E.coli, S. aureus, and P. aeruginosa. Overall, the findings suggest that GS-loaded AXSA films hold potential for further development as antibacterial wound dressing material.Biopolymer-based antibacterial films are attractive materials for wound dressing application because they possess chemical, mechanical, exudate absorption, drug delivery, antibacterial, and biocompatible properties required to support wound healing. Herein, we fabricated and characterized films composed of arabinoxylan (AX) and sodium alginate (SA) loaded with gentamicin sulfate (GS) for application as a wound dressing. The FTIR, XRD, and thermal analyses show that AX, SA, and GS interacted through hydrogen bonding and were thermally stable. The AXSA film displays desirable wound dressing characteristics: transparency, uniform thickness, smooth surface morphology, tensile strength similar to human skin, mild water/exudate uptake capacity, water transmission rate suitable for wound dressing, and excellent cytocompatibility. In Franz diffusion release studies, >80% GS was released from AXSA films in two phases in 24 h following the Fickian diffusion mechanism. In disk diffusion assay, the AXSA films demonstrated excellent antibacterial effect against  Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacteroides fragilis) are one of the most predominant reasons for the prolongation of wound healing (in the inflammatory stage) [A wound can be defined as a breakage in intact tissue (particularly skin), caused by physical, thermal, chemical, or mechanical trauma, or may result from complicated pathological conditions ,2. Immedy stage) ,11,12,13y stage) ,15. The Numerous kinds of dressing materials have been in human use since ancient times, with the primary aim being to cover the wound to provide protection against the environment and prevent bleeding and further injury ,12,16. WSeveral natural, semi-synthetic, and synthetic polymers have been explored to develop film dressings, as reviewed recently by Savencu et al. . Among tPlantago ovata (psyllium) seed husk (PSH), is a branched polysaccharide that mainly contains xylose backbone and arabinose side chains with a minor percentage of rhamnose and uronic acids [Arabinoxylan (AX), isolated from the husk of ic acids . AX is aic acids ,33. In oic acids ,33. Howeic acids . Therefoic acids ,35. To tIn this study, novel GS-loaded AXSA films were fabricated for application as an antibacterial wound dressing. The thickness, mechanical properties, morphology, water transmission, water uptake, chemical nature, thermal degradation behavior, drug release, antimicrobial, and cell viability were investigated to assess the suitability of AXSA films for antibacterial wound dressing application.2 granules (anhydrous), pen-strep solution , cellulose acetate membranes , MTT -2,5-diphenyl-tetrazolium bromide), polystyrene petri dishes (10 cm × 20 mm), and gentamicin sulfate were procured from MilliporeSigma . MRC-5 cells (ATCC CCL-171) were cultured in RPMI-1640 culture medium supplemented with FBS . Three bacterial strains S. aureus (ATCC 25923), E. coli (ATCC 8739), and P. aeruginosa (ATCC 9027) were all cultured in Mueller-Hinton broth (MHB) and Agar . Arabinoxylan (AX), containing Xylp (72.5%), Araf (21.8%), Rhap (2.3%), and uronic acids (1.4%) was isolated from Plantago ovata (PO) seeds husk  as reported previously [Sodium alginate (SA)  , glycerol (Gly). PBS tablets, CaCleviously ,32.w/w) were prepared in distilled water by high-speed stirring (1000 rpm) at 40 °C for 12 h. Then, AX  and glycerol (2.5%) were added to SA dispersion and stirred until homogenized gel was obtained. The resultant AXSA film casting gels were sonicated to eliminate air. After that, to cast blank AXSA films, 25 g of AXSA gels were poured into polystyrene plates and kept for drying in an oven at 40 °C for 48 h. While for GS-loaded AXSA films, GS (0.1% w/w) was added in homogenized AXSA gels and films were cast as described for blank films. After drying, films were peeled from plates, and their physical appearances  were visually observed. The films were kept in a desiccator until further analysis. The composition and codes of AXSA film formulations are displayed in The AXSA films were fabricated by solvent casting technique . BrieflyThe cross-sectional thicknesses of all dried films were measured at five randomly selected points. The thickness measurements were performed using a micrometer with 0.01 mm accuracy .2 (desiccant). Then, caps with a 1 cm opening were fitted on the film containing vials. Then, these vials were weighed and kept in a desiccator at 85 ± 3% RH and 25 ± 2 °C. The change in the weights of the vials was noted at constant time intervals up to 24 h. WVTR was calculated according to the formula described earlier [The WVTR of AXSA films was measured following the previously reported procedure with some modifications . Briefly earlier . −1 until breakage. Finally, tensile strengths (TS) and percent elongations at break (% EAB) were calculated according to the formula described previously [Mechanical tests were performed using a 5 kN load cell (LS5) tensile testing machine . For this purpose, dumbbell-shaped specimens (0.5 cm width and 3 cm length) of AXSA films were prepared. To protect the film from damage due to grip, the film’s area in contact with the grip was covered with double adhesive tape. The films were stretched at a crosshead speed of 10 mm·mineviously .The surface morphology of gold sputter-coated AXSA films (with and without GS) was observed using a Quanta 250-FEG scanning electron microscope (SEM)  at 5 kV operating voltage and 1000× and 2000× magnifications. −1 resolution in the wavenumber range of 4000 to 550 cm−1.ATR-FTIR spectroscopic analyses were performed with an FTIR-7600 spectrophotometer . Transmittance spectra were recorded at 4 cmθ range of 10 to 80° by operating at 30 mA, 40 kV, and 2° min−1 scanning speed.X-ray diffraction (XRD) analyses were performed with Shimadzu X-ray diffractometer  using CuKα radiations. The X-ray diffractograms were recorded in the 2−1 heating rate and 20 mL. min−1 nitrogen gas flow.The TGA analyses were performed with Shimadzu TGA-50 analyzer . TG curves were obtained by heating weighed AXSA films from 30 to 600 °C at 20 °C. min−1 heating rate. The changes in the heat flow of AXSA films were recorded from 30 to 450 °C under 20 mL.min−1 nitrogen gas flow.The DSC curves were acquired on DSC3 STAR instrument , operated at 20 °C. min−1). Briefly, to create an environment similar to exudating wounds, 25 mL of gelatin solution was allowed to solidify in the polystyrene plates for 18 h at 25 °C. Then, pieces of AXSA films (~25 mm diameter) were placed over solidified gelatin, and expansion in the size of the films was measured using a ruler at predefined intervals of time. The percent expansion was calculated according to the previously described formula [The exudate absorbing profile of the AXSA films was investigated by measuring the expansion of films over a gelatin solution . The GS release profile was plotted as percent cumulative release versus time [The release profile of GS from GF2 and GF3 films was studied with DHC-6T Franz diffusion cell  using 10 mL release media  in the receiver compartment. Cellulose acetate membranes were placed over the receiver compartment, and then weighed GS-loaded AXSA films (~1.5 cm diameter) were mounted over these membranes. GS release experiments were performed at 37 °C, with constant stirring of the release media. Aliquots were withdrawn from the receiver compartment at pre-programmed time points. The concentration of GS in the aliquots was determined according to the Zero order(1)First order(2)Higuchi(3)Korsmeyer–Pappas(4)where tQ and Q0 are the amounts of GS released from AXSA films at time t and 0, while K0, K1, HK, and kK represent kinetic constants and n is the release exponent.To determine GS release kinetics and mechanism of release from AXSA films, the following kinetic models were fitted to the release