B2 phenol) regarding its potential use as a differential biomarker. For that, we performed 1D 1HNMR (TOCSY), UV-Vis absorption spectra in different organic solvents, voltammetry profile (including a scan-rate study), and TD-DFT calculations that including NBO analyses, to provide valuable information about B2 structure and luminescence. In our study, we found that the B2 structure is highly stable, where the presence of an intramolecular hydrogen bond (IHB) seems to have a crucial role in the stability of luminescence, and its emission can be assigned as fluorescence. In fact, we found that the relatively large Stokes Shift observed for B2 (around 175 nm) may be attributed to the stability of the B2 geometry and the strength of its IHB. On the other hand, we determined that B2 is biocompatible by cytotoxicity experiments in HeLa cells, an epithelial cell line. Furthermore, in cellular assays we found that B2 could be internalized by passive diffusion in absence of artificial permeabilization at short incubation times (15 min to 30 min). Fluorescence microscopy studies confirmed that B2 accumulates in the endoplasmic reticulum (ER) and Golgi apparatus, two organelles involved in the secretory pathway. Finally, we determined that B2 exhibited no noticeable blinking or bleaching after 1 h of continuous exposure. Thus, B2 provides a biocompatible, rapid, simple, and efficient way to fluorescently label particular organelles, producing similar results to that obtained with other well-established but more complex methods.In this study, we explored new properties of the bioinspired pyridine benzimidazole compound  B2 shows luminescent emission at room temperature, with a large Stokes shift  , biofilms of Lactobacillus kunkei and L. rhamnosus, and epithelial cell lines (SKOV-3 and HEK-293), as assessed by confocal microscopy . Furthermore, we found that B2 uptake can be detected at short incubation times, apparently by passive a diffusion mechanism. Finally, we found that B2 provides a rapid (30 min), simple (no cell permeabilization is required), biocompatible, and efficient way to fluorescently label both ER and Golgi apparatus, producing similar results to that obtained with other well-established methods. We also provide evidence that B2 is a good candidate to be used as a new, differential, antibody-free fluorophore for organelles belonging to the cell secretory pathway, in time-lapse experiments or short videos with continuous exposure, even at low temperatures.Recently, we demonstrated that 2,4-di-tert-butyl-6-phenol : 2961 (νOH), 2904 and 2868 (νNH), 1626 (νCe = N), 1526 (νC = C). 1HNMR : δ = 1.33 ; 1.43; 7.40 , 7.69 , 8.02 , 8.36 , 8.99 , 13.60. UV/VIS:  λ nm (ε mol−1 dm3 cm−1): 332 (13.32 × 103), 294 (19.09 × 103), 284 (15.51 × 103);  λ nm (ε mol−1 dm3 cm−1): 327 (12.91 × 103), 292 (18.67 × 103), 282 (14.88 × 103);  λ nm (ε mol−1 dm3 cm−1): 332 (11.30 × 103), 294 (15.44 × 103), 284 (12.23 × 103). Rf: 0.38 (ethylacetate as solvent).The general procedure for the synthesis was previously reported, , using tetramethylsilane as internal standard. FTIR techniques were recorded in an UATR spectrum Two Perkin Elmer spectrophotometer.NMR spectra were recorded on a Bruker AVANCE 400 spectrometer operating at 400 MHz, at 25°C. Samples were dissolved in deuterated dimethyl sulfoxide . Spots were visualized with short wave ultraviolet light (λ = 254 nm) using Spectroline LongLife TM Filter. Melting points were determined on a Stuart 10 Scientific melting point apparatus SMP3 (UK) in open capillary tubes.Purity of 6) as supporting electrolyte in CH3CN, was used. Prior to each experiment, the working solution was purged with high purity argon, and an argon atmosphere was maintained during the whole experiment. A polycrystalline non-annealed platinum disc (2 mm diameter) was used as working electrode. A platinum gauze of large geometrical area, separated from the cell main compartment by a fine sintered glass, was used as counter electrode. All potentials quoted in this work were referred to an Ag/AgCl electrode in tetramethylammonium chloride to match the potential of a saturated calomel electrode (SCE), at room temperature. All electrochemical experiments were performed at room temperature on a CHI900B bipotentiostat interfaced to a PC running the CHI 9.12 software that allowed experimental control and data acquisition.For electrochemical experiments, a working solution containing 0.01 mol/L of the respective compound together with 0.1 mol/L tetrabutylammonium hexafluorophosphate  code  supplemented with 10% v/v fetal serum bovine (FBS), 1 mM sodium pyruvate and 1% v/v penicillin-streptomycin. Cells were incubated at 37°C and 5% CO2, changing the culture medium every 2–3 days, and propagated when they reached between 80 and 90% confluence.The HeLa cell line (ATCC® CCL-2™)  was grown in 25 cm5 cells per well) in which a 12-mm diameter coverslip was previously added, and allowed to acclimate for 24 h. Each well was then washed 3 times with sterile 1 × PBS and then the different concentrations of B2  and DMSO vehicle  were added to each well and incubated for 15 and 30 min at 37°C with 5% CO2. It is important to underline that permeabilization procedures are not necessary. Subsequently, each well was washed 3 times with sterile 1 × PBS and coverslips were deposited on the slides using 5 μl of Fluoromount® mounting medium. Each slide was left to dry in the dark at room temperature overnight and were sealed with acrylic paint.HeLa cells were seeded in a 24-well culture dish , Spinning Disk Olympus (DSU) system, coupled to an ORCA-R2 camera  and CellSens Dimension software v1.9 . Fluorescence emission was obtained by excitation with a xenon lamp. Emission was collected with a DAPI filter (450 to 500 nm).5 cells/well) were seeded in 24-well plates and incubated for 24 h. To evaluate the cellular uptake of compound B2, concentrations of 25 μg/mL and 50 μg/mL were prepared in culture medium, added to each well and incubated for 15 and 30 min. In addition, to evaluate whether the uptake of B2 depends on energy, cells were incubated with the same treatments at 4°C, a temperature where no endocytic processes occur  and resuspended in 500 μl PBS. Finally, the fluorescence intensity of the cells was quantified through a BD Accuri™ FACSARIA II flow cytometer using the FlowJo 7.6.1 software.2 flasks in a 5% CO2-humidified atmosphere at 37°C. Passage took place every 2–3 days. All cell culture supplies were purchased from Sigma-Aldrich. Toxicity was determined using the 3--2,5- diphenyltetrazolium bromide (MTT) cell viability assay after 15 min, 30 min, 1 h, and 24 h of incubation with B2. MTT is a yellow compound that, when reduced by active mitochondria, produces purple formazan crystals that can be measured spectrophotometrically  containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were maintained in 75 cmE. coli TOP10 (ThermoFisher)For propagation of the desired vector, it was introduced into the chemically competent 5 cells per well) in which a glass cover was previously added to each well and allowed to acclimate for 24 h. Subsequently, the entire culture medium was removed, and each well was washed 3 times with sterile 1 × PBS. The different combinations of DNA and Lipofectamine 3000® Transfection Reagent were performed according to the manufacturer's instructions. After all components were added to the cell culture , it was incubated for 6 h at 37°C with 5% CO2. The content of each well was removed and washed 3 times with 1 × sterile PBS. Then, 500 μL of complete DMEM medium was added to each well and incubated until 48 h of treatment were completed. Finally, cell transfection was checked using an inverted-light microscope and a BX-53 epifluorescence microscope.HeLa cells were seeded in a 24-well culture dish  and the percentages of vehicle DMSO (12.5 and 6.125%) were added for 15 min with 5% CO2 and at 37°C. Each well was then washed 3 times with sterile 1 × PBS and fixed for 5 min with 4% PFA in PBS , washed 3 times with PBS and each coverslip was mounted on slides using 5 μl of Electron Microscopy Sciences. Each slide was allowed to dry in the dark at room temperature overnight and then sealed with acrylic paint. Finally, all samples were observed using the Olympus BX-61-DSU epifluorescence microscope.HeLa cells were seeded in a 24-well culture dish with 5 × 10p < 0.05.All values of analyzed data are presented as mean standard error (SE) from three biological replicates. Statistical analysis included was one-way ANOVA followed by multiple comparison test (Tukey). Differences among groups were considered statistically significant when B2  and π → π* transitions, respectively. No significant shifts were observed for B2 in the different solvents used , produced by interaction with the solvent, would lead to changes in the absorption spectra. This was not observed for B2 under the tested conditions  and the first excited singlet state (S1); in a second step, absorption and emission bands were calculated using TDDFT. All the calculations where performed using COSMO model for solvent with the parameters of DMSO . We found that the IHB energy is 6.23 kcal/mol for S0, and 6.13 kcal/mol for S1. These values are in agreement with the reported values in similar compounds harboring IHB , HOMO-1 → LUMO (π → π*), and HOMO → LUMO (π → π*). The band located experimentally at 332 nm (DMSO), theoretically calculated at 339 nm, corresponded to a HOMO → LUMO transition. Both the HOMO and LUMO composition involves the IHB   with B2  for 30 min, prior to fluorescent microscopy. Beforehand, we characterized the B2 staining properties using a confocal microscope, with laser excitation at 405 nm, and emission collected with a long-pass filter in the range of 425 to 525 nm  and DAPI filter  . Interestingly, we observed that B2 produced a punctuate pattern inside cells , highlighting the potential of this compound as a relatively quick biomarker, even using common equipment, such as a xenon or mercury lamp, and a DAPI filter. Moreover, it is clear from these experiments that chemical derivatizations are not required for B2 to be used as biomarker. Finally, the B2 staining protocol can be performed without the need of additional permeabilization steps, underlining its simplicity. In this sense, DMSO has been known to enhance cell membrane permeability of drugs or DNA. Studies exploring DMSO applicability to promote plasma membrane permeability using different molecules in living cells, demonstrated that DMSO can increase cell permeabilization, in a both concentration- and time-depending manner , and shorter times of incubation , we speculate that the DMSO could contribute to the entry of B2 into epithelial cells, with minimal effects on cell morphology. Nevertheless, the potential impact of the DMSO in cellular compartments, at the concentrations and incubation times proposed in this study for B2 staining protocol, must be explored in future analyses.To further characterize B2 potential as biomarker, we characterized its cellular uptake in epithelial cells. For that, we performed flow cytometry assays of cells stained under three different conditions: (1) Two B2 concentrations that allow cell staining (25 and 50 μg/mL); (2) two incubation times with B2: 15 min, suboptimal for staining, and 30 min, optimal for staining; and (3) two temperatures (4 and 37°C). We determined its cellular uptake under the last condition because it is particularly valuable to assess the entry mechanism, since active endocytic processes are inhibited at 4°C -based (d6) compounds as potential biomarkers that were unable to enter cells at 4°C, even after prolonged incubation periods (more than 2 h) . Normally, biomarkers can be modified to be used as differential dye through