ALI score was determined as 0 to 5 grades: 0, no injury and appears normal; 1, minimal (injury up to 25% of the field); 2, mild (injury between 25 and 50% of the field); 3, moderate (injury between 50 and 75% of the field); and 4, severe . Tissue sections were examined by a pathologist blinded to the experiment.The right inferior lobe of lung was fixed at 10% neutralized formalin for 24 h. The lung tissues were dehydrated and embedded in paraffin for 24 h. The paraffin blocks were cross cut into 4 μg/mL streptomycin (Gibco). Cells were grown at 37°C in an atmosphere containing 5% CO2 of 95% humidity. Cells were seeded in 6-well plates and treated with 1, 10, 100, 250, 500, and 1000 μg/mL of SSHT in the presence of LPS 1 μg/mL for 24 h. DEX was treated at the 1 μg/mL concentration to raw 264.7 cells for 24 h.Raw 264.7 cells were grown in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% v/v fetal bovine serum (Gibco), 2 mM glutamine, 100 IU/ml penicillin, and 100 ß-glycerophosphate, 10 mM NaF, and 5 mM Na3VO4) containing the protease inhibitor cocktail and centrifuged at 24 g for 5 min. The supernatants obtained from the homogenate are used to analyze IκB-α and phospho-IκB-α. For nuclear protein extraction, a nuclear lysis buffer  containing a protease inhibitor cocktail was added to the pellet. Nuclear protein was centrifuged at 15,928 g for 10 min to detect NF-κB.Whole left lobe of lung tissues containing the left-upper lobe and the lower lobe was homogenized using a radioimmunoprecipitation assay (RIPA) lysis buffer  containing protease inhibitor cocktail  for extracts of whole proteins. The lysed proteins were centrifuged at 15,928 g for 10 min at 4°C and collected supernatant was used to determine mitogen-activated protein (MAP) Kinase, ERK1/2, SAPK/JNK, and p38. To extract cytoplasmic proteins, frozen lung tissues were homogenized with cytoplasmic buffer  were separated into 10% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked for 1 h in TBS containing 0.1% Tween (TBS-T) and 5% bovine serum albumin. The membrane was incubated overnight at 4°C with primary antibodies. The following antibodies were used in this study: ß-actin , ERK1/2 , phospho-ERK1/2 , SAPK/JNK , phospho-SAPK/JNK , p38 , phospho-p38 , NF-κB , IκB-α , and phospho-IκB-α . Anti-mouse IgG  and anti-rabbit IgG  secondary antibodies were incubated room temperature for 1 h. The blots were visualized by Davinch-Chemi  with enhanced chemiluminescence kit .The amount of proteins was quantified using the Bradford protein assay. Protein samples (20 μg RNA at 45°C for 1 hour and incubated at 95°C for 5 min. PCR was performed using Maxime PCR PreMix (Invitrogen) and synthesized cDNA template. The sequence of the primers used was as shown in For RNA extraction, the lung tissues and raw 264.7 cells were homogenized in TRIzol reagent , according to the manufacturer's instructions. The optical density was read at a wavelength of 260 nm to quantify RNA. To synthesize complementary DNA, Maxime RT PreMix (Invitrogen) was mixed with 1 p < 0.05 was taken to indicate statistical significance.Significance was determined by one-way analysis of variance (ANOVA) and Tukey's multiple comparison tests. In all analyses, p < 0.05). SSHT treatment at all concentrations  significantly decreased the histology scores by 34.5%, 46.2%, and 42.2% (p < 0.05), respectively, while DEX treatment showed 51.8% reduction (p < 0.05) of ALI score in LPS-sensitized mice  in lung tissue was assessed by RT-PCR. Expression of IL-6 was increased 3.18 times (p < 0.05) in LPS-treated group compared to the normal group. SSHT treatment at the concentrations of 0.55 and 5.55 g/kg decreased expression of IL-6 to 34.1% and 55.4% of LPS-treated group, respectively (p < 0.05). The levels of TNF-α increased 3.56 times of LPS-treated group than that in the normal group (p < 0.05). Expression of TNF-α was decreased 58.5% in treated with 5.55 g/kg of SSHT (p < 0.05). In contrast with the other cytokine expression levels, expression of IFN-γ was 0.57 times decreased in LPS-treated group compared with the nontreated CTR group (p < 0.05). SSHT treatment with 5.55 g/kg increased the expression of IFN-γ 35.94% compared to LPS group (p < 0.05).To examine whether SSHT treatment inhibits LPS-induced inflammation in ALI model, proinflammatory cytokines IL-6, TNF-PS group  (p < 0.0α in LPS-sensitized raw 264.7 cells were 2.8 and 3.0 times increased compared to nontreated cells (p < 0.05), while LPS significantly decreased the mRNA level of IFN-γ in raw 264.7 cells (p < 0.05). In the SSHT-treated cells in the presence of LPS, the expressions of IL-6 and TNF-α were markedly decreased. 