data:S. aureus, E.coli, and P. aeruginosa) were uniformly spread over MHA using a glass spreader. After that, GS-loaded AXSA films (GF2 and GF3), GS standard, and blank AXSA film (F2) were placed over the bacterial cultures, and plates were incubated for 24 h at 37 °C. Clear zones of inhibition formed around the samples were measured.Antibacterial activities of the GS-loaded (GF2 and GF3) films were estimated using the agar disk (filter paper) diffusion method. For this purpose, AXSA films were cut into ~6.8 mm with filter paper backing and sterilized by UV exposure for 60 min. Autoclaved MHA (40 mL) was poured into glass petri plates and allowed to solidify. Then, standardized inoculums (20 µL) of bacterial strains  and incubated for another 48 h. After that, culture media in the 96-well plates was replaced with AXSA films extract, except the control (untreated) well, and incubated for 48 h. Finally, according to the previously reported method, an MTT assay was performed to determine the percent cell viability (% CV) [The effect of AXSA films on the viability of MRC-5 cells was investigated by indirect MTT assay. For this purpose, extracts of the blank (F2) and GS-loaded (GF2) AXSA films were prepared by incubating weighed films in culture media for 24 h under aseptic conditions, as reported previously . MRC-5 cy (% CV) . p was found to be less than 0.05 by one-way ANOVA. The results are expressed as mean ± standard deviation (SD).Statistical analysis of the data was performed using GraphPad Prism software. The differences were considered statistically significant when w/w) AX and SA, prepared by high-speed stirring, were easy to pour, and free from air bubbles and polymer aggregates. This 5% (w/w) gel concentration for casting AXSA films was selected based on previous studies on alginate composite films [w/v), SA (3% w/v), and AXSA (AX 2% and SA 3%) films without glycerol were prepared and compared with F2 film (with 2.5% glycerol), as shown in The AXSA gels containing 5% (te films . Howeverte films . Therefote films . To studGentamicin sulfate (0.1%) was incorporated into AXSA film formulation to fabricate GS-loaded films. The selection of GS dose was based on the previous reports where 0.1% GS was reported to be safe and effective for wound healing applications ,40,41. MThe mean thicknesses of F2, GF2, F3, GF3, F4, and GF4 AXSA films were 227 ± 6, 253 ± 6, 263 ± 11, 273 ± 6, 276 ± 14, and 276 ± 9 µm, respectively, as shown in −2.day−1, respectively. In blank AXSA films, WVTR of the F2 and F3 films was significantly higher than in F3 films, while in GS-loaded films, WVTR of the GF2 and GF3 films was significantly higher than that of GF4 films. Moreover, the WVTR of GS-loaded films was less than that of the blank AXSA films, but the difference was statistically insignificant. The WVTR of the film dressings usually depends on the chemistry of the polymers, thickness, composition, and porosity [−2·day−1, and the WVTR of injured skin may be up to 4274 g·m−2·day−1 [The results of the WVTR of the AXSA films (blank and GS-loaded) are presented in porosity . The WVTporosity . Moreoveporosity . Similarporosity ,43. The porosity . The WVT−2·day−1 . Hence, The mechanical properties (TS and EAB) of the AXSA films are presented in SEM was used to investigate the surface microstructure and smoothness of the blank and GS-loaded AXSA films, as presented in 2, –COOH, C–O–C (glycosidic linkage), and C–O–H  are present at around 3650–3300 cm−1, 2930 cm−1, 1660 cm−1, 1450 cm−1, 1160 cm−1, and 1055 cm−1, respectively [−1), Amide I (1645 cm−1), Amide II (1540 cm−1), and C–O–C (1050 cm−1) [−1 and 1450 cm−1 to 1620 and 1425 cm−1, indicating the hydrogen bonding interaction between polysaccharides. The shift in the vibrational frequency of hydroxyl groups from 3423 cm−1 (SA) and 3360 cm−1 (AX) to 3350 cm−1 (in films) confirms that hydrogen bonding is present in the films due to the interaction between hydroxyl, carboxylic, and amide groups of AX, SA and GS [The ATR-FTIR spectra of AX, SA, GS, and AXSA films are presented in ectively . The spe50 cm−1) . On the A and GS . The hydθ 13°, 56°, 20.6°, 20.1°, 29° and 36.4° [θ 13° to 38°, whereas AX did not show any sharp peaks attributed to its amorphous nature. The intensities of the semi-crystalline peaks of the SA were reduced in the AXSA films (F3 and F4). This reduction in the peak intensities may originate from the hydrogen bonding interaction of the SA with other amorphous components of the films . Previously, Ramakrishnan et al. 2021 suggested a similar decrease in the SA crystallinity due to hydrogen bonding for SA/gum kondagogu blended films [The results of the XRD analyses of AX, SA, F3 (blank film), and GF3 (GS-loaded film) are presented in nd 36.4° . The pured films .rangeT) ~125–300 °C (weight loss (∆W) = 99.3%) with maximum weight loss temperature (maxT) at 256.2 °C. The TG and DTG curves show that AX exhibited degradation at rangeT < 120 °C (∆W = 5.1%), 260–320 °C , 325–395 °C , and > 395 °C (∆W = 21.7%) attributed to the loss of water, AX backbone and complete pyrolysis [rangeT < 105 °C (∆W = 5.7%), 265–415 °C , and >415 °C (∆W = 12.5%) due to water loss, fragmentation of polysaccharide backbone of SA and complete degradation [rangeT < 120 °C (∆W = 13.7%), 245–295 °C , 295–370 °C , and >370 °C (∆W = 35.5%), which shows that thermal decomposition of GS starts at 245 °C [The thermal degradation and stability of the AXSA films were investigated by TGA. The TG and DTG curves of the blank  and GS-loaded  films are presented in radation . Similart 245 °C . The F2 and GF2 films exhibited similar thermal degradation events at Trange <120 °C (∆W up to 8%), 125–245 °C (∆W up to 19.8%), 250–430 °C ∆W up to 47.1%), and >430 °C (∆W up to 18.8%). Similarly, F3 and GF3 films show similar degradation events at Trange <120 °C (∆W up to 7.2%), 140–310 °C (∆W up to 36.6%), 310–410 °C (∆W up to 28.8%), and >410 °C (∆W up to 20.6%). These results suggest that AXSA films undergo four main events of thermal degradation corresponding to the degradation events of the individual components of these films. However, the effect of the composition of the AXSA films was observed in the TGA of the films. In F3 and GF3 films, the second degradation event was broader than F2 and GF2 due to the higher AX contents in the F3 and GF3 films, resulting in the merging of the degradation step for Gly and the first step for AX. Similarly, the second degradation step was slightly broader in GS-loaded films than in the blank films due to the merging of degradations steps for Gly and the first degradation step of GS in GS-loaded films. The TG curves of F4 and GF4 also exhibited similar four-step thermal degradation behavior. It was observed that due to the plasticizer’s effect, the onset temperature for polysaccharide (AX and SA) degradation shifted to a lower temperature in films vs. pure polysaccharides. These shifts in the TG and DTG curves for the AXSA films indicate the miscibility of the components .1%, and ,32,33.The results of the DSC analyses of the AXSA films  are depicted in Water uptake (or hydration) is a vital characteristic of polymeric materials designed for wound dressing application. After application to the wounds, the dressings should absorb water (exudate) and expand in size while maintaining their integrity and shape . HydratiThe expansion profile of AXSA films shows that films hydrated (uptake water) and expanded rapidly during the first hour of contact with solidified gelatin. After that, the expansion of the AXSA films slowed down. Among the blank AXSA films, F2 exhibited the highest expansion, followed by F3 and F4, respectively. Similarly, the expansion of GF2 films was highest among the GS-loaded AXSA films. Moreover, all AXSA films maintained their shape after 24 h. Higher expansion of the F2 films can be attributed to greater SA content in these films. The carboxylic group of SA converts to carboxylate ions in water, resulting in electrostatic repulsion among the polymeric chains, leading