conjugation with antibodies, relatively big and complex molecules that usually preclude cell uptake; thereby, additional permeabilization steps are required. By contrast, B2 cytotoxicity in epithelial cells by MTT assays. MTT is a yellow compound that, when reduced by functioning mitochondria, produces purple formazan crystals that can be measured spectrophotometrically  and different incubation times . It is important to remark that the optimal staining protocol requires 50 μg/mL of B2, and 30 min of incubation  compared with the vehicle alone , values that represent no toxicity for cellular models . DMSO increases cyclic adenosine monophosphate (cAMP), a second messenger involved in cell death by apoptosis , whereas more prolonged times  are not recommended due to higher cytotoxicity levels, not biocompatible for these kind of studies  . It has been reported that endoplasmic reticulum (ER) exhibit a similar staining pattern ], to stain the ER; and Sialyl-RFP [Sialyl transferase signal anchor sequence—red fluorescent protein (583 nm)], to stain the Golgi apparatus . Unlike B2, KDEL-GFP and Sialyl-RFP need to be expressed directly by the cells, thereby a transfection protocol must be performed. Transfection consists in introducing purified nucleic acids, normally produced in bacteria, into eukaryotic cells to express heterologous proteins, such as KDEL-GFP or sialyl-RFP. Complete transfection protocol can take two or more days.As shown in Figure s Tsien, . These rB2 accumulates in subcellular compartments from the secretory pathways , revealing these organelles as efficiently as KDEL-GFP or Sialyl-RFP, but with a simpler method. B2 accumulation in the ER and in the Golgi apparatus is probably due to its affinity for certain proteins inside these organelles, probably glycoproteins. Zhang et al. demonstrated that two fluorophores based on Ir (III) present high affinity for the Golgi apparatus -based fluorophores, B2 is a simpler molecule exhibiting better quantum yields (φ = 0.21 in acetonitrile) ,  is able to stain the ER at 10 μg/mL. Nevertheless, at lower concentrations , DiOC6(3) stains mitochondria instead, letting unclear which are the limit concentrations to stain one organelle or another , whereas lower concentrations appear to be unable to stain (as mentioned above), allowing a better identification.On the other hand, it has been reported that DiOCB2 provides a simple and efficient way to differentially stain ER and Golgi apparatus in epithelial cells, showing that B2 can be considered a differential fluorescent dye.Thus, unlike other fluorophores which only have cytoplasmic or perinuclear distribution  exhibits photobleaching similar to that of the rhodamine , simple (since no cell permeabilization is required), biocompatible (low cytotoxicity under staining conditions), and efficient way to fluorescently label both ER and Golgi apparatus, producing similar results to that obtained with other well-established methods, but without photobleaching or blinking. Altogether, our results show that B2 is suitable to be used for differential labeling of ER and Golgi apparatus, in time-lapse experiments or short videos with continuous exposure, even at low temperatures.In this work, we explored new features of the luminescent compound Dedicated to Professor Juan Manuel Manríquez on the occasion of his retirement.FL: Cytotoxicity and uptake experiment, discussion of biological experiment and paper writing; JF: Discussion of all the experiments, and paper writing; AC: Synthesis, characterization, discussion of all the experiments, and paper writing; CZ: Optical and other chemical characterizations; DP-H: Theoretical calculations; MG: Electrochemical studies; RP: Discussion of Biological experiments; MP: Discussion of NMR, TOCSY and FTIR experiments; RA-P: Discussion of theoretical calculations; CO: Microscopy and discussion of Biological experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Geoffroea decorticans (chañar) grows in the arid lands of northern Chile. It has been used as a food plant since prehistoric times. Phenolic-enriched extracts (PEEs) of Chilean chañar fruits were assessed for their chemical composition, antioxidant properties and inhibition of pro-inflammatory and metabolic syndrome-associated enzymes. Phenolic profiles were determined by HPLC-DAD-MS/MS. The PEEs of G. decorticans showed a strong effect towards the enzymes COX-1/COX-2, with inhibition percentages ranging from inactive to 92.1% and inactive to 76.0% at 50 µg PEE/mL, respectively. The IC50 values of the PEEs towards lipoxygenase and phospholipase A2 inhibitory activity were between 43.6–96.8 and 98.9–156.0 μg PEE/mL, respectively. Samples inhibited α-glucosidase (IC50 0.8–7.3 μg PEE/mL) and lipase (9.9 to >100 μg PEE/mL). However, samples did not inhibit α-amylase. The HPLC-DAD-MS analysis of the PEEs allowed the tentative identification of 53 compounds, mainly flavonol glycosides and procyanidins. The procyanidin content of the Chilean G. decorticans pulp was positively correlated with the antioxidant activity and the inhibition of the enzyme α-glucosidase. These results indicate that the Chilean chañar fruit contains bioactive polyphenols with functional properties.The native tree  Geoffroea decorticans (Gill. ex Hook. et Arn.) Burkart (Fabaceae), popularly known as chañar, is a characteristic species of South America, including the arid northern Chile − base peak at m/z 579, with the consecutive neutral loss of 132 and 162 Da, being assigned as kaempferol pentoside hexosides. Triglycosylated kaempferol derivatives were identified in peaks 22 and 27. Peak 22 showed a [M − H]− base peak at m/z 741, with the consecutive loss of 162, 132 and 162 Da, and was tentatively identified as kaempferol dihexoside pentoside, while peak 27 showed a [M − H]− base peak at m/z 755, with the consecutive loss of 162 and 308 Da, being identified as kaempferol hexoside rutinoside. The last kaempferol derivative (peak 52) showed the neutral loss of 176 Da, and was tentatively identified as kaempferol glucuronide. Peak 35 showed a [M − H]− base peak at m/z 623, with the consecutive loss of 162 and 176 Da, being identified as kaempferol hexoside glucuronide. Peak 11 showed a base peak at m/z 615 leading to a MS2 at m/z 285, and was assigned as an unknown kaempferol derivative.Ten kaempferol derivatives were identified by the main fragment ion at −m/z 771, 625, 609, 595, 477, and 463 with main fragment ion at m/z 301. Peak 41 was identified as quercetin hexoside by the loss of 162 Da. Peak 43 showed the neutral loss of 176 Da and was tentatively identified as quercetin glucuronide. The dihexoside of quercetin were identified by the consecutive loss of two hexoses units (peak 23), while a pentoside hexoside derivative (peak 32) was also identified by the neutral loss of 132 and 162 Da. The main peak identified in the total ion chromatogram (peak 39) showed a parent ion of m/z 609, with MS2 base peak at m/z 463 and 301, being identified as quercetin rutinoside. The identity of this compound was confirmed with the co-injection of a rutin standard (data not shown). The triglycosides of quercetin were identified by the loss of two hexoses and one rhamnose (peaks 24 and 33).Eight quercetin glycosides were identified at [M − H]m/z 315 Da. Some diglycosides were identified by the consecutive loss of 132 and 162 Da . In addition, isorhamnetin rutinoside (peak 50) was identified by the base peak at m/z 623 and the neutral loss of 308 Da. The triglycosides of isorhamnetin were also identified, including peak 45 that showed the neutral loss of 132 and 308 Da, tentatively identified as isorhamnetin pentoside rutinoside, and peak 47, which showed the neutral loss of two 162 and 146, and was assigned as isorhamnetin dihexoside rhamnoside. Six isorhamnetin derivatives were identified by the base peak at 40) was identified by the base peak at m/z 285, with MS3 fragmentation to 241 and 175, characteristic of luteolin aglycone − at m/z 577 and MS/MS fragmentation of 289 is compatible with (epi) catechin- (epi) catechin dimer, tentatively identified as a B-type procyanidin according to the fragmentation pattern − ion at m/z 863 with fragmentations to m/z 711, 695, 573 and 451. This fragmentation pattern is compatible with A-type procyanidin trimer with two A-type linkages [21 shows the [M − H]− base peak at m/z 939 with the loss of , and the consecutive fragmentation pattern of a procyanidin trimer. Hence, the compound 21 was assigned as a procyanidin trimer derivative. Compound 19 showed a base m/z peak at 1153, with three consecutive loss of 289 Da, and was assigned as a B-type procyanidin tetramer [G. decorticans quantified by colorimetric means [G. decorticans flour [G. decorticans fruits. A broader analysis that includes the identification of mean degree of polymerization by means of thiolytical depolymerization will be carried out in future studies.Peaks hexoside . The MS  pattern . Compoun1 UV max 0 nm, wittetramer . Condens9 and 10 were characterized as vanillic acid mono- and diglycosides, respectively, according to their fragmentation patterns. Peak 9 shows a base peak at m/z 329, with the neutral loss of a hexose from the mother ion, leading to vanillic acid. The compound was identified as a vanillic acid hexoside. On the other hand, peak 10 shows the consecutive loss of a hexose (−162 Da) and a pentose (−132 Da) leading to the base peak at m/z 167. The compound was identified as a vanillic acid hexoside pentoside. The compounds have been previously identified in Argentinean G. decorticans fruits [18 shows a parent ion of m/z 337, with MS2 base peak at m/z 191. The hierarchical scheme for the identification of phenolic acids allowed us to tentatively identify this compound as 5-p-coumaroylquinic acid [48 and 53 that showed the same base peak at m/z 515. The fragmentation pattern of compound 48 lead to MS2 peaks at m/z 353, 191 and 135, compatible with 3,5-dicaffeoylquinic acid [53 showed MS2 peaks at m/z 353 and 173, and was tentatively identified as 4,5-dicaffeoylquinic acid [Peaks s fruits . The MS 50 of the crude PEE from Diego de Almagro (12.1 µg PEE/mL) was higher than all the single fractions pools, suggesting a possible synergy of the different constituents in the fractions . In the DPPH discoloration assay, the SCractions .50 of 24.1 µg/mL. Hereinafter, the antioxidant capacity started to decline. On the other hand, the activity of the Diego de Almagro PEE towards α-glucosidase showed a strong inhibition with an IC50 of 4.7 µg/mL. Separation of the PEE by Sephadex LH-20 led to fraction pools with higher inhibitory effect, ranging from 0.4 to 8.0 µg/mL , CUPRAC  and the scavenging of superoxide anion . The total flavonoid content did not correlate with any of the bioactivity assays performed. However, the total proanthocyanidin content showed remarkable correlations (p < 0.01) with all the bioactivity assays, with the exception of the inhibition of LOX (p > 0.05). Condensed tannins isolated from several fruits have been previously correlated with the inhibitory effect against α-glucosidase [G. decorticans was reported [G. decorticans fruits may be mainly responsible for the bioactivities assayed; however, a quantification of individual monomers and polymers should be done in order to establish a more precise correlation.A Pearson correlation was used to evaluate the relationship between the individual parameters and to establish their relative importance in determining the bioactivity of the cosidase , and thecosidase . A dose-reported . Our obs3, amberlite XAD7 HP, α-amylase from porcine pancreas , α-glucosidase from Saccharomyces cerevisiae , DPPH , DMAC , 3,5-dinitrosalicylic acid, catechin, caffeic acid, CuCl2, hypoxanthine, indomethacin, linoleic