500 and 1000 μg/mL of SSHT significantly inhibited the IL-6 levels, while 250, 500, and 1000 μg/mL of SSHT significantly reduced the TNF-α levels, respectively (p < 0.05). Moreover, 1000 μg/mL of SSHT significantly increased the IFN-γ expression in LPS-sensitized raw 264.7 cells (p < 0.05).Additionally, the expressions of IL-6 and TNF-.7 cells  . IκB-α phosphorylation was reduced 18.6% and 24.3% by 0.55 and 5.55 g/kg of SSHT treatment, respectively (p < 0.05). Moreover, expression of nuclear NF-κB was also decreased 63.5%, 67.9%, and 88.9% by SSHT treatment (p < 0.05).Iectively  (p < 0.0p < 0.05). Administration of SSHT at all concentrations  had significantly dose-dependently decreased the ERK phosphorylation by 13%, 26.4%, and 56.8% (p < 0.05). Phosphorylation of JNK was significantly increased by 2.65 times in LPS-treated group compared with the CTR group (p < 0.05), whereas 0.55 and 5.55 g/kg of SSHT treatment markedly reduced LPS-induced phosphorylated JNK by 33.7% and 41% (p < 0.05). In LPS-sensitized mice, phosphorylation of p38 was 1.12 times increased in comparison with normal mice (p < 0.05). SSHT 5.55 g/kg-treated group showed a significant 15.7% reduction of p38 phosphorylation (p < 0.05) (LPS-induced 1.36 times increase of ERK phosphorylation in lung tissues of mice ( < 0.05) .New finding of efficacy of medicine would be helpful to expand its medical indication as a prescription for health insurance. Recommended daily intake dose of SSHT to human is 9 g/day/60 kg, converted to 5.55 g/kg of SSHT in mice. In this study, 0.05, 0.55, and 5.55 g/kg of SSHT were administered to LPS-induced ALI mice. LPS causes histological changes including alveolar hemorrhage, shrinkage of alveolar spaces, and alveolar wall thickening . Those sB. falcatum Linne polysaccharides, a major herb of SSHT, suppressed LPS-induced proinflammatory cytokines production including IL-6, IL-1β, IFN-β, and TNF-α in peritoneal macrophages of mice [S. baicalensis Georgi consisting of SSHT has been studied to decrease TNF-α and IL-1β levels in bronchoalveolar lavage fluids of LPS-induced ALI mice [α level [α, and IFN-γ in ALI model were investigated to find the underlying mechanism of SSHT on the recovery of alveolar wall thickening. Proinflammatory cytokines such as IL-6 and TNF-α are considered key components of the acute inflammatory response [α produced by initiating inflammatory response in macrophages undergo to activate NF-κB pathway and MAP kinase [γ as a primary activator by stimulating innate and adaptive immunity clears intracellular pathogens and produces enzymes to inhibit viral infections [α mRNA levels in the lung tissues and raw 264.7 cells were increased by exposure of LPS and significantly decreased by the pretreatment or cotreatment with SSHT. Those results demonstrate that SSHT might regulate the counterbalance of inflammatory responses by downregulating IL-6 and TNF-α and upregulating IFN-γ. of mice . AdditioALI mice . Total s[α level . Based oresponse –31. SomeP kinase . IL-6 isP kinase . IFN-γ afections , 35. IL-κB is translocated from the cytoplasm to the nucleus by inflammatory cytokines such as IL-6 and TNF-α [κB is regulated by IκB-α proteolytic degradation [κB and MAP kinases in LPS-sensitized lung tissues could ameliorate ALI [κB and the phosphorylation of IκB-α in LPS-sensitized lung tissue were increased, but they were markedly decreased in the SSHT-treated mice. SSHT also significantly reduced the phosphorylation of ERK1/2, SAPK/JNK, and p38 in the lungs. In addition to the results from IL-6 and TNF-α, SSHT attenuated the activation of MAP kinase and NF-κB by inhibition of inflammatory cytokines such as IL-6 and TNF-α (NF-nd TNF-α –40. The radation . Additioradation . Inhibitrate ALI . The expnd TNF-α .α production, following the decreases of transcription factors including NF-κB and MAP kinases. Inhibition of inflammatory responses by SSHT in lung would be helpful to ameliorate LPS-induced ALI. Taken together, SSHT might be beneficial for treating ALI. Since the administration of 5.55 g/kg of SSHT to mice is converted to daily intake 9 g/day/60 kg dose for human, Jungwoo Pharmacy Sosiho-tang Soft Ext. is applicable for treating lung inflammation. Optimal dosage range of SSHT might be 9 g/day/60 kg, a daily intake dose for human in present. Further studies regarding the maximal efficacy and toxicity of SSHT are needed.In conclusion, SSHT alleviated the inflammatory IL-6 and TNF-