to the film expansion . TherefoThe GS release profile from AXSA (GF2 and GF3) films, investigated by Franz diffusion cell, is presented in 2R” values indicate the best fitting model. The results , first-order (0.543 and 0.5856), and Higuchi (0.6804 and 0.7015) equations, were less than those obtained with the Korsmeyer–Peppas equation (0.9788 and 0.9451). These results suggest that the Korsmeyer–Peppas model is the best-fitted model (“R2” close to 1) for GS release from GF2 and GF3 films. The values of n (release exponent) for the Korsmeyer–Peppas model were 0.4603 and 0.4982 for GF2 and GF3 films, respectively. According to Korsmeyer–Peppas, for thin films, n values below 0.5 indicate that Fickian diffusion is the mechanism governing drug release [n values of GS release from the AXSA films indicate that Fickian diffusion was the predominant mechanism, with the rate of GS release being limited by solvent penetration rate. A similar release mechanism was reported by Pires et al. for extract release from chitosan and alginate membranes intended for wound dressing [The drug release from the dressing material depends on various factors, such as the solubility of the drug in the release media, relaxation, swelling and erosion of the polymeric network, and diffusion rate of the drug ,38,52. T results  indicate release . Therefow/w) GS solution were used as a positive control. The F3 (blank) films formed no clear inhibition zones, suggesting an insignificant antibacterial effect of AXSA films against the tested bacterial strains. However, opaque regions were observed around F2 films due to bacterial growth in the expanded blank AXSA films. In contrast, positive control (GS std.) and GS-loaded AXSA films (GF2 and GF3) exhibited significant antibacterial effects against both Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria. The values of inhibition zones for GF2 and GF3 were slightly higher than for the positive control. The antibacterial effect of the films was slightly higher against E. coli compared to the other two test strains, but this difference was statistically insignificant. The antibacterial effect of the AXSA films was better than the effect of recently reported chitosan-SA/GS nanofibrous wound dressing [The results of the antibacterial effect of GS solution, F3, GF2, and GF3 films are depicted in dressing . These rThe indirect cell viability assay results of AXSA films against MRC-5 are depicted in The findings of this study demonstrate that the optimized AXSA films possess various desirable characteristics for antibacterial wound dressings. FTIR, XRD, and TGA analyses suggested that AX, SA, Gly, and GS were finely blended and formed hydrogen bonds. The prepared AXSA films were smooth, flexible, and transparent, which can aid in inspecting wounds without removing the dressing. The skin-like mechanical strength and flexibility of AXSA films make them suitable for their application on wounds in problematic body areas like joints. The moderate water uptake (expansion) and transmission (WVTR) of these films could be helpful for protecting moderately exudating wounds from dehydration and to maintain a moist environment for healing. The AXSA films released more than 80% of the loaded GS in 24 h with the initial rapid release (in the first hour), which can be used to control infections. The inhibition zones formed by the GS-loaded AXSA films in Gram-positive and Gram-negative bacteria were similar to those of GS solution, suggesting that these films can be used to control bacterial growth in infected wounds. The AXSA films also demonstrated excellent cytocompatibility in MTT assay, suggesting their safety for in vivo applications. Overall, we demonstrated that the AXSA films are a potential candidate for antibacterial wound dressing applications.


Candida albicans is the most common species isolated from nosocomial bloodstream infections. Due to limited therapeutic arsenal and increase of drug resistance, there is an urgent need for new antifungals. Therefore, the antifungal activity against C. albicans and in vivo toxicity of a 1,3,4-oxadiazole compound (LMM6) was evaluated. This compound was selected by in silico approach based on chemical similarity. LMM6 was highly effective against several clinical C. albicans isolates, with minimum inhibitory concentration values ranging from 8 to 32 µg/mL. This compound also showed synergic effect with amphotericin B and caspofungin. In addition, quantitative assay showed that LMM6 exhibited a fungicidal profile and a promising anti-biofilm activity, pointing to its therapeutic potential. The evaluation of acute toxicity indicated that LMM6 is safe for preclinical trials. No mortality and no alterations in the investigated parameters were observed. In addition, no substantial alteration was found in Hippocratic screening, biochemical or hematological analyzes. LMM6 (5 mg/kg twice a day) was able to reduce both spleen and kidneys fungal burden and further, promoted the suppresses of inflammatory cytokines, resulting in infection control. These preclinical findings support future application of LMM6 as potential antifungal in the treatment of invasive candidiasis. Candida spp. is the most common cause of nosocomial bloodstream infections by fungal, responsible for over 90% of these cases Nbenzamide (LMM6) was purchased from Life Chemicals Inc. . Dimethyl sulfoxide (DMSO) was used to prepare LMM6 stock solution (50 μg/mL). Prior to each experiment, LMM6 was solubilized with non-ionic surfactant from Sigma–Aldrich  for 24 h at 35 °C. Two controls were considered: negative (only medium without inoculum) and positive (medium plus inoculum). The LMM6 MIC values were determined by measuring the absorbance at 405 nm in a SpectraMax ® Plus 384 plate reader  and defined as the lowest concentration able to inhibit the growth ≥ 80% in relation to positive control. For conventional antifungal agents, MIC values were determined visually: for azoles (FLC and ITC), it was defined as the concentration which resulted in 50% reduction of fungal growth and for polyenes (AMB) and echinocandins (CAS), it was defined as the concentration that was not visible fungal growth in relation to the positive control. The conventional antifungals MICs were interpreted according to the M27-S4 document [The minimum inhibitory concentration (MIC) of LMM6 (0.5–256 μg/mL), AMB (0.032–16 µg/mL), CAS (0.032–16 µg/mL), FLC (0.125–64 µg/mL) and ITC (0.032–16 µg/mL) was determined for 30 clinical isolates and reference strain, based on broth microdilution method, according to document M-27A3 (CLSI) with modifications . The LMMdocument .The minimum fungicidal concentration (MFC) of LMM6 was determined for all clinical isolates and reference strain by subculture in SDA. The plates were incubated at 35 °C for 24 h. The MFC was defined as the lowest LMM6 concentration at which no colony growth was visible. A quantitative analysis of LMM6 antifungal activity was also performed for each concentration LMM6 tested. After 24 h, aliquots were serially diluted in phosphate buffered saline (PBS), subcultured on SDA medium without the compound and incubated at 35 °C for 24 h for colony-forming units (CFU) counting.3 cells/mL) in RPMI-1640. Two controls were prepared: drug-free (only culture medium) and conventional antifungal . At predetermined time points , aliquots were obtained from each condition tested, serially diluted in PBS, plated on SDA and incubated at 35 °C for 24 h for CFU determination. Mean counts (log10 CFU/milliliter) were plotted as a function of time for each concentration of LMM6 or FLC tested. The fungicidal activity was defined as a ≥ 99.9% (3 log10) reduction in numbers of CFU and fungistatic activity was defined as a < 99.9% reduction in growth compared to the control [LMM6 at concentrations of 16, 32, 64 and 128 μg/mL were incubated in 24-well plates at 35 °C with yeast suspension  after 24 h. The analysis of the combination of the compounds was carried out calculating the fractional