acid (L-5900), lipase from porcine pancreas type II , Na2CO3, nimesulide, 4-nitrophenyl-α-d-glucopyranoside, nitro blue tetrazolium salt, p-nitrophenyl palmitate, sodium acetate, soy lipooxygenase-15 , starch, 2,4,6-tri(2-pyridyl)1,3,5-triazine (TPTZ), triton X-100, tris, and ursolic acid. From Merck : Trolox , FeCl3∙6H2O, neocuproin, potassium sodium tartrate and HPLC-grade methanol. Ammonium acetate was from JT Baker . The sodium and potassium phosphate salts were from Scharlau Chemicals . Orlistat was from Laboratorio Chile . Culture medium, antibiotics and fetal bovine serum were obtained from Invitrogen Corp. . Ultrapure water was obtained using a Barnsted EasyPure water filter .The following reagents were purchased from Sigma-Aldrich : AlClG. decorticans were collected in seven different locations of the Región de Atacama, Chile, namely, in the Provincia de Chañaral, Copiapó and Huasco (2O (7:3) under reflux for 15 min, two times. The dry fruit:solvent ratio was 1:3 w/v. The procedure was carried out in a flask fitted with a condenser, changing the solvent each time. The combined extracts of each sample were filtered, dried under reduced pressure and lyophilized. After freeze-drying, the extracts were dissolved in water, filtered and adsorbed into Amberlite XAD-7 resin. The resin was washed with water, and the phenolics were desorbed with MeOH to afford the PEE solution. This solution was dried under reduced pressure and lyophilized for analysis and bioactivity assays. Samples were stored refrigerated and in the dark to prevent oxidation.Fruits of d Huasco C. From t3 method [The TP was determined by the Folin-Ciocalteu method as previously described . The res3 method . The res3 method  and the The ferric-reducing antioxidant power (FRAP) assay was carried out according to a previously described method . The cupG. decorticans PEEs was evaluated against the free radicals DPPH and superoxide anion. The discoloration of the DPPH radical was carried out according to literature [50, μg PEE/mL).The scavenging capacity of the terature . The scaterature . Results50 (μg PEE/mL), corresponding to the concentration of the sample that inhibits the activity of LOX by 50%. Caffeic acid was used as a positive control.LOX inhibitory activity was determined following a previously reported methodology . The ass2 produced was reduced to the more stable PGF2α with stannous chloride and determined by enzyme immune assay (EIA) at 415 nm. Results were expressed as a percentage of inhibition of PGF2α production. The commercial anti-inflammatory compound nimesulide was included as the reference compound [COX inhibitory activity was determined using a commercial kit . Enzyme and samples (50 μg PEE/mL) were pre-incubated during 10 min and then arachidonic acid was added. The reaction was conducted for exactly 2 min at 37 °C and halted in boiling water for 5 min. The PGH50 (μg PEE/mL). Ursolic acid was used as the reference compound [The sPLA2 inhibitory activity was determined using a colorimetric kit . Enzyme and samples (200–100 μg PEE/mL) were mixed, and then the substrate (diheptanoyl Thio-PC) was added, and incubated during 15 min at 25 °C. Then, DTNB (Ellman’s reagent) was added and absorbance was read at 415 nm. Results are expressed as a percentage of inhibition or ICcompound .p-nitrophenyl-α-d-glucopyranoside (5 mM) into the wells. The mixture was incubated for 15 min at 37 °C. Then, absorbance was measured at 415 nm in a microplate reader . Results are expressed as IC50 values (μg PEE/mL). Acarbose was used as a positive control [The α-glucosidase inhibition assay was carried out as previously described . Briefly50 values (μg PEE/mL).The α-amylase inhibition assay was carried out as described in literature . Brieflyp-nitrophenyl palmitate assay [p-nitrophenyl palmitate (0.08% w/v) was prepared in 5 mM sodium acetate buffer (pH 5.0) containing 1% Triton X-100. This solution was heated in boiling water for 2 min for a better dissolution and cooled to room temperature. The assay mixture was 100 mM Tris buffer (pH 8.2), extracts, lipase and substrate solution. The mixture was incubated for 2 h at 37 °C and absorbance was read at 400 nm. All samples were assayed at 100 µg/mL as the maximum concentration in sextuplicate. Orlistat was used as the reference compound [50 values (μg PEE/mL) or as percentage of inhibition.The assay was carried out using the te assay . PorcineG. decorticans PEEs were analysed by HPLC-DAD to compare their phenolic profile. Taking into account the results of the bioactivity assays and the sample availability, the PEEs from Alto del Carmen and Copiapó were selected for the HPLC-MSn studies. The HPLC system used was from Shimadzu , and consisted of a LC-20AT pump, a SPD-M20A UV diode array detector, CTO-20AC column oven and LabSolution software. A MultoHigh 100 RP 18–5 µ (250 mm × 4.6 mm)  column was used. The HPLC conditions included a linear gradient solvent system with 1.0 mL/min flow and 30 °C, consisting of two solvent systems: H2O–formic acid–acetonitrile (ACN)  and H2O–formic acid–ACN . Initial conditions were 97% A and 3% B. Then, the solvent ratio was changed to 63:27 A:B in 100 min and returned to the initial conditions (97:3 A:B) at min 105. The column was allowed to stabilize for additional 10 min in the same gradient (95:5 A:B) for the next injection. The UV spectra from the chromatograms were recorded from 200 to 600 nm for peak characterization. The n system . Ionization was performed at 3000 V assisted by nitrogen as nebulizing gas at 50 psi and as drying gas at 365 °C, with a flow rate of 10 L/min. Negative ions were detected using full scan (m/z 2000–20) and normal resolution . The trap parameters were set in ion change control (ICC) using manufacturer default parameters, and maximum accumulation time of 200 ms. The mass spectrometric conditions for analysis were: electrospray needle, 4000 V; end plate offset, −500 V; skimmer 1, 56.0 V; skimmer 2, 6.0 V; capillary exit offset, 84.6 V; capillary exit, 140.6 V. Collision induced dissociation (CID) spectra were obtained with a fragmentation amplitude of 1.00 V (MS/MS) using helium as the collision gas and was automatically controlled through the SmartFrag option. The same column and solvent system used in HPLC-DAD chromatography were used and the compounds were monitored at 280 nm.The HPLC-ESI-MS/MS analyses were recorded using an Agilent 1100 liquid chromatography system  connected through a split to an Esquire 4000 Ion Trap LC/MSG. decorticans, a Sephadex column chromatography was applied. For this purpose, the PEE from Diego de Almagro (2.5 g) was dissolved in MeOH:H2O 7:3 (v/v) and was permeated on a Sephadex LH-20 column  using MeOH:H2O 7:3 as the eluent. Some 48 fractions of 20–40 mL each were collected. TLC analysis was carried out in silica gel plates  using as the mobile phase the mixture ethyl acetate:acetic acid:H2O (10:2:3 v/v/v), revealed with diphenylboric acid-ethanolamine complex in MeOH and visualized under UV/visible light. Fractions with similar TLC patterns were pooled into twelve sub-fractions as follows: 1–9; 10–14; 15–19; 20–25; 26–27; 28–30; 31–33; 34–37; 38–42; 43–44; 45–46 and 47–48. Fractions were submitted to the DPPH and α-glucosidase assays, as well as HPLC-DAD and HPLC-MS analyses.In order to get a more comprehensive analysis of the polyphenol composition of l-glutamine, 1.5 g/L sodium bicarbonate, 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin and 100 µg/mL streptomycin. Cells were cultured in a humidified incubator with 5% CO2 in air at 37 °C. For the subsequent experiments, cells were plated at a density of 2.5 × 104 cells/mL.Human lung fibroblast MRC-5 (ATCC CCL-171) were grown as monolayers in minimum essential Eagle medium (MEM) with Earle’s salts and supplemented with 2 mM Confluent cultures of MRC-5 cells were treated during 24 h with medium containing the different PEEs at concentrations ranging from 0–500 µg/mL. The samples were dissolved in medium supplemented with 2% FBS. Cells treated with medium only were used as controls. Cell viability was determined at the end of the incubation by means of the MTT reduction assay . Resultsp < 0.05). To assess the relationship between the bioactivities and composition, Pearson’s correlation coefficients were calculated with 95% confidence, using the SPSS 14.0 software for Windows .All experiments were carried out in triplicate. All data are presented as mean values ± standard deviation (SD). Statistical significant differences within each sample were determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (G. decorticans fruit PEE include (epi)-catechin derivatives, procyanidin dimer, trimer and tetramers, ellagic acid derivatives and flavonoids glycosides. The total proanthocyanidin content of the Chilean G. decorticans pulp was positively correlated with the antioxidant activity and the inhibition of the enzyme α-glucosidase. The compounds tentatively identified in the active antioxidant fractions from the whole PEE of Chilean chañar includes procyanidin B-type dimer, procyanidin A trimer, glycosides of kaempferol, isorhamnetin and quercetin, (epi)-catechin hexosides, as well as dicaffeoylquinic acids. The high inhibitory activity of the PEE on the enzyme α-glucosidase and the fact that chromatographic separation of the whole extract yielded fraction pools with even higher inhibitory effect on the enzyme than the whole PEE encourages further work to isolate and identify the active α-glucosidase inhibitors from the fruit. Our findings support a different chemistry for the western Andean population of G. decorticans. Additional research is needed to study the genetic similarities between both populations of this native food plant.The constituents of the Chilean


While remanent polarization, coercive field, and loop area of the same sample were 0.9 μC/cm2, 9.78 kV/cm, and 24.5 μC/cm2·V, respectively. Our results showed that a combination of graphene and ferroelectric ceramic additives are an excellent approach to significantly advance the performance of dielectric and ferroelectric properties of piezoelectric polymers for broad applications including energy storage.To address the limitations of piezoelectric polymers which have a low dielectric constant andto improve their dielectric and ferroelectric efficiency for energy storage applications, we designed and characterized a new hybrid composite that contains polyvinylidene fluoride as a dielectric polymer matrix combined with graphene platelets as a conductive and barium titanite as ceramic ferroelectric fillers. Different graphene/barium titanate/polyvinylidene fluoride nanocomposite films were synthesized by changing the concentration of graphene and barium titanate to explore the impact of each component and their potential synergetic effect on dielectric and ferroelectric properties of the composite. Results showed that with an increase in the barium titanate fraction, dielectric efficiency ofthe nanocomposite was improved. Among all synthesized nanocomposite films, graphene/barium titanate/polyvinylidene fluoride nanocomposite in the weight ratio of 0.15:0.5:1 exhibited thehighest dielectric constant of 199 at 40 Hz, i.e., 15 fold greater than that of neat polyvinylidene fluoride film at the same frequency, and possessed a low loss tangent of 0.6. However, AC conductivity and ferroelectric properties of graphene/barium titanate/polyvinylidene fluoride nanocomposite films were enhanced with an increase in the graphene weight fraction. Graphene/barium titanate/polyvinylidene fluoride nanocomposite films with a weight ratio of 0.2:0.1:1 possessed a high AC conductivity of 1.2 × 10 Dielectric constant of the resulting composite was 110 while its tanδ was 0.016 at 10 kHz [3/PVDF) composite possessing ε′ of 7 and tanδ as low as 0.03 [2 composites with polyvinyl alcohol (PVA) and poly(methyl methacrylate) (PMMA) with ε′ of 24.6 and 26.8 for both composites, respectively [Dielectric and piezoelectric polymer materials have attracted significant attention to replace piezoelectric ceramic materials used in various applications such as medical, automotive industry, and consumer electronics dueto their lightweight, flexibility, low-cost and low-acoustic impedance, and high piezoelectric constant ,2,3. Apaa (TiO2) ,11,12,13t 10 kHz . Ravindr as 0.03 . Sugumar3O4) [Another strategy to address the limitations of piezoelectric polymers is to add a small amount of conducting fillers into the polymer matrix to achieve high ɛ′. Some conducting fillers including multiwalled carbon nanotubes (MWCNT) , nano ir3O4) , silver 3O4) , and gra3O4)  etc. hav3O4) . The con3O4) ,23.3/PVDF composites by adding MWCNT filler. Synthesized three-phase composite showed enhanced dielectric efficiency with ε′ of 151 and tanδ of 0.08 at 100 Hz [3 reported by Zhang et al. possessed ε′ equal to 40.8 and tanδ of 0.055 at 100 Hz showing improvement in dielectric efficiency because it used Ag with a higher conductivity [3 and PVDF possessing ε′ and tanδ equal to 65 and 0.35, respectively at 1 MHz that demonstrated a promising potential of combined graphene/ceramic additives to advance performance of polymer dielectrics [To further advance the properties of dielectric polymer materials, the combination of both ceramic and conducting fillers has been explored to design ternary polymer composites. Yao et al. increased ε′ of BaTiOt 100 Hz . Anotheructivity . Wang etlectrics . AlthougIn this paper, we present the use of graphene as a conducting element to design three-phase graphene/barium titanate/polyvinylidene fluoride (G/BT/PVDF) nanocomposites with high ε′, low tanδ, and enhanced ferroelectric properties and explore performance for energy storage and capacitors applications. To demonstrate this concept, we selected graphene as the conducting filler because of its controllable conductive properties, size and 2D unique structure, high mechanical strength, and high electrical conductivity (σ) ,26. Amon4, Sigma-Aldrich, Gillman, Australia), hydrochloric acid , sulfuric acid , phosphoric acid , hydrogen peroxide , N,N–Dimethylforamide , barium titanate(IV)  and polyvinylidenefluroride  were of analytical grade and were used without further purification.Commercial natural graphite rocks  were purchased from an Australian mining site. Potassium permanganate (97% KMnOGraphene oxide (GO) was synthesized by improved method , and it Different G/BT/PVDF nanocomposite films of uniform thickness of 550 μmwere synthesized by the solution casting technique using different graphene content while keeping the concentration of BT (100 mg) and PVDF (1 g) constant. We explored several different G/BT ratios to analyzetheir influence on dielectric and ferroelectric properties and found an optimized composite formulation for future work. After characterization and calculating ε′ and tanδ of the above synthesized films, asuitable fraction of graphene with high ε′ and low tanδ (G:BT:PVDF = 0.15:0.1:1) was selected and further samples were synthesized with varying concentrations of BT while keeping the concentration ratios of graphene and PVDF (0.15:1) constant. The scheme of the designed composites with their key results is presented in −1 was used to identify the functional groups of synthesized nanocomposite films. Thermal stability was studied by using a thermal gravimetric analyzer  under air where the samples were heated from room temperature (RT) to 900 °C at a heating rate of 10 °C min−1. Dielectric characterization of the synthesized G/BT/PVDF nanocomposite films was carried out using precision impedance analyzer  at RT between 20 Hz to 1 MHz. Films were plated between copper electrodes and measurements were done at RT. Ferroelectric characterization of all samples was made by using Radiant Technologies Multiferroic testing system. Samples were placed between copper electrodes at RT.All samples including PVDF and ternary G/BT/PVDF nanocomposite films were analyzed to study their morphology by field emission scanning electron microscopy . Structural investigations were made by using X-ray diffraction  and Raman spectroscopy by using Raman microscope  equipped with LabSpec 6 software  A 532 nm laser line from He–Ne source was used for excitationand 600 grooves per mm grating was used. Acquisition time was 5 s and the accumulation time was 3 s. Fourier transform infrared (FTIR) spectroscopy  in transmittance mode and range 400–4000 cmMorphology of the synthesized G/BT/PVDF nanocomposite films was characterized by FESEM as shown in XRD analysis was carried out to investigate the crystal structure of the PVDF film and synthesized G/BT/PVDF nanocomposite films. −1 belong to the tetragonal structure of BT nanoparticles [−1 corresponds to beta (β) and gamma (γ) phases of PVDF. The GBP-III nanocomposite shows a band at 839 cm−1 confirming that PVDF is in β phase. The well-defined D and G bands of graphene appeared at 1333 cm−1 and 1557 cm−1 in the Raman spectra of GBP-III while these bands appear at 1333 cm−1 and 1546 cm−1, respectively in Raman spectra of GBP-IX. The intensity ratio of D and G bandsId/Ig was calculated to be 0.82 and 1.02 for GBP-III and GBP-IX, respectively. Id/Ig ratio shows that defects in graphene structure are more pronounced in GBP-III than in GBP-IX as the former has relatively higher graphene fraction than later [Raman spectra of neat PVDF film, GBP-III, and GBP-IX were recorded to confirm the presence of graphene, BT, and PVDF in synthesized G/BT/PVDF nanocomposite films. Results of Raman spectroscopy are in accordance with those of XRD except that it also shows graphene bands. Raman spectra are shown in articles . A very an later .−1 [−1 suggests the presence of β phase of PVDF [−1 is also indicating β phase PVDF. Two weak peaks at 2849 cm−1 and 2917 cm−1 correspond to C–H stretching of PVDF [−1 is present [−1 is indicative of entrapped remnants of DMF in nanocomposite films [FTIR spectra of all synthesized samples were recorded to further confirm the formation of G/BT/PVDF nanocomposites. FTIR spectra of G/BT/PVDF nanocomposites are shown in −1 . The pea of PVDF . The pea of PVDF . A chara present . But it te films .Thermal gravimetric analyses (TGA) of graphene, BT, neat PVDF film, and all synthesized G/BT/PVDF nanocomposite films were carried out to study their thermal stability. TG curves show that BT is stable over a high temperature range; even at 900 °C it is stable and does not decompose. Graphene decomposes with a rise in temperature and decomposes up to 95% around 600 °C. It was observed that incorporation of BT in the polymer improves thermal stability of the polymer from GBP-V to GBP-X. It is due to the high surface area contact of BT with the polymer matrix that results in the restriction of PVDF degradation . As BT fDielectric constant (ε′) of neat PVDF and all synthesized G/BT/PVDF nanocomposites was plotted against frequency as shown in At lower frequency, tanδ is low which may be due to the presence of ionizable hydroxyl group (OH) at the heterogeneous interface as well as the fact that an extendedtime is available for diploes to collide . Not onlac) of PVDF and all nanocomposite films is shown in ac of 1.2 × 10−4 (S/m) at 40 Hz. While a further increase in graphene might enhance the σac and ε′, a further rise in graphene content was not carried out due to the expected increase in tanδ. However, when BT content was further increased, it caused segregation of graphene sheets as shown in FESEM images in ac of GBP-IV. GBP-IX possesses the highest conductivity among nanocomposites with varying BT contents (GBP-V to GBP-X). It is due to the distance between graphene sheets reducing with increasing BT content to a certain extent. Here, tunneling conductance occurs through neighboring graphene sheets [AC conductivity  and loop area of PVDF film were 1.29 kV/cm and 0.00146 μC/cm2·V, respectively, as shown in The polarization electric field (P-E) hysteresis loop was studied to further analyze thegeneration of charge in the synthesized nanocomposite films. Analyses were carried out at RT. The thickness of each film was 550 μm, and the area of the electrode was 0.786 cm2, 9.78 kV/cm, and 24.5 μC/cm2·V, respectively. It is the highest among samples with different graphene content as shown in 2, 8.4 kV/cm, and 0.025 μC/cm2·V, respectively. Although these values are not much larger when compared to GBP-IV, they are still much better than that of neat PVDF. An increase in ferroelectric properties by increasing the BT content can be explained as follows:When BT content is low, there are fewer mobile charge carriers. Thus, energy dissipation is also low, and loop area is also low, but when BT fraction is increased, the number of charge-carrying particles is also increased. It results in increased energy dissipation and an enhanced loop area [It is evident from the ferroelectric analysis that by increasing graphene content while keeping BT fraction constant, ferroelectric properties are enhanced. Pr, Ec, and loop area of GBP-IV with maximum graphene fraction (200 mg) are 0.9 μC/cmoop area . A sligh2, 9.78 kV/cm, and 24.5 μC/cm2·V, respectively) among neat PVDF and all synthesized G/BT/PVDF nanocomposites. Our results demonstrate that the combination of graphene and ferroelectric ceramic additives is an excellent approach to enhance significantly the performance of dielectric and ferroelectric properties of piezoelectric polymers and can be used for broad applications, specifically, to be suitable for high performing energy storage and supercapacitors.In this paper, we designed and explored a concept to enhance dielectric and ferroelectric properties of piezoelectric polymers for energy storage applications by combining conductive graphene and ferroelectric ceramic (BT) additives. New hybrid composite films of G/BT/PVDF were synthesized with variations in dosage and ratio of graphene and BT to explore their influence on dielectric (ε′ and tanδ) and ferroelectric  values. In the first set of experiments, we kept the concentration of BT and PVDF constant and changed the concentration of graphene, until it reached a value with high ε′ and low tanδ. The optimized graphene fraction which showed good dielectric properties was selected for synthesis of another series of G/BT/PVDF nanocomposite films. In the second experiment, the concentration of BT was increased, andthe concentration of graphene and PVDF was fixed. GBP-VIII nanocomposite film with graphene, BT, and PVDF fraction (0.15:0.5:1) was found to have the highest ε′ values (199 at 40 Hz) and lowesttanδ (0.6) among all prepared samples, that is, it is highly suitable as a dielectric candidate for capacitors. Regarding the ferroelectric properties of prepared G/BT/PVDF nanocomposite films, results showed that GBP-IV (G:BT:PVDF = 0.2:0.1:1) has the best ferroelectric properties (Pr, Ec, and loop area of GBP-IV are 0.9 μC/cm