inhibitory concentration (FIC) index, which is defined as the sum of FICA + FICB . Drug interactions were classified as FIC values < 0.5 indicate strongly synergistic effect, FIC < 1 synergistic effect, FIC = 1 additive effect, 1 < FIC < 2 no effect and FIC > 2 antagonistic effect [Synergistic interaction between LMM6 and conventional antifungals  were performed against one clinical isolate  and reference strain by checkerboard assay . The inhc effect . In addic effect . Interac5 cells/mL was added in 96-well plates (200 μL) and incubated at 37 °C on a shaker at 120 rpm for 2 h to allow adhesion phase. After the incubation, the medium was aspirated from the wells and non-adherent cells were removed by washing with sterile PBS. Subsequently, 200 μL of LMM6 without surfactant F-127, at concentrations 16, 32 and 64 μg/mL were added to form biofilm and incubated at 37 °C/120 rpm/24 h. Untreated biofilm containing only RPMI 1640 medium was used as control. Biofilm inhibition was evaluated by total biomass , viability cells (determination of CFU) and scanning electron microscopy (SEM). Before any evaluation, the total medium was removed from wells and the biofilm washed once with PBS to ensure the removal of unadhered cells and the residual of LMM6.The anti-biofilm activity was determined as previously described by Tobaldini-Valerio et al. . A cell v/v) was added to the wells (100 μL) for 5 min. The wells were washed twice with sterile distilled water and 200 μL of acetic acid (33% v/v) was added to dissolve the stain. The absorbance at 570 nm was measured in a SpectraMax ® Plus 384 plate reader .To assess biofilm total biomass, the wells were fixed with methanol 100% for 15 min. After methanol removal, the 96-well plates were dried at room temperature. CV  diluted in 2% paraformaldehyde and in 0.1 M sodium cacodylate buffer . The samples were dehydrated in an ethanol series  and coated with gold  for observation using a scanning electron microscope FEI Quanta 250  at 5000× magnification . The imaAll animal procedures were performed according to Brazil’s National Council for the Control of Animal Experimentation (CONCEA) and approved by the Ethics Committee for Animal Use of the State University of Maringá, PR, Brazil (protocol number CEUA 3855010719). The animals were kept with free access to water and food, in a controlled animal facility having a constant temperature of 22–24 °C, relative humidity of 50–60% and a 12 h light/dark cycle.The LMM6 acute toxicity in a single dose was evaluated according to the guide for conducting non-clinical toxicology and pharmacological safety studies necessary for drug development . InbredThe animals were monitored by Hippocratic screening at times 0, 15, 30, 60, 120, 240 min and daily in which were observed clinical and behavioral parameters that included general appearance, motor coordination , muscle tone , reflexes, lethargy, central nervous systems activity  and autonomic nervous system , over a period of 14 days post treatment. Body weight of mice was recorded daily during the whole experiment. After the 14th day, mice were humanely anesthetized for blood collection and then euthanized with isoflurane vaporizer. The organs, brain, heart, kidneys, liver, lungs and spleen were removed, weighed and the relationship between organ weight and body weight (RW) of each mice was established for all groups. Relative weight (RW) = organ weight (g)/body weight on sacrifice day × 100 .Blood collection was done from retro-orbital sinus in microtubes with and without Ethylenediaminetetraacetic acid (EDTA 10%) for hematological and biochemical analysis, respectively. The blood without the EDTA was left at 37° C to coagulate, centrifuged at 5000 rpm, 20 °C for 5 min to obtain serum and stored at −80 °C. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE), urea (UR) and glucose (GLU) were determined according to the manufacturer’s instructions using standard diagnostic kits  and a semi-automatic biochemistry analyzer BIOPLUS-2000 .Hematology parameters were performed using standard hematological manual methods ,70. The 5 yeast cells) of reference strain by the lateral tail vein, the mice were randomly divided into three experimental groups with 6 animals each: LMM6 (treated with LMM6 compound 5 mg/kg), FLC (treated with fluconazole 5 mg/kg) and Control . The antifungal treatment started after 3 h of infection and it was conducted twice a day for 5 days via intraperitoneal. The animals were anesthetized for blood collection and then humanely euthanized by isoflurane vaporizer. The left kidney and spleen were aseptically removed for determination of fungal burden. The organs were weighed and then macerated with 1 mL of lysis buffer . The homogenates were serially diluted, plated on SDA and incubated for 24 h at 35 °C for CFU counts. Mean CFU counts were normalized by the weight of tissue sample (g).Systemic candidiasis model was established in female inbred Balb/c mice (n = 18), 6–7-week-old with ± 20 g of weight, according to previously described protocols ,73. AfteTM Cytometric Bead Array (CBA) Mouse Inflammation Kit  as per the manufacturer’s instructions and was analyzed on BD FACSCalibur™  flow cytometer. The following cytokines were measured: Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Interleukin-17a (IL-17a), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). The results for the standard curves of each cytokines and samples were generated using FCAP Array software v3.0 .The kidney homogenates were centrifuged at 14,000 rpm, 4 °C for 15 min and the supernatant transferred to microtubes containing protease inhibitor . Blood collected from mice retro-orbital sinus was left at 37 °C to coagulate and centrifuged at 5000 rpm, 20 °C for 5 min to obtain serum. The samples were stored at −80 °C prior to analysis. Analysis of the kidney supernatant and blood for detection systemic and local cytokines, respectively, were conducted by BDImmediately after euthanasia, the right kidney of mice was collected and immersed in paraformaldehyde 4% for fixation during 24 h. The organs were preserved in 100% ethanol until processing. Posteriorly, the samples were embedded in paraffin, sectioned longitudinally at 5 μm and stained with hematoxylin eosin (HE) and Grocott-Gomori (GG) for detection of inflammatory areas and fungi in situ. Histopathology images from tissues stained were obtained using a binocular light microscope (Motic BA310- camera Moticam 5), at × 200 and × 600 magnification. In qualitative analysis of fungal cells and inflammatory infiltrates, at least 20 fields of three histological sections were analyzed for each group.t-test and the other assays with one-way analysis of variance (ANOVA) using the Bonferroni. Values of p < 0.05 were considered statistically significant.The data were evaluated as the mean ± standard deviation (SD) using Prism 6.0 software . Reduction of kidney fungal burden on in vivo treatment was analyzed using unpaired Student’s C. albicans is described. These findings suggest important therapeutic potential of LMM6 due to its fungicidal ability, inhibition of biofilm formation, synergistic effect with conventional drugs and capacity to reduce renal fungal burden in a murine model of disseminated candidiasis. Immunomodulatory activity also increases the protective effects of LMM6 against C. albicans infection and maintains homeostasis. LMM6 compound has no hepatotoxic or nephrotoxic effects and does not interfere with the biochemical and hematological parameters in the mice, being safe for future applications as antifungal agent or in association with conventional drugs for the treatment of candidiasis.In conclusion, this is the first time the antifungal activity of LMM6 both in vitro and in vivo against Kioshima ES, Svidzinski TIE, Bonfim-Mendonça PS, et al. Composição farmacêutica baseada em compostos 1,3,4-oxadiazólicos e seu uso na preparação de medicamentos para tratamento de infecções sistêmicas. Brazil patent BR 10 2018 009020 8. 03 May 2018.


Bombyx mori (B. mori), an important economic insect, is famous for its silk. B. mori silk is mainly composed of silk fibroin coated with sericin. Among them, the silk fibroin heavy chain protein has the highest content and the largest molecular weight, which is encoded by the silk fibroin heavy chain (FibH) gene. At present, apart from the complete sequence of the FibH of the B. mori strain p50T, there are no other reports regarding this protein. This is mainly because the special structure formed by the GC-rich repetitive sequence in FibH hinders the amplification of polymerase and the application of Sanger sequencing. Here, the FibH sequence of Dazao, which has 99.98% similarity to that of p50T, was obtained by means of CEO. As far as we know, this is the first complete FibH sequence of the Chinese B. mori strain. Additionally, the methylated CG sites in the FibH repeat unit were identified.B. mori FibH gene, which is composed of many repeat units with a long and GC-rich sequence up to 17 kb and is not easily amplified by means of a polymerase chain reaction (PCR). CEO has four steps: the dephosphorylation of genomic DNA, the Cpf1 targeted cleavage of FibH, adapter ligation, and ONT sequencing. Using CEO, we determined the fine structure of B. moriFibH, which is 16,845 bp long and includes 12 repetitive domains separated by amorphous regions. Except for the difference of three bases in the intron from the reference gene, the other sequences are identical. Surprisingly, many methylated CG sites were found and distributed unevenly on the FibH repeat unit. The CEO we established is an available means to depict highly repetitive genes, but also a supplement to the enrichment method based on Cas9.To study the evolution of gene function and a species, it is essential to characterize the tandem repetitive sequences distributed across the genome. Cas9-based enrichment combined with nanopore sequencing is an important technique for targeting repetitive sequences. Cpf1 has low molecular weight, low off-target efficiency, and the same editing efficiency as Cas9. There are numerous studies on enrichment sequencing using Cas9 combined with nanopore, while there are only a few studies on the enrichment sequencing of long and highly repetitive genes using Cpf1. We developed Cpf1-based enrichment combined with ONT sequencing (CEO) to characterize the  B. mori silk fibroin [Tandem repeat is a common repetitive DNA sequence in eukaryotes, which are arranged consecutively in the genome, and has been called junk or even selfish DNA because most of these DNA sequences cannot encode proteins ,2. In fa fibroin ,14,15, d fibroin ,16,17. V fibroin .Since the invention of Sanger sequencing in 1977, new sequencing technologies have emerged. Third-generation sequencing not only allows long-read lengths but also provides more information on DNA methylation and is therefore preferred by researchers over other techniques ,21,22. TAcidaminococcus sp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), and Fransicella novicida U112 (FnCpf1) [CRISPR/Cas9 is an acquired immune defense system in bacteria and archaea that can be used against invasive viruses and any foreign DNA . Today, (FnCpf1) ,28,29. U(FnCpf1) . Cpf1 cu(FnCpf1) . At the (FnCpf1) ,33. Cpf1(FnCpf1) ,36,37,38FibH may be an important factor affecting the mechanical properties of cocoon silk, but there are only a few studies on this gene, which is mainly because the FibH sequence is highly repetitive and not easily amplified by traditional PCR. This study uses the B. mori as a model to precisely characterize the sequence of FibH, which will provide a reference for the analysis of other large and highly repetitive genes. At the same time, CEO is also an important supplement to the Cas9-based enrichment sequencing method [Cocoon silk protein is mainly composed of sericin-coated fibroin, which includes silk fibroin heavy chain protein (FibH), silk fibroin light chain protein (FibL), and glycoprotein P25 ,40. Among method ,45,46,47B. mori pupa ground in liquid nitrogen was incubated in an extraction buffer  with RNase A at 37 °C for 1 h, and it was then digested with proteinase K (100 µg/mL) at 55 °C overnight to completely degrade the proteins. Next, it was extracted twice with saturated phenol, once with Tris saturated phenol-chloroform-isoamyl alcohol (25:24:1), and once with chloroform. Finally, ethanol was added to the extracted supernatant to precipitate the gDNA. Carotenoids are often precipitated with B. mori pupal gDNA. Therefore, to obtain high purity gDNA, we repeated the extraction process again and extracted the gDNA three times with phenol and chloroform. The DNA pellet was resuspended overnight with EB  at 4 °C, and it was then centrifuged at 10,080 g for 10 min at 4 °C to obtain the supernatant and a viscous lower suspension. The former was transparent, while the latter was turbid and viscous. The DNA concentration in the supernatant and the DNA suspension was determined using Nanodrop and Qubit. The supernatant DNA was used for the subsequent experiments.To reduce the cost and to increase the yield of gDNA extraction, we adopted the classical phenol extraction method. Briefly, a B. mori pupal gDNA as DNA templates in a 40 µL reaction mixture containing 20 µL PrimeSTAR Max Premix (2×)  and 1.2 µL 10 uM forward and reverse primers. The thermal cycle program was as follows: 98 °C for 4 min, 30 cycles of 98 °C for 10 s, 55 °C for 15 s, 72 °C for 30 s, and 5 min of final extension at 72 °C, using a S1000 Thermal Cycler . Each PCR product was mixed with 8 µL 5× DNA Loading Buffer with GelRed , separated by 1% agarose gel via electrophoresis, and purified using a gel extraction kit  according to the manufacturer’s manual. The purified DNA was immediately cleaved by Cpf1/crRNA or stored at −20 °C.The forward and reverse primers  were synE. coli BL21(DE3) and was purified [We mixed 2.5 μL 10 μM crRNA  and 2.5 μL 10× cutsmart buffer  in 20 μL DNase/RNase-free water . After heat denaturation at 90 °C for 6 min, we placed it on ice immediately. According to the method in the previous study, the Cpf1 protein expressed purified . Then 1.https://imagej.nih.gov/ij/ (accessed on 1 January 2020)) to estimate the cleavage efficiency of the four Cpf1/crRNA complexes.To verify the availability of the four Cpf1/crRNA complexes, the purified DNA was cleaved by the Cpf1/crRNA complex for 1 h at 37 °C and then separated by 1% agarose gel via electrophoresis. The cleavage reaction was composed of 1 µL Cpf1/crRNA complex, 1 µL purified DNA, 1 µL 10× cutsmart buffer, and 7 µL DNase/RNase-free water. The DNA bands were analyzed by ImageJ  were mixed well and incubated at 37 °C for 30 min followed by the thermal inactivation of the enzyme at 80 °C for 5 min. After cooling to 25 °C, 1 μL of 1 mM dNTP , 0.5 μL of 10 mM dATP , 5 μL each of the 4 Cpf1/crRNA complexes, 1 μL 10× cutsmart buffer, and 6.5 μL DNase/RNase-free water were added to the system after being cooled to 25 °C and were mixed well. Next, 1 μL Klenow Fragment (3′→5′ exo-)  was added and mixed well. CRISPR digestion, end repair, and poly-A tail addition were performed at 37 °C. The enzymes were inactivated at 67 °C for 7 min. The reaction solution was purified using 1× Ampure XP magnetic beads , washed twice with 75% ethanol, dried, and 62 μL EB was added . Next, 60 μL DNA, 5 μL AMX , 10 μL quick T4 DNA ligase , and 25 μL LNB  were mixed well and incubated at 25 °C for 30 min to add adaptors. The reaction solution was purified using 0.4× Ampure XP magnetic beads, washed twice with LFB , and then dissolved with 26 μL EB . Next, 75 μL SQB  and 51 μL LB  were added to 24 μL DNA and mixed thoroughly. The sequencing was conducted according to the protocols created by Nanopore PromethIon .http://silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi (accessed on 5 November 2019)) [http://crispr.cos.uni-heidelberg.de/ (accessed on 1 December 2019)) [https://pypi.org/project/ont-pyguppy-client-lib/ (accessed on 2 March 2020)) to obtain the original data. The reads with a quality value less than 7 were filtered out, and high-quality data  were used for subsequent analysis. The ONT Reads were aligned to the silkworm reference genome using Minimap2 (v 2.17-r941) [https://github.com/nanoporetech/medaka (accessed on 23 March 2020)) was used to align the ONT reads to the assembled sequence for error correction on the assembled sequence to obtain a consensus sequence. Based on the reference genome sequence, nanopolish software (Available online: https://github.com/shiliyan/nanopolish (accessed on 15 April 2020)) was used to detect the methylation of the target region.Based on the silkworm reference genome (Available online: r 2019)) , the crRr 2019)) . In addir 2019))  to check17-r941) , and the17-r941)  was used17-r941)  was usedB. mori gDNA was dephosphorylated with CIAP to block the 5′ end of all the linearized DNA. The dephosphorylated gDNA was then digested with the Cpf1/crRNA complex to release the target region. Then, the sticky ends left by Cpf1 cleavage were filled by a poly-A tail, and the adaptors were ligated. Finally, the complete sequence of the target gene was obtained through ONT sequencing and bioinformatic analysis.To precisely characterize large, highly repetitive, and less complex genes, we established the CEO strategy . The strB. mori pupa. Because carotenoids are easily extracted from B. mori pupae along with gDNA and affect subsequent experiments, they were therefore removed by repeating the extraction process. Suspension and supernatant DNA were obtained by high-speed centrifugation at 4 °C, and the supernatant DNA was used for subsequent enrichment. The DNA fragments were 23–200 kb (FibH (KWMTBOMO15365) and met the needs for ONT library construction.ONT sequencing is known for its long read-length, which is determined by the integrity of the gDNA. To obtain high-quality gDNA, it was extracted using phenol-chloroform from a 3–200 kb A, which FibH  from SilkBase (Available online: http://silkbase.ab.a.u-tokyo.ac.jp (accessed on 2 June 2020)) and the other (AF226688.1) from NCBI. The annotated KWMTBOMO15365 has 4 introns, which is different from AF226688.1. We analyzed all introns and found that, with the exception of intron 1 (971bp) of KWMTBOMO15365, the sequence or partial sequence of the other three introns were identical to the repetitive unit, which implied that KWMTBOMO15365 was probably incorrectly annotated. Therefore, we defined that the reference gene is encoded by two exons . The methylation frequency of each motif and their methylation frequency at different positions on the FibH gene were counted  activity and the degree of dephosphorylation of gDNA. It is necessary to compare the enrichment efficiency of these methods under the same conditions to study genes.As we all know, the CRISPR/Cas system is an acquired immune response used by bacteria and archaea to prevent the invasion of foreign DNA, such as plasmids and phages . Among tIn addition to silk protein genes, genes with tandem repeats also include many disease-related genes, such as SAMD12 , BRCA1 , and VHL


Warionia saharae Benth. & Coss. (Asteraceae) is an endemic species of North Africa naturally grown in the southwest of the Algerian Sahara. In the present study, this species’ hydromethanolic leaf extract was investigated for its phenolic profile characterized by ultra-high-performance liquid chromatography coupled with a diode array detector and an electrospray mass spectrometer (UHPLC-DAD-ESI/MS). Additionally, the chemical composition of W. saharae was analyzed by gas chromatography–mass spectrometry, and its antioxidant potential was assessed through five in vitro tests: DPPH● scavenging activity, ABTS●+ scavenging assay, galvinoxyl scavenging activity, ferric reducing power (FRP), and cupric reducing antioxidant capacity. The UHPLC-DAD-ESI/MS analysis allowed the detection and quantification of 22 compounds, with taxifolin as the dominant compound. The GC–MS analysis allowed the identification of 37 compounds, and the antioxidant activity data indicate that W. saharae extract has a very high capacity to capture radicals due to its richness in compounds with antioxidant capacity. The extract also showed potent α-glucosidase inhibition as well as a good anti-inflammatory activity. However, weak anti-α-amylase and anticholinesterase activities were recorded. Moreover, an in silico docking study was performed to highlight possible interactions between three significant compounds identified in W. saharae extract and α-glucosidase enzyme. It is recognized that medicinal plants have been used since the dawn of civilization to treat a wide range of ailments ,2. TheirWarionia saharae Benth. & Coss. is an endemic species grown in Beni Ounif Zousfana and Bechar (south-west of Algerian Sahara). It was also found in various regions in the south of Morocco [W. saharae are used in folk medicine to treat many health problems, among them inflammatory diseases, gastrointestinal disorders, and epileptic crises [Asteraceae is one of the most prominent plant families. It covers 23,000 species distributed across more than 1,620 genera. Among them,  Morocco ,12. The c crises ,14.Warionia saharae, until now, did not arouse researchers’ interest, and few works have been published about this species. These works were focused on different types of W. saharae extracts: aqueous extract [W. saharae chloroform and ethyl acetate extracts and were identified using UV, IR, and NMR methods [W. saharae leaves have not been studied. extract , essenti extract ,17, and  extract . Their a extract ,18, anti extract ,19, anti extract ,20 activW. saharae extract.Furthermore, this species’ extracts and secondary metabolites remain less explored regarding their anti-inflammatory, cytotoxic, and enzyme inhibition activities. Therefore, our study will be the pioneer in this field. Simultaneously, evaluating the extracts’ antioxidant and anti-inflammatory potentials and their inhibition ability toward key enzymes involved in diabetes and neurodegenerative diseases  was also an objective. Molecular docking experiments investigate the most energetically favorable interactions between bioactive compounds and enzymes . In thisThis research is expected to provide a better understanding of the chemical composition and the health benefits of this plant, which could contribute to new perspectives for developing new and potent pharmacological agents.n analysis of W. saharae hydromethanolic  extract allowed the identification, for the first time, of 24 compounds. The UHPLC-DAD-ESI-MS/MSAmong the 24 metabolites, hydroxycinnamic acid derivatives and flavonoids were identified, which are secondary metabolites usually associated with anti-inflammatory properties. Actually, the quantification shows around 76 µg/mg extract of hydroxycinnamic acid derivatives and around 80 µg/mg extract of flavonoids .O-dicaffeoylquinic acid  can be highlighted because of its quantity , were also observed. Again, their identification was confirmed by literature data, and peaks 4, 5, and 6 were assigned to 5-O-CQA, 3-O-CQA, and 4-O-CQA, respectively [The other dicaffeoylquinic acids, isomers 3,5- peak 9, , but we ectively ,23,24. Mectively .W. saharae by 10 derivatives, 7 aglycones, and 3 glycosides. These results are consistent with those previously reported because we also found luteolin, esculetin, taxifolin, and quercetin derivatives, although Mezhoud et al. [The flavonoid family is represented in the hydromethanolic extract of d et al.  mainly r− at m/z 303, MS2 fragment ion at m/z 285, and UV spectrum data also in agreement with a taxifolin derivative [2 fragmentation [2 fragment at m/z 300 obtained by losing the methyl group can be highlighted [− at m/z 477 and MS2 fragment at m/z 315, which corresponds to the loss of a glycosyl group, was assigned to isorhamnetin-O-hexoside , alcohols (128.520 µg/mg), carboxylic acids and esters (124.6 µg/mg), and fatty acids (11.364 µg/mg). The peak characteristics and the identification of the compounds are presented in GC–MS analysis is the most suitable technique to assess plant extracts′ lipophilic profile ,35. It rW. saharae, so this research provides new knowledge about this plant′s fatty acids and important alcohols  scavenging activity, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS●+) scavenging assay, galvinoxyl● scavenging assay, ferric reducing power (FRP), and cupric reducing antioxidant capacity (CUPRAC). The results obtained by the different tests, expressed in IC50 and A0.50, respectively, are shown in The antioxidant activity of The antioxidant reaction with free radicals involves different mechanisms: the hydrogen atom transfer (HAT) method, in which the free radical removes one hydrogen atom from the antioxidant, and single electron transfer (SET), in which the antioxidant provides an electron to reduce compounds such as metals, carbonyls, and radicals. An antioxidant reaction can also involve both methods (HAT and SET) .W. saharae extract was assessed through five different trials , data of DPPH● scavenging activity show that the W. saharae hydromethanolic extract exhibited high antioxidant potential (IC50 = 7.12 ± 0.09 µg/mL) close to positive controls Trolox, BHT, and ascorbic acid, 5.12 ± 0.21, 6.14 ± 0.41, and 4.39 ± 0.01 µg/mL, respectively, but more potent than BHA (12.99 ± 0.41 µg/mL) with qTukey = 5.99.According to ANOVA , in the ABTS●+ scavenging assay, there is not a significant difference between Trolox and ascorbic acid and between BHT and BHA . The studied extract exhibits high antioxidant activity (IC50 = 4.19 ± 0.35 µg/mL), very close to those of