Toxorhynchites amboinensis, which is a non-blood feeding species proposed as a biological control agent against pest mosquitoes. Using a derived cell line (TRA-171), we demonstrate that these mosquitoes possess a functional RNAi response that is active against a mosquito-borne alphavirus, Semliki Forest virus. As observed in vector mosquito species, small RNAs are produced that target viral sequences. The size and characteristics of these small RNAs indicate that both the siRNA and piRNA pathways are induced in response to infection. Taken together, this data suggests that Tx. amboinensis are able to control viral infections in a similar way to natural arbovirus vector mosquito species. Understanding their ability to manage arboviral infections will be advantageous when assessing these and similar species as biological control agents.Arthropod vectors control the replication of arboviruses through their innate antiviral immune responses. In particular, the RNA interference (RNAi) pathways are of notable significance for the control of viral infections. Although much has been done to understand the role of RNAi in vector populations, little is known about its importance in non-vector mosquito species. In this study, we investigated the presence of an RNAi response in  Toxorhynchites (Diptera: Culicidae) mosquitoes or “elephant mosquitoes” are the largest mosquitoes on the planet, with a wingspan surpassing 12 mm for some species [Aedes aegypti, which is a key vector for many important human arboviruses. As a result, various species, such as Tx. splendens, Tx. rutilus, and Tx. amboinensis, have been proposed as biological control agents against pest species [ species ,4.Toxorhynchites being exclusively nectarivorous, they are not considered to be natural vectors for arboviruses. However, previous work has demonstrated that several species of Toxorhynchites are susceptible to important arboviruses and, as such, have a role as artificial hosts for their detection and propagation. Tx. amboinensis, Tx. brevipalpis, Tx. rutilus rutilus, Tx. theobaldi, and Tx. splendens have previously been shown to be susceptible to a number of flaviviruses including dengue virus (DENV) serotypes 1–4, Japanese encephalitis virus (JEV), yellow fever virus (YFV), and Zika virus (ZIKV) [Tx. amboinensis and Tx. brevipalpis generate greater titers of DENV compared to Ae. albopictus or their derived cell line, C6/36 [Tx. amboinensis were also shown to be susceptible to JEV and allowed it to replicate to high titres [Tx. amboinensis have been shown to efficiently propagate alphaviruses (chikungunya (CHIKV), Ross River (RRV), and Venezuelan equine encephalitis (VEEV) viruses) and bunyaviruses (La Crosse (LACV), San Angelo (SAV), and Keystone (KEYV)) viruses [As a consequence of adult s (ZIKV) ,6,7. Fure, C6/36 ,8. Tx. ah titres . In addi viruses ,9,10.Toxorhynchites to facilitate virus propagation and isolation in vitro. Cell cultures derived from Tx. amboinensis have been established which show comparative levels of sensitivity as the adults and commonly used vector cell lines to DENV and other arboviruses [Toxorhynchites mosquitoes.Several continuous cell lines have been derived from oviruses ,12,13,14Toxorhynchites spp. may become exposed to arboviruses by predating on vertically infected larvae [Drosophila melanogaster model, although an increasingly detailed picture of mosquito immunity in vector species is now emerging which highlights a number of key differences [D. melanogaster [D. melanogaster, the pathway involves PIWI proteins Piwi, Aub, and Ago3. However, Ae. aegypti lack orthologues of Aub and Piwi, but express Ago3 and an additional 7 PIWI family proteins, Piwi1-7 [1). These are loaded into the Piwi complex and are further processed to produce mature piRNAs with an adenine at the 10th nucleotide position (A10). The mature piRNAs are bound by Ago3 and target complementary antisense RNA transcripts to produce more piRNAs. Therefore, a typical characteristic of ping-pong derived piRNAs is not only the A10 and U1 bias but also a high frequency of 10 nt complementarity to opposing small RNAs.Despite their usability for the propagation of arboviruses, nothing is known about the antiviral responses in this mosquito genus. In nature, d larvae , and it ferences ,19,20,21ferences ,37,38,39ferences . It alsonogaster . In D. m Piwi1-7 . The patTx. amboinensis-derived TRA-171 cells. Our observations indicate that these cells possess a functional RNAi response that is effective against Semliki Forest virus  infection. We used deep sequencing analysis to show the production of both vsiRNAs and virus-specific piRNAs derived from SFV. In addition, silencing assays showed that RNAi responses are induced by the presence of sequence-specific dsRNA against both viral RNAs and mRNAs transcribed from transfected plasmid DNA. This evidence suggests that Tx. amboinensis is able to mount a classical RNAi immune response against viral infections in a similar manner to what is known for mosquito vector species.In this study, we describe an active antiviral immune response in Tx. amboinensis-derived TRA-171 cells (European Collection of Authenticated Cell Cultures (ECACC), 90120514) were grown in media prepared in house consisting of L-15 (Leibovitz) growth culture medium  mixed 1:1 with Mitsuhashi and Maramorosch basal media prepared in house (CaCl22H2O (250 mg/lt), MgCl26H2O (125 mg/lt), KCl (250 mg/lt), NaHCO3 (150 mg/lt), NaCl (8750 mg/lt), NaH2PO4H2O (250 mg/lt), d-glucose (500 mg/lt), lactalbumin hydrolysate (8125 mg/lt), and yeastolate (0.75 mL/lt)) supplemented with 10% tryptose phosphate broth , 10% fetal bovine serum , 0.05% bovine serum albumin (BSA) , 1% non-essential amino acids (Sigma-Aldrich), and penicillin-streptomycin . D. melanogaster-derived S2 cells [2. Baby hamster kidney (BHK-21) [2.S2 cells ,42 were (BHK-21)  cells weFFLuc) (SFV4(3H)-FFLuc) or Renilla luciferase (RLuc) (SFV4(3H)-RLuc) inserted between duplicated nsP2 cleavage sites at the nsP3/4 junction, were grown and titered by plaque assay in BHK-21 cells, as described previously [The prototype molecular clone of SFV, SFV4, and two derived reporter viruses either expressing Firefly luciferase  using a PCR product template flanked by T7 RNA polymerase promoter sequences. pIZ-Fluc [FFLuc and RLuc, respectively. An eGFP-derived dsRNA was taken as a control. This sequence was obtained from a gel-purified PCR product using peGFP-C1  as a template. Primer sequences can be found in dsRNA molecules against either pIZ-Fluc  and pRL-32P rCTP , 1 µL T7 RNA polymerase (Ambion), 1 µL RNase inhibitor (Invitrogen), and 3 µL RNase/DNase free H2O. The reaction was incubated for 1 to 3 h at 37 °C before heating to 65 °C for 5 min and allowing it to gradually cool to room temperature. Following this, 2 µL of DNase I and 1 µL of RNase A were added and the reaction was incubated for a further 30 min at 37 °C. The dsRNA could then be purified by running on an 8% native acrylamide gel.Internally radio-labelled dsRNAs were prepared by combining 5 µL 114 nt eGFP PCR product with T7 polymerase sites, 4 µL 5× Transcription buffer (Ambion), 2 µL DTT , 1 µL rNTPs  (Promega), 3 µL α-5 cells per well of a 24-well plate 24 h prior to transfection with Dharmafect 2  following the manufacturer’s instructions. For plasmid transfection experiments, each well was co-transfected with 100 ng pIZ-Fluc, 100 ng pAcIE1-Rluc  with either 1 ng of dsRNA (FFLuc-specific or eGFP control dsRNA), or 2 ng siRNAs (FFLuc-specific or Hygromycin B resistance gene control siRNA [Cells were seeded at a density of 2.2 × 10ol siRNA ). Cells RLuc or eGFP-specific control dsRNA. After 24 h, cells were infected with SFV4(3H)-RLuc at a multiplicity of infection (MOI) of 0.005. Cells were lysed 24 h post infection (p.i.) and luciferase activities were determined.For infection experiments, viral reporter gene transcripts were silenced by transfecting 50 ng The cells were lysed in Passive Lysis Buffer (Promega) and luciferase expression was determined with either the Renilla-Glo Luciferase assay system (Promega) or the Dual Luciferase assay system (Promega) and a GloMax luminometer.5 per well of a six-well plate. RNA extraction was performed using 1 mL TRIzol (Life Technologies) as per the manufacturer’s instructions, with the addition of glycogen as a carrier. DNA libraries consisting of small RNAs between 15–40 nt were gel purified and sequenced using the Illumina Hiseq 4000 platform at BGI Tech . Data analysis was carried out as described previously by aligning sequence reads to the SFV4 reference genome (Genbank accession number: KP699763) [https://www.ncbi.nlm.nih.gov/sra) under the accession number: PRJNA486770.The cells were grown at a density of 9 × 10P699763) ,37. A maP699763) . The mapP699763) . Using p5 and 1 × 106 cells per well of a six-well plate respectively. Following a 24 h incubation, the media was removed. The cells were re-suspended in sterile PBS and centrifuged for 5 min at 1500 rpm. The supernatant was removed and the procedure repeated a second time as described. Following this, the pellet was re-suspended in 200 µL 1× lysis buffer (10 mM MgAc (Sigma-Aldrich) and 150 mM Hepes-KOH (pH 7.5)) and homogenized using a micro-pestle. A further centrifugation step  was performed to remove cell debris, after which 5 µL of the supernatant was transferred to a fresh tube. To this was added, 3 µL 32P labelled dsRNA, 1 µL H2O, and a 3 µL creatine mix (consisting of 1 µL DTT (1 M), 10 µL creatine phosphate (12 mg/100 µL) , 20 µL 5× lysis buffer (10 mM MgAc and 150 mM Hepes-KOH (pH 7.5)), 20 µL glycerol, 2 µL RNase Inhibitor (Promega), 2 µL ATP (100 mM) , 0.3 µL 20 mg/mL creatine phosphate kinase in 1× storage buffer  in 2× storage buffer (ice-cold 40 mM tris-acetate (pH 6.8), 200 mM KAc, 0.2 mM EDTA, 20 mM β-mercaptoethanol) diluted 1:1 in an equal volume of 100% ice-cold glycerol), and 4.7 µL RNase/DNase free H2O). The reactions were incubated at 28 °C overnight. Subsequently, 200 µL 2× PK buffer (200 mM Tris (pH 7.5), 300 mM NaCl, 5 mM EDTA, 2% SDS), 1 µL glycogen (10 mg/mL) , and 0.3 µL proteinase K (10 mg/mL) (Sigma-Aldrich) were added and each reaction was incubated for a further 10 min at 65 °C. Following this, 200 µL phenol/chloroform/isoamylalcohol (25:24:1) (Ambion) was added. Reactions were vortexed for 15 sec and centrifuged for 10 min at 10,000 rpm. The aqueous phase was transferred to a new tube containing 450 µL ice-cold 96% EtOH and centrifuged at 13,000 rpm for 10 min. The supernatant was removed and the pellet washed with 400 µL ice cold 70% EtOH. The samples were further centrifuged as described and the supernatant was removed. The resulting pellets were air dried for 5–10 min prior to resuspension in 15 µL 2× RNA gel loading buffer (Thermo Fisher Scientific). The samples were boiled for 5 min at 65 °C before placing on ice for 2 min. On completion, the samples were loaded onto a 0.75 mm 12% denaturing acrylamide gel with 0.96% urea chilled by submerging the tank in ice-cold water. Electrophoresis was carried out at 200 V. The gel was then transferred to a gel dryer and allowed to dry at 80 °C for 2 h. The resulting bands were detected by exposing the gel to a phosphor imaging screen for ≥16 h and viewed using a personal molecular imager .TRA-171 and S2 cells were seeded at 9 × 10t-test.Statistical analysis was performed using GraphPad Prism. Data was analyzed using an unpaired, two-tailed http://dx.doi.org/10.5525/gla.researchdata.703. Small RNA sequencing data can be found under the accession number described above.Source data for the figures can be found at Tx. amboinensis-derived TRA-171 cells are known to be permissive to infection by CHIKV [RLuc or FFLuc) (RLuc at a high (10) or low (0.01) MOI and incubated for 24 h prior to lysing. As anticipated, proportional SFV replication was detected at each MOI used (FFLuc) at MOI 10 and its replication efficiency was monitored at regular intervals over 120 h. Both viral titres  A, which MOI used B. To undl titres C and lucl titres D peaked l titres E. These ll lines ,49. This32P internally radio-labeled dsRNA and incubated overnight before isolating the small RNAs. Extracts were run on an acrylamide gel alongside size markers; input dsRNA (114 nt) and siRNAs (21 nt), as well as an extract from S2 cells as a positive control. Both samples showed the detection of input dsRNA in addition to discernible bands at the size expected for 21 nt siRNAs -RLuc. The infections were performed at either a high (10) or low (0.005) MOI. The cells were lysed 24 h p.i. and luciferase expression was assessed. A decrease in the relative luciferase activity was observed in cells treated with RLuc-specific dsRNA when compared to those that received control eGFP-specific dsRNA at both MOIs, which directly indicates a decrease in viral replication and, therefore, a reduction in virus production , although further studies are needed to confirm their presence . ISVs laD. melanogaster as a model for RNAi studies [Toxorhynchites is one of three mosquito genera, along with Malaya and Topomyia, which do not require a blood meal during their adult life stages to initiate egg development and, as such, do not exhibit host-seeking behavior [Toxorhynchites may be at risk of an arbovirus infection due to ingesting vertically infected larvae [Studies in mosquito vector species have shown that RNAi is the predominant antiviral response against infections ,19,20. S studies ,67,68,69behavior ,71. Theid larvae . TherefoTx. amboinensis-derived TRA-171 cells. SFV infection induced the production of 21 nt vsiRNAs derived from both the viral genome and antigenome, which is indicative of Dcr2 cleavage of dsRNA [Bunyaviridae and Flaviviridae [This study identifies the presence of a functional RNAi response within of dsRNA . A similof dsRNA ,37,39,72iviridae ,19,20,21iviridae ,28,37,3910/U1 bias and a 10 nt overlap between the 5′ ends of piRNAs from different strands, which has been previously described in aedine cells [10 and U1 bias and a 10 nt overlap between the 5′ ends of different strands suggests that TRA-171 cells encode PIWI clade proteins.In addition, larger classes of SFV-derived small RNAs between 24–29 nt were detected which presented with the hallmark characteristic ping-pong motif of piRNAs; an Ane cells ,32,37. Cne cells ,32. HoweAe. aegypti show that Ago3, Piwi5, and, to a lesser extent, Piwi6 participate in the production of viral-derived piRNAs [Tx. amboinensis, it was not possible to identify RNAi effector-encoding sequences and, therefore, we can only speculate if these proteins are present in the Toxorhychites genome. However, the results presented here are a strong indicator for the presence of RNAi machinery comparable to that of aedine species. Previous research has shown that arboviruses are able to replicate to high levels within Toxorhynchites mosquitoes and their derived cell lines. The data shown here for SFV replication and production is congruous with these findings. Further work will be required to determine the exact temporal and spatial mechanisms, as well as the proteins involved in the Toxorhynchites immune response, which permit enhanced arbovirus replication.Our findings show that the exo-siRNA pathway can be artificially induced in TRA-171 cells by the transfection of long dsRNA molecules, which leads to sequence-specific silencing. Similarly, our results also show that sequence-specific siRNAs are capable of achieving efficient gene silencing, which supports the presence of a natural antiviral RNAi pathway within TRA-171 cells. Key mediator proteins of the exo-siRNA pathway, such as Dcr2 and Ago2, are highly conserved between mosquito and drosophila and the effector mechanisms are considered to be similar . Previoud piRNAs ,36. GiveToxorhynchites cells which successfully modulates SFV infection in a manner similar to that of natural arboviral vector species. Only once we fully understand their ability to manage infections will we be able to make an informed decision regarding the suitability of certain species as biological control agents against vector mosquito species. Our data also expands on the current knowledge of ISVs, TEs, and EVEs in mosquitoes that may influence pathogen transmission in vector populations and highlights their interactions with the exo-siRNA and piRNA pathways.In conclusion, we have demonstrated the presence of an active antiviral RNAi response in


Actinobacteria, nature’s main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in  Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production. The worrying worldwide escalation of antimicrobial resistance is limiting the treatment options for numerous infectious diseases . TherefoActinobacteria have long been the largest natural source of antibiotics  in the phylum Actinobacteria and a paradigm of secondary-metabolite-producing microorganisms  uptake and metabolism and of degradation of chitin to GlcNAc, DasR from the GntR family (–Streptomyces (–Postgenomic-era advances in the regulation of antibiotic production in R family –10. GlcNptomyces –13 and ptomyces –.Actinobacteria (–Bacillus subtilis in complex with GlcN-6P have been reported (Actinobacteria have been described: N-acetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase, NagA (EC 3.5.1.25), which deacetylates GlcNAc-6P formed by phosphorylation of acquired extracellular GlcNAc by a phosphotransferase system  oligosaccharides and, at a lower scale, of monomeric GlcN  into the GlcN metabolism, experimental evidence for this activity in that medically important phylum is still missing. Here, we identify and characterize a unique GlcN kinase from the soil bacterium Streptacidiphilus jiangxiensis, closely related to Streptomyces spp., the classical antibiotic producers  –18. More–bacteria –13, 19. reported , 21. Howe system –25, and  system –, 27. Homric GlcN . Phosphocholerae  and in tkarensis , and theroducers . This Gl archaea , 30. TheActinobacteria prompted a careful analysis of its genomic context, which revealed a tight association with two other uncharacterized genes, a putative sugar isomerase (SIS) of the AgaS superfamily (COG2222) and a putative glycosyltransferase (GT) with homology to members of the GT1 family  and UTP–glucose-1-phosphate uridylyltransferase (UDPGP), thus completing a potential pathway for sugar activation and transfer. Furthermore, in several Actinobacteria, this operon was predicted to be included in larger biosynthetic gene clusters (BGCs), suggesting a role in the production of complex secondary metabolites  was identified, in addition to the canonical maltokinase gene . In each1 family . In Streabolites . Since tS. jiangxiensis maltokinase paralogue, here SjGlcNK, a sequence profile-based search for homologues was performed using the Fold and Function Assignment System (FFAS03) ,Mycolicibacterium vanbaalenii   (317DFD319 (in vitro (mK) for glucose and GlcN suggested that SjGlcNK is better described as a GlcN kinase (EC 2.7.1.8) with a strong preference for using GlcN (mK = 8 ± 1 mM) over glucose (mK > 100 mM) as the substrate  . As expetry 4U94 , FFAS sctry 4O7O , FFAS scNESV124)  and the VGQI306)  and 317D17DFD319  loops co17DFD319 ; Fig. 2Ain vitro , indicatin vitro . GlcN wain vitro ; Fig. S2ubstrate .10.1128/mBio.00239-19.1FIG S1Streptacidiphilus jiangxiensis (UniProtKB entry A0A1H7TQR5) was aligned with those of MvMak from Mycolicibacterium vanbaalenii (UniProtKB entry A1TH50), MtMak from Mycobacterium tuberculosis (UniProtKB entry O07177), and MsMak from Mycolicibacterium smegmatis (UniProtKB entry A0R6D9). Strictly conserved alignment positions are shown in inverted type on a red background. Secondary structure elements for SjGlcNK are represented above the alignment. The catalytic and DFD loops, the P-loop, and the DVV and AVVKW motifs are labeled in red. The residues that participate in GlcN and maltose binding are indicated by green and orange hexagons, respectively. Download FIG S1, PDF file, 2.2 MB.Multiple amino acid sequence alignment of SjGlcNK with homologous mycobacterial maltokinases. The amino acid sequence of SjGlcNK from Copyright © 2019 Manso et al.2019Manso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00239-19.2FIG S21H-NMR spectra of the enzymatic product of SjGlcNK. (A) Sample from enzymatic reaction mixture purified by thin-layer chromatography. (B) GlcN-6P standard (Sigma-Aldrich) in D2O. (C) Sample from spectrum A spiked with an aliquot of standard B. The alignment between the signals in samples A and B is perfect. The spiking of sample A with the standard revealed no new signals and a slight increase of those already present in the sample, thus confirming the identity of the sample compound as GlcN-6P. Download FIG S2, PDF file, 0.1 MB.Copyright © 2019 Manso et al.2019Manso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The crystal structure of SjGlcNK was solved at 1.98-Å resolution  and reve10.1128/mBio.00239-19.3FIG S34U94 ) N-terminal caps (two views rotated by 180° around y), (B) intermediate subdomains, and (C) C-lobes (two views rotated by 90° around y). Secondary structure elements are labeled. Download FIG S3, PDF file, 2.5 MB.Overall structure of SjGlcNK and structural comparison with MvMak. (A) Structural superposition of SjGlcNK  crystal forms, A and B, which differ considerably in the dimensions of the respective unit cells 1097-0134(19980201)30:2%3C144::AID-PROT4%3E3.0.CO%3B2-N). Download FIG S4, PDF file, 1.0 MB.The two crystallographic forms, A and B, of SjGlcNK comprise six conformational states of the enzyme. (A) Cartoon representation of two  and four  molecules found in the asymmetric units of the two SjGlcNK crystal forms. The individual structures are labeled with Roman numerals. (B) Superposition of the Cα traces of the six SjGlcNK monomers (colored as in panel A), by alignment of the C-lobe subdomains. The orientations of the N-lobe in the open and closed conformations are related by a rotation of 23.8° around a hinge axis (dashed line) as determined by DynDom  and X-ray crystallography. In solution, SjGlcNK is a monomer , adoptin10.1128/mBio.00239-19.5FIG S5gR values (±SD) are indicated for each experiment. (C) Plot of Guinier gR at several concentrations, for the same combinations of protein and ligand(s) as in panel A. Download FIG S5, PDF file, 0.3 MB.Ligand-induced conformational transformation of SjGlcNK in solution probed by SAXS. (A) SAXS profiles extrapolated to infinite dilution of SjGlcNK (a) in absence of substrates (red) and in presence of (b) 200 mM GlcN (blue), (c) 50 mM glucose (violet), (d) 1 mM ATP (orange), (e) 200 mM GlcN and 1 mM ATP (green), and (f) 50 mM glucose and 1 mM ATP (yellow). The curves are offset on the log scale. The scattering calculated for a combination of open and closed conformations (ratio given above the curves) is shown as dashed lines. (B) Guinier plots of the scattering data shown in panel A. Copyright © 2019 Manso et al.2019Manso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00239-19.8TABLE S1Table S1, DOCX file, 0.1 MB.Small-angle X-ray scattering results for SjGlcNK with and without ligands. Download Copyright © 2019 Manso et al.2019Manso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the i), with the two hydrolysis products clearly identifiable in the electron density map. The ADP moiety is located in a cleft between the two lobes of SjGlcNK, which adopts a closed conformation  (contoured at 4σ) around the ADP, inorganic phosphate (Pi), magnesium ions (Mg1 and Mg2), and GlcN is shown as a green mesh. (B) Ligand binding induces the reorientation of the side chain of D317. Magnesium binding sites are shown for the nucleotide-free SjGlcNK structure (left) and for the phosphorylation transition state (right) found in the GlcN-ADP-Pi quaternary complex. Download FIG S6, PDF file, 1.5 MB.Crystal structure of the complex between SjGlcNK, GlcN and ATP. (A) The structure of the complex reveals the transition state of the phosphoryl transfer reaction of ATP to GlcN. Although a residual, noninterpretable positive electron density was observed near the active site of the closed state VI in  of apo-SCopyright © 2019 Manso et al.2019Manso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the i, two magnesium ion cofactors (Mg1 and Mg2), and GlcN—were captured midreaction, providing a detailed mechanistic insight into phosphoryl transfer   –44. The e (PKA) –. The absAlthough K133 establishes hydrogen bonds with the α and β phosphate groups of ADP in the quaternary complex, this interaction is not key for anchoring the nucleotide to the active site . As obse405QEVR408 in helix α12 (426KAVY429 in MtMak) also plays a part in determining substrate specificity. Noteworthy is the role of Q405, stabilized by polar contacts with R408, in providing specificity to this family of GlcN kinases. The only difference between GlcN and glucose resides at position 2, where the amino group of GlcN is replaced by a hydroxyl. In SjGlcNK, the amino group of GlcN is within hydrogen-bonding distance of the side chains of Q405 (K426 in MtMak) and E409 , MsGlcNK. The substrate specificity of MsGlcNK was also investigated, revealing that it phosphorylates GlcN with a mK very similar to that of SjGlcNK, being inactive toward maltose  and is bacteria . In addibacteria . Interes maltose . Thus, t10.1128/mBio.00239-19.7FIG S7M. smegmatis, MsGlcNK, phosphorylates GlcN. Similarly to SjGlcNK, MsGlcNK displays preference for GlcN over maltose as the substrate. The enzymatic activity was measured as ADP release from ATP using the ADP-Glo kinase assay kit (Promega) . Kinase reactions were performed in 100 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.1 mg ml−1 BSA with [MsGlcNK] = 1 μM, [ATP] = 2.5 mM, and various concentrations of GlcN (red dots) and maltose (black dots), upon incubation at room temperature for 5 min. Fitting the data to the Michaelis-Menten equation (red line) resulted in a mK of 14 ± 2 mM, very similar to that of SjGlcNK (mK = 8 ± 1 mM). Part of the amino acid sequence of MsGlcNK is displayed, with the residues matching the proposed consensus sequence Q-x(2)-RE-x(2)-YA-x(3)-LP-x-W for actinobacterial glucosamine kinases highlighted in red. Download FIG S7, PDF file, 0.2 MB.The putative maltokinase from Copyright © 2019 Manso et al.2019Manso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Actinobacteria is activated through derepression of DasR (Actinobacteria: (i) deacetylation of GlcNAc-6P, formed by phosphorylation of extracellular GlcNAc by the PTS system  by the ABC transporter CsnEFG  creates cholerae  and one karensis  and propin vitro. The molecular determinants for substrate specificity of SjGlcNK were unveiled by the crystal structure of a productive complex with GlcN and comprise a discrete number of highly conserved residues from two short motifs, 300D-x-H302 in the catalytic loop and 405Q-x(2)-RE-x(2)-YA-x(3)-LP-x-W420 in helix α12, which we propose as consensus sequences for GlcN phosphorylation in Actinobacteria. In accordance, the SjGlcNK homologue from M. smegmatis, MsGlcNK, also annotated as a maltokinase and displaying the consensus sequence for GlcN binding and processing (see Streptomyces coelicolor A3(2) postulated to play a role in the phosphorylation of acquired chitosan-derived GlcN oligosaccharides (255Q-x(2)-RE-x(2)-AA263, although with the tyrosine residue replaced by alanine.Both SjGlcNK and related orthologues were annotated as maltokinases or aminoglycoside phosphotransferases of unknown function. Despite a three-dimensional structure very similar to that of mycobacterial maltokinases, SjGlcNK was unable to phosphorylate maltose or aminoglycosides sing see . Interescharides , display10.1128/mBio.00239-19.9TABLE S2Actinobacteria as determined with the ConSurf server . Download Table S2, DOCX file, 0.1 MB.GlcN kinases proposed as homologues of SjGlcNK in Copyright © 2019 Manso et al.2019Manso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Corynebacteriales, Propionibacteriales, Frankiales, Streptosporangiales, Micromonosporales, Micrococcales, and Streptomycetales from the phylum Actinobacteria  and Mycobacteroides (including the rapidly growing pathogens Mycobacteroidesabscessus and Mycobacteroideschelonae), which contain the most clinically relevant mycobacterial species  database (genome ID 235985.3). Paralogue detection and homology between paralogues were determined based on the KEGG SSDB database (https://www.kegg.jp/kegg/ssdb). For genomic context analyses and identification of BGCs, genome assemblies of Actinobacteria containing SjGlcNK homologues were recovered from the NCBI database and analyzed with the antibiotics and secondary metabolite analysis shell, antiSMASH version 4.2.0  and retrieved from the PATRIC , obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Germany), was cultivated at 28°C for 6 days in ISP2 agar medium at pH 5.5 . Mycolicibacterium smegmatis MC2155 (ATCC 700084), obtained from LGC Standards S.L.U. (Spain), was cultivated for 5 days at 30°C in a glycerol-based agar medium  ( pH 7.0) .S. jiangxiensis and M. smegmatis proteins were expressed in Escherichia coli strain BL21(DE3). Cells were precultured at 37°C for 4 to 5 h in lysogeny broth (LB) supplemented with 50 mg liter−1 kanamycin. Six milliliters of the preculture were used to inoculate 2-liter flasks containing 750 ml of the same culture medium. When cultures reached an OD600 of 0.6 to 0.8 after ∼6 h of shaking at 37°C, protein production was induced by adding 500 μM isopropyl-β-d-thiogalactopyranoside, and expression proceeded for ∼16 h at 20°C. Cells were harvested by centrifugation at 4°C for 15 min at 3,500 × g, and the resulting pellets were resuspended in 20 mM sodium phosphate pH 7.4, 500 mM NaCl, 20 mM imidazole, and frozen at −80°C. Upon thawing, 1 mM PMSF, 1 mM DTT, and 1 mg ml−1 DNase were added to the cell suspension, and the cells were disrupted by sonication on ice (4 min at 25% amplitude). Cell lysates were centrifuged at 4°C for 30 min at 39,200 × g, and the supernatant was loaded onto a 5-ml nickel NTA agarose column (Agarose Bead Technologies) equilibrated with 20 mM sodium phosphate pH 7.4, 500 mM NaCl, 20 mM imidazole (buffer A). Bound protein was eluted with buffer A containing 500 mM imidazole. The purity of the eluted fractions was evaluated by SDS-PAGE, and those containing recombinant protein were pooled and dialyzed overnight at 4°C against 20 mM Bis-Tris propane (BTP) pH 7.4, 50 mM NaCl, 0.1 mM DTT, 0.1 mM EDTA. The protein solution was concentrated to 5 ml in a 30-kDa molecular weight cutoff centrifugal ultrafiltration device (Millipore) and loaded onto a Sephacryl S300 HR 26/60 size exclusion chromatography column , with a mobile phase adequate to the downstream use of the protein (see below). Fractions containing pure protein were combined, concentrated by ultrafiltration, flash frozen in liquid N2, and stored at −80°C . Protein concentrations were determined using the extinction coefficient at 280 nm calculated with the ProtParam tool (http://web.expasy.org/protparam/). Recombinant MvMak was produced and purified as previously described  and developed with one of two solvent systems: acetic acid-ethyl acetate-water-ammonia 25%  or ethanol-water . Sugars were visualized by spraying with α-naphthol–sulfuric acid solution and charring at 120°C.Enzyme activity was assessed in 50-μl mixtures containing 50 mM BTP pH 8.0, 10 mM MgCl2, 20 mM GlcN, 10 mM ATP, and 120 μg SjGlcNK) was incubated overnight at 37°C and separated by TLC. The mixture was spotted on Silica 60 gel plates (Merck) and developed with a methanol-chloroform-acetic acid-water  solvent system. The marginal lanes of the TLC plates were stained to identify the spot corresponding to the SjGlcNK product, and the remaining unstained lanes were scrapped in the same region. The product was extracted from the silica gel with ultrapure water, lyophilized, and further purified on a Sephadex G10 column  with a water flow of 1 ml min−1, followed by a second lyophilization step before NMR analysis. All NMR spectra were acquired on an Avance III 400 spectrometer from Bruker operating at a central proton frequency of 400.13 MHz equipped with a BBI(F)-z H-X-D (5-mm) probe at 25°C with presaturation of the water signal.To confirm the identity of the SjGlcNK product, a 2-ml reaction mixture  were deposited with the Structural Biology Data Grid (PDB) under accession numbers 6HWJ , 6HWK , and 6HWL (SjGlcNK-GlcN-ADP-Pi complex). SAXS data were deposited at the Small Angle Scattering Biological Data Bank (SASBDB)   under coAdditional methods are described in 10.1128/mBio.00239-19.9TEXT S1Text S1, DOCX file, 0.1 MB.Supplemental methods. Download Copyright © 2019 Manso et al.2019Manso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


TaPR-1-1 within the pathogenesis-related (PR) protein family was selected for detailed characterization. Although previous studies had shown that PR genes function during pathogen attack, our results demonstrated that TaPR-1-1 expression was also induced by freezing, salinity, and osmotic stresses. Overexpression in yeast and Arabidopsis showed that TaPR-1-1 conferred tolerance to these stresses. We concluded that screening cDNA yeast libraries following abiotic stress is an efficient way to identify stress-tolerance genes.Abiotic stress significantly impacts growth and yield of crop plants. It is imperative for crop improvement to discover and utilize stress-tolerant functional genes. In this study, genes responding to abiotic stresses, such as freezing, salt and osmotic stress, were screened from a cDNA yeast library that was constructed from the drought- and heat-tolerant wheat variety Hanxuan 10. After screening for surviving clones we isolated 7,249, 4,313 and 4,469 raw sequences, corresponding to 4,695, 2,641 and 2,771 genes following each treatment. Venn diagrams revealed 377 overlapping genes. GO analysis suggested that these genes were mainly involved in the metabolic and stress signal pathways. KEGG pathway enrichment analysis indicated that the isolated genes predominantly belonged to pathways concerning energy and metabolism. Overlapping gene  The Food and Agriculture Organization (FAO) estimates are that the demand for food will increase greatly by 20503. Therefore, identification of functional genes that respond to stress has become an important research objective and this also could provide sources of stress resistance for breeding programs5. During the last few decades numerous functional factors thought to have a role in abiotic stress response have been isolated; these include components of the SOS  pathway, abscisic acid (ABA) pathway, kinases, phosphatases, ion transporters, and transcription factors8.Abiotic stresses, such as salinity, drought and freezing, significantly impact wheat production11. Screening of yeast libraries that express heterologous cDNA is also an effective method to identify functional genes15. Because cellular responses to stress are conserved in eukaryotes, yeast is a much easier model for genetic research than plants, and heterologous proteins in yeast are biologically active, and undergo similar behavior to plants in respect of protein folding and glycosylation17. According to Zhu18 stress signaling pathways in plants evolved from yeast and mammalian energy sensing indicating that many stress tolerance components should be conserved.Methods for isolating genes include map-based cloning, yeast two hybrid analysis, transcriptomics analysis, proteomics analysis, biochemical methods, and cloning by homologyE. coli, Agrobacterium or yeast. For example, a mannose-1-phosphate guanyl transferase gene from a rice cDNA yeast library was identified as source of salinity response, the Dbf2 kinase gene from an Arabidopsis cDNA yeast library was identified as an essential part of stress tolerance, and SR-like splicing proteins were isolated from a cDNA yeast Arabidopsis library developed following salt treatment22. However, there are no similar reports for wheat and wheat cDNA yeast libraries are needed to identify stress-related genes. Similar studies in other plant species have not reported enrichment analyses for genes detected in screens. With release of the wheat genome sequence, screening cDNA yeast libraries to identify wheat stress-tolerance genes is feasible25.Several genes involved in abiotic stress response were identified by screening plant cDNA libraries expressed in 26. Some studies showed that PR protein participated in disease resistance- responses mediated by salicylic acid28. Other studies suggested that PR proteins also function in abiotic stress. For example, AtPR protein had a role in seed germination under salt stress29, and AtPR1, AtPR2, and AtPR5 functioned in response to drought stress30. Rice pathogenesis-related 1a protein/sperm coating protein (OsSCP) enhanced abiotic stress tolerance32. Spinach and peanut PR10 had a role in the stress signaling pathway35. These studies indicated that PR proteins function not only in responses to biotic stress, but also in response to abiotic stress. There are 23 cloned TaPR-1 genes in wheat, and all contain intron-free open reading frames37. Their functions are not very clear.Pathogenesis-related protein (PR-1) was first discovered in tobacco leaves infected by tobacco mosaic virusTaPR-1-1, one of the genes responding to all three treatments was selected for further investigation. Overexpression of TaPR-1-1 in yeast and Arabidopsis revealed various functions in stress tolerance.In the present research stress-related genes screened from wheat cDNA yeast libraries following three separate abiotic treatments were subjected to GO and KEGG analyses. Drought and heat tolerant common wheat variety Hanxuan 10 was used as the plant material for construction of the cDNA yeast library. Two-week-old Hanxuan 10 seedlings were separately treated at low temperature (4 °C), 250 mM NaCl, 16.1% PEG6000, and sprayed with 50 μM ABA solution. Seedling samples were harvested at 0, 0.5, 1, 3, 6, 12 and 24 h post treatments. Total RNAs were extracted with Trizol reagent . After pooling equal quantities of RNAs from each treatment the pooled mRNA was purified with a FastTrack MAG mRNA Isolation Kit . The ‘SuperScript III First-Strand Synthesis System’  was used for reverse transcription. A wheat cDNA library was prepared using a CloneMiner II cDNA Library Construction Kit  following the manufacturer’s instructions. Three kinds of reading frame adapters  vector were diluted with SD/-Leu liquid medium, spread on an SD/-Leu medium plate, and treated at −20 °C for 3, 6, 7 or 8 days, and incubated at 30 °C for 3 days. For salinity treatment, the cDNA library yeast and control yeast were incubated on SD/-Leu medium supplemented with 1.2, 1.4, 1.6 or 1.8 M NaCl for 5 days. For osmotic stress treatment, the cDNA library yeast and control yeast were incubated on SD/-Leu medium supplemented with 3.2, 3.4, 3.5 or 3.6 M sorbitol for 5 days. The final screening conditions were as follows: for the freezing treatment the library was incubated on SD/-Leu medium at 30 °C for 3 days following treatment at −20 °C for 8 days; for salinity treatment, the library was incubated on SD/-Leu medium supplemented with 1.8 M NaCl for 5 days; for osmotic stress treatment, the library was incubated on SD/-Leu medium supplemented with 3.6 M sorbitol for 5 days.Clones surviving after treatment were selected for PCR using pGADT7 vector primer T7 and 3′ AD. The PCR products were purified and sequenced by T7 sequencing primer in an ABI3730 DNA analyzer. This provided raw sequence data for the stress-related genes.http://www.geneontology.org). KEGG pathways for the isolated genes were retrieved (http://www.kegg.jp/kegg/kegg1.html) and R function ‘phyper’ was used for enrichment analysis41.Clean sequences were obtained after removing poor quality sequences, empty vector regions, poly A regions, and incorrectly translated sequences from the data (Qv < 15). The sequences were then mapped to the Release-32 version of the wheat reference genome (ftp://ftp.ensemblgenomes.org/pub/release-32/plants/fasta/triticum_aestivum/) using the blat tool, and corresponding genes were detected in the wheat genome database. Venn diagrams were made to show the numbers of genes isolated following each treatment. The GO terms for isolated genes were extracted (TaPR-1-1 (TRIAE_CS42_5BL_TGACv1_404207_AA1290140), TaMYC2 (TRIAE_CS42_4DL_TGACv1_344028_AA1142670) and TaHSP70 (TRIAE_CS42_5AL_TGACv1_374650_AA1205560) full length cDNA were separately cloned into the pGBKT7 (BD) vector all at the Nde I and BamH I enzyme sites and transformed to yeast strain AH109 (Supplementary Table http://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree was constructed by the maximum likelihood method in Molecular Evolutionary Genetics Analysis (MEGA) software version 5.2.Various pathogenesis-related protein 1 genes were obtained for different plant species by protein BLAST analysis  with 3% (w/v) sucrose and 0.8% agar in plates and cultured in an incubator with 16 h light (21 °C)/8 h darkness (19 °C).Surface-sterilized 44. Two-week old Arabidopsis seedlings were treated with cold acclimation at 4 °C for 2 days and were then placed in a freezing chamber at −10 °C for 12 h. After freezing treatment, the seedlings were transferred to 4 °C in the dark for 12 h, and then were incubated with 16 h light (21 °C)/8 h darkness (19 °C). After 3 days of recovery, survival rates were recorded. Ion leakage was determined following boiling. Briefly, 1 g of two-week-old seedling tissue was placed in 10 ml water, and conductivity of the liquid was measured after shaking for 30 min. Conductivity was measured again after the solution was boiled for 10 min. Ion leakage was measured as the ratio of conductivity before and after boiling.Freezing tolerance assays were performed as described with some modificationsArabidopsis seedlings were transferred to MS solid medium containing 200 mM NaCl and grown for an additional 7 days in vertical culture. Main root lengths were recorded and chlorophyll contents were measured as follows: the seedlings were harvested, weighed and ground into powder in liquid nitrogen; chlorophyll was extracted in N,N-dimethylformamide (DMF) followed by centrifugation, and absorbance of the supernatant was measured using a spectrophotometer with wavelength settings of 646.8 nm and 663.8 nm, and normalized to fresh weight45.Seven-day-old Surface sterilized seeds were planted on MS medium and MS medium supplemented with 300 mM sorbitol. Green cotyledon ratios were scored after 7 days.7 cells/ml  of the cDNA library was 3.0 × 10Abiotic stress-related genes were isolated from the wheat cDNA yeast library, three stress conditions were optimized and imposed, including the freezing, salinity (NaCl) and osmotic (sorbitol) treatments. Yeast survival rates declined with longer periods of freezing, and higher concentrations of NaCl and sorbitol , empty vector regions, poly A regions, and incorrectly translated sequences, 6,697, 4,031 and 3,736 clean sequences were obtained from each treatment library. As shown in Fig. R2 were >0.93. On checking the last 100 sequenced clones, 60–70% of sequences were similar to clones already obtained. This indicated that most stress-related genes had been captured from these libraries. Under freezing, NaCl, and sorbitol treatments, the most frequently screened sequence was TRIAE_CS42_U_TGACv1_642768_AA2122990 detected in 28, 25 and 21 clones, respectively.After sequencing of 7,249, 4,313, and 4,469 clones, 4,695, 2,641 and 2,771 genes were isolated from the respective libraries following freezing, salinity and osmotic stress treatments. The relationships between clone number and gene number for the three treatments are shown in Supplementary Fig. In GO annotation, the isolated genes from the freezing-treated library were distributed across 22 biological processes, 20 cellular components, and 12 molecular functions; isolated genes from the salinity treated library were distributed across 23 biological processes, 15 cellular components, and 12 molecular functions; and isolated genes from the osmotic treated library were distributed in 23 biological processes, 16 cellular components, and 12 molecular functions. Overlapping genes from the three libraries were distributed across 20 biological processes, 14 cellular components, and 8 molecular functions  leucine zipper transcription factor; its expression can be ABA-induced and it takes part in the stress signal response pathway. Abiotic stresses usually cause protein dysfunction, while heat-shock protein (HSP) assists in protein refolding under stress conditions. The three genes were separately cloned into pGBKT7 vector with an ADH1 promoter, and transformed into yeast strain AH109, hence different from the AD vector and Y187 yeast strain used in the library screening. As shown in Fig. TaPR-1-1, TaMYC2 and TaHSP70, have function in stress tolerance and identifying stress related genes from this yeast screening is effective.Three overlapping genes, TaPR-1-1 was selected for further study. Previous reports demonstrated that PR proteins participated in abiotic stress response in Arabidopsis and rice, but their functions in wheat were not clear. Four clones of TaPR-1-1 were identified in all three treatments. Three clones contained an intron-less 495 bp coding region encoding a polypeptide of 164 amino acids with a predicted molecular mass of 17.6 kDa and pI of 8.57, whereas the fourth contained the same CDS, but lacked 72 bp at 5′ end corresponding to an N terminal 24 amino acids. Through ‘SignalP 4.1 Server’ analysis, this N terminal 24 amino acids was predicted to be a signal peptide , but was strongly induced by NaCl and PEG. TaPR-1-1 expression reached its highest level of 4-fold that of the control after exposure to salinity stress for 6 h. Expression of TaPR-1-1 was also induced by osmotic treatment using PEG, and a double peak expression pattern was identified. The first small peak of about 4-fold the level of the control appeared after 0.5 h, then expression gradually increased until 12 h and 24 h when it reached 7-8-fold the control level. These results indicated that TaPR-1-1 responded to abiotic stress.Many studies showed that TaPR-1-1 gene was cloned into expression vector pCAMBIA1300 with the 35S promoter, and more than 20 transgenic Arabidopsis lines were obtained. Expression levels of TaPR-1-1 in 8 transgenic lines are shown in Supplementary Fig. TaPR-1-1 were selected for presentation of results. A previous study showed that PR1 proteins participated in freezing tolerance in winter rye47. Treated at −10 °C for 12 h, about 40% of the control seedlings were dead, whereas survival rates in the transgenic lines were more than 70%  control, , PR-3 (chitinase) and PR-5 (thaumatin-like protein), inhibit microbial growth through enzymatic activity57. PR-1 proteins that contain a CAP-derived  peptide 1 have been shown to confer stress tolerance58. However the actual mechanism of TaPR-1-1 in abiotic stress tolerance and whether TaPR-1-1 takes part in tolerance to both abiotic and biotic stress through the same pathway remains to be addressed.Most studies oness Figs , 6–8. HeTaPR-1-1 detected in all three stress treatments through overexpression in Arabidopsis and yeast. Together, this study provides insights on screening for stress-responsive functional genes that might be useful in dissecting and improving abiotic stress tolerance in crop plants.In this study, we screened a wheat cDNA yeast library, and identified 4,695 freezing-related genes, 2,641 salinity stress-related genes, and 2,771 osmotic stress-related genes. The gene function in stress tolerance was confirmed by study of an overlapping gene Dataset