the standards.According to the HSD Tukey test following ANOVA  exhibits very high activity, statistically similar to both standards BHT and Trolox .This high antioxidant potential was also noted when analyzing the data of the galvinoxyl assay. Thus, W. saharae was determined using FRP. In this assay, the antioxidant activity causes the reduction of the Fe3+/ferricyanid complex to the ferrous form. The FRP of extracts is linked with reductants, which exert antioxidant action by donating electrons and reacting with free radicals to convert them to more stable products [4,12 = 1446.84, p < 0.001, qTukey > 4.65). Data show that W. saharae extract activity (A0.5 = 31.21 ± 0.14 µg/mL) was weak compared to the used standards . Reducing capacity detected by cupric acid activity was significantly higher compared to that by the FRP assay.On the other hand, the reduction potential of the hydromethanolic extract of products ,49. A sip > 0.05). A significant difference was noted with ANOVA and HSD Tukey tests , which unveil the best activity of the W. saharae extract (A0.5 = 3.84 ± 0.16 μg/mL) as compared to all tested standards: Trolox, BHA, BHT, and ascorbic acid .The CUPRAC assay enables total antioxidant capacity (TAC) measurements of a plant extract using the copper (II)–neocuproine reagent in ammonium acetate buffer and based on an electron transfer mechanism . AccordiW. saharae extract showed a greater capacity to capture radicals. The results of the DPPH● activity are in agreement with those reported in previous works on the ethyl acetate extract of the Algerian W. saharae (IC50 = 6.98 µg/mL) [50 = 182 µg/mL) [W. saharae using the ABTS●+ scavenging assay, galvinoxyl assay, FRP, and cupric reducing antioxidant capacity.Overall, present data indicate that 8 µg/mL) , but are2 µg/mL) . The nat●, ABTS●+, and galvinoxyl scavenging data suggest that the components present in W. saharae extract act as excellent scavenging free radicals through a mechanism of electron or hydrogen donation to stabilize the free radicals, and should be able to protect biological matrices from free radical-mediated oxidative degradation [The DPPHradation ,52.W. saharae was as effective as the positive standards in reducing Cu2+ to Cu+ but had a moderate effect in reducing the Fe3+ to Fe2+. The good reducing behavior of W. saharae extract suggests that its constituents, mainly chlorogenic acids, flavonoids, and sugars, are capable of terminating a chain reaction by eliminating free radical intermediates and hence are considered antioxidants [W. saharae contains a significant amount (around 215 μg/mg).According to the CUPRAC and FRP data, oxidants . A signioxidants . TherefoW. saharae reveals that phenolic acids are the most dominant group identified in this species. In addition, many compounds detected in the W. saharae hydromethanolic extract are known to possess great antioxidant capacity. For example, simple hydroxycinnamic acids have been reported to possess potent antioxidant activity against DPPH● and ABTS●+ [O-caffeoylquinic acid being the major contributor [Furthermore, phenolic acids’ and flavonoids’ antioxidant potentials are closely related to their chemical structure  with doud ABTS●+ . Chlorogtributor .O-caffeoylquinic acids exhibit high antioxidant activity using conventional antioxidant assays. The same authors proposed that their antioxidant mechanisms might include electron transfer, H+ transfer, and Fe2+ chelation.Li et al.  reportedW. saharae and may contribute to its strong antioxidant activity. A previous study reported that in antioxidant assays, taxifolin scavenges HO●, DPPH●, and ABTS●+ efficiently and enhances the relative Cu2+ and Fe3+ reducing levels [Taxifolin (peak 14) is the major compound present in the hydromethanolic extract of g levels . Zu et ag levels  also meng levels  from an Although it is present in lower amounts, luteolin has received increasing attention due to its various biological activities and pharmacological effects, including antioxidant activity , and hasW. saharae with its strong antioxidant power.The high antioxidant potential recorded in this study may be linked to the presence of these compounds that could act individually or combined to provide the hydromethanolic extract of W. saharae was evaluated by the NO● scavenging method. NO● is one of the main chemical mediators involved in inflammatory events. Regarding its ability to scavenge NO●, the relevant anti-inflammatory activity of W. saharae extract (IC50 = 76.66 ± 4.16 µg/mL) is better than that of ascorbic acid (IC50 = 144.380 ± 7.035 µg/mL) but lower than that of curcumin (IC50 = 7.49 ± 0.58 µg/mL). To the best of our knowledge, no anti-inflammatory effect had been previously described for W. saharae extract. Thus, this is the first report describing the anti-inflammatory potential of this species.The anti-inflammatory effect of W. saharae extract are probably responsible for the anti-inflammatory property observed in this study, and flavonoids can be highlighted [W. saharae extract, was previously found to possess significant anti-inflammatory activity against carrageenan-induced edema involving the exudative phase of inflammation and also against formaldehyde-induced arthritis [The phenolic compounds highly present in hlighted ,68. Taxirthritis . These rW. saharae extract and the standard inhibitors tested toward α-glucosidase, α-amylase, and acetylcholinesterase expressed in IC50 or the percentage of inhibition are shown in The enzyme inhibitory activities of the W. saharae species toward these two enzymes was evaluated in this study.The α-glucosidase and α-amylase inhibitors are used to treat diabetes mellitus as they slow down the digestion of carbohydrates in the small intestine and thus reduce the rapid rise in blood sugar after eating ,71. The p < 0.001), W. saharae extract expressed significant strong activity against the α-glucosidase enzyme with an IC50 = 23.52 ± 6.33 μg/mL lower than that of acarbose (IC50 = 405.77 ± 34.83 mg/mL). However, low inhibitory activity against α-amylase was recorded (38.41 ± 5.51% inhibition at the concentration of 4.2 mg/mL).According to an ANOVA test . Thus, the weak inhibitory activity of W. saharae extract toward AChE activity could be related to the chemical composition of this species since we considered that the major substance in the plant extract is usually responsible for its biological activity.We also evaluated the inhibitory effect of the acetylcholinesterase enzyme here. At the highest dose of the extract (1 mg/mL in this assay), the enzyme′s inhibition was observed (28.578 ± 2.979%). The inhibitory action on AChE in the in vitro assay proved to be lower than the donepezil used as a positive control  [W. saharae extract could not be sufficient to inhibit this enzyme strongly. Some of them, like 4,5-di-O-caffeoylquinic, one of the major compounds in W. saharae extract, showed AChE inhibition under 50% [Taxifolin, highly present in the 1 μg/mL)  despite 1 μg/mL) . Althoug1 μg/mL) , their pnder 50% .W. saharae extract could, in part, explain these results.In addition, some phenolic compounds may act as activators of AChE. Earlier studies demonstrated that caffeic acid was shown to increase AChE activity in in vitro studies . Some auW. saharae extract. An in silico study was performed to further screen the mechanisms of interaction between α-glucosidase and the three major compounds in W. saharae extract interact with α-glucosidase, engaging different amino acids , respectively.Based on the docking test’s interaction data, the major constituents of no acids . Taxifol50 of 0.038 mg/mL [Herein, it can be observed that the best docking pose obtained was for taxifolin docked to α-glucosidase. Different interactions, such as three hydrogen bonds with Asn715, Ala687, and Gly690, π–cation, and π–alkyl with Ile691 to the aromatic cycle and many other Van Der Waals interactions can be observed. Taxifolin, which has shown the best binding affinity within the major compounds, was previously reported as a competitive inhibitor of the α-glucosidase enzyme with an IC38 mg/mL .O-dicaffeoylquinic acid and 1,3-O-dicaffeoylquinic acid, and the α-glucosidase active site , 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS●+), trichloroacetic acid (TCA), potassium ferricyanide, and neocuproine, were used for the determination of the antioxidant activities and were purchased from Sigma Chemical Co. . α-Glucosidase from Saccharomyces cerevisiae, 4-nitrophenyl α-D-glucopyranoside (pNPG), α-amylase from porcine pancreas, β-nicotinamide adenine dinucleotide (β-NADH), phenazine methosulphate (PMS), nitrotetrazolium blue chloride (NBT), and ascorbic acid were obtained from Sigma . Acarbose was purchased from Fluka  and potato starch from Fisher . Sodium nitroprusside, sulfanilamide, and 3,5-dinitrosalicylic acid (DNS) were obtained from Acros Organics . Folin–Ciocalteu reagent, Na2CO3, and gallic acid were purchased from Panreac . Acetylcholinesterase (AChE) from Electrophorus electricus, 5,5-dithiobis(2-nitrobenzoic acid), acetylthiocholine iodine, and donepezil were purchased from Sigma-Aldrich .The following chemicals: butylated hydroxyanisole (BHA), hydroxytoluene (BHT) and α-tocopherol, 1,1-diphenyl-2-picrylhydrazyl .® water purification system . All other chemicals were of analytical grade.Formic acid and acetonitrile HPLC-grade solvents were purchased from Panreac . Ultrapure water was obtained by a Direct-QN,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (99%), trimethylsilyl chloride (TMSCl) (99%), eicosane (99%), hexatriacontane (99%), citric acid (99.5%), cinnamic acid (99%), palmitic acid (99%), mannose (99%), D-mannitol (98%), 5-cholesten-3β-ol (99%), tetradecanol (98%), and 1-palmitoylglycerol (99%) were purchased from Sigma-Aldrich.Solvents used for GC–MS analysis were purchased from Panreac and Acros Organics and were of analytical grade. Other chemicals such as palmitic acid (99%), W. saharae were collected from Djebel Antar . A voucher specimen was deposited in the Herbarium of the VAREN laboratory of Mentouri University of Constantine, Algeria, under the reference number VAREN/CWS04/05. The leaves were shade air-dried at room temperature (25 °C) before they were ground to a fine powder. The powder was then macerated at room temperature with a 80:20 mixture of methanol:water (v/v) for 72 h three times. The filtrates were concentrated under reduced pressure using a rotary evaporator at 30 °C. The residual water was freeze-dried in a freeze dryer and then stored at 4 °C until future use.Aerial parts of 2CO3 solution (20% w/v) was added. The mixture was then incubated in the darkness for 60 min, and absorbance was read at 760 nm. The standard curve of gallic acid was obtained under the same conditions as above, and the total phenolic content was measured as μg equivalents of gallic acid per mg of extract (μg GAE mg of extract).Total phenolic content was determined using the Folin–Ciocalteu method . An aliq3 (2%) solution. The mixture was incubated at room temperature for 10 min, and then the absorbance was measured at 415 nm. The standard curve of quercetin was obtained under the same conditions as above, and the total phenolic content was measured as μg equivalents of quercetin per mg of extract (μg GAE/mg of extract).The total flavonoid content of the plant’s crude extract was determined according to the method described by Türkoğlu et al.  with a sW. saharae was determined using an Ultimate 3000  apparatus equipped with a binary pump, an automatic sampler, and a diode array detector . The MS analysis was performed using a Thermo LTQ XL mass spectrometer , which was equipped with an electrospray ionization interface (ESI). The separation was carried out with a Hypersil Gold  C18 column  at room temperature 25 °C. The extract was prepared at a 1 mg/mL concentration and the injection volume was 10 µL. The solvents used were formic acid in water (A) and acetonitrile (B). The flow rate was set at 2 mL/min. Spectra were recorded in negative mode. MS and MS/MS data were processed using the Thermo Xcalibur Qual Browser data system .Phenolic composition of hydromethanolic extract of W. saharae was analyzed by GC–MS using a Shimadzu Gas Chromatograph QP2010 Ultra. Three aliquots of the W. saharae extract (20 mg) were submitted to silylation procedure according to the method described by Freire et al. [−1). The following standards: eicontane, palmitic acid, tetradecanol, D-(+)-mannose, mannitol, and cholestanol, were used to elaborate calibration curves. Compounds were identified by comparing their mass spectra with mass spectra in the Wiley and NIST libraries and those available in the literature.The lipophilic composition of the hydromethanolic extract of e et al. . Silylat●) free radical-scavenging activity was evaluated by a slightly modified method [● was prepared in ethanol, and 160 µL of this solution was added to 40 µL of sample solutions diluted in methanol at different concentrations. Twenty-five minutes later, the absorbance was measured at 517 nm. The scavenging capability of DPPH● was calculated using the following equation. The results were given as IC50 (mg/mL) corresponding to the concentration of 50% inhibition.S refers to the absorbance of sample.The 1,1-diphenyl-2-picrylhydrazyl  was calculated from the graph of DPPH● scavenging effect percentage against sample concentration. BHA, BHT, Trolox, and ascorbic acid were used as antioxidant standards for comparison of the activity.The sample concentration providing 50% free radical scavenging activity (IC●+) scavenging activity was obtained by spectrophotometric analysis according to the slightly modified method proposed by Re et al. [●+ was regenerated by adding 2.45 mM potassium persulfate to an aqueous solution of 7 mM ABTS●+, which was stored in the dark at room temperature for 12 h. Before use, the ABTS●+ solution was diluted until an absorbance of 0.700 ± 0.025 at 734 nm with ethanol was obtained. Then, to each well, in a 96-well plate, 160 µL of this solution and 40 µL of sample solutions diluted in methanol at different concentrations were added. After 10 min, the percentage inhibition at 734 nm was calculated for each concentration relative to a blank absorbance (methanol). The scavenging capability of ABTS●+ was calculated using the following equation:The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) free cation-radical  was calculated from the graph of ABTS●+ scavenging effect percentage against sample concentration. BHA, BHT, Trolox, and ascorbic acid were used as antioxidant standards for comparison of the activity.The sample concentration providing 50% cation radical scavenging activity  was calculated by relating the inhibition percentages to the concentrations of the extracts. The antioxidant capacity of the extract was compared with ascorbic acid, BHT, BHA, and Trolox used as antioxidant standards and prepared under the same conditions.The concentration of the sample providing 50% inhibition (IC3Fe(CN)6 in 100 mL H2O) were mixed with 10 μL plant extract at different concentrations and then incubated 20 min at 50 °C. A mixture of 50 mL trichloroacetic acid (TCA) (10%), 40 mL H2O, and 10 μL FeCl3 (0.1%) was then added. The absorbance of the reaction mixture was then recorded at 700 nm. Increased absorbance of the reaction mixture indicated increased FRP. The results were given as A0.50, which corresponds to the concentration producing 0.500 absorbance.The FRP assay was carried out according to the method of Oyaizu  with sliW. saharae extract at different concentrations was added to a solution composed of 50 µL Cu Cl2, 2H2O (10 mM), 50 µL neocuproine (7.5 mM), and 60 µL ammonium acetate buffer solution . The reaction mixtures were incubated in the dark for 1 h. The absorbance was then measured at 450 nm.The cupric ion reducing antioxidant capacity (CUPRAC) assay was determined by the method described by Apak et al. . A total0.50 (µg/mL) corresponding to the concentration indicating 0.500 of absorbance. The reduction capacity of the extracts was compared with that of BHA, BHT, Trolox, and ascorbic acid prepared under the same conditions.The results were calculated as A• following the method described by Afonso et al. [The in vitro anti-inflammatory activity was determined by the ability of the extract to scavenge NOo et al. . A total● was calculated using the following equation:The scavenging ability of NO● scavenging activity (IC50) was calculated from the graph of NO● scavenging effect percentage against sample concentration.The sample concentration providing 50% NOd-glucopyranoside (PNPG) solution used as substrate. A total of 100 µL of the α-glucosidase enzyme solution was then added to the mixture. The absorbance was measured at 405 nm at 37 °C, for 20 min every minute. Acarbose was used as reference.α-Glucosidase inhibition activity was measured by the method described by Neto et al.  with briThe α-glucosidase inhibitory activity was expressed as percentage inhibition and was determined according to the following formula:IC values (concentration of inhibitor required to inhibit 50% of enzyme activity) were determined.w/v) starch solution dissolved in 20 mM phosphate buffer . After incubation at 37 °C for 5 min, the reaction was started with the addition of 200 µL of α-amylase solution. A total of 200 µL of the mixture was then mixed with 600 µL of 3,5-dinitrosalicylic acid (DNS) reagent.α-Amylase inhibition activity was determined according to the method previously described by Wickramaratne et al.  with sliA second aliquot of 200 µL of the first mixture was also collected 15 min later and mixed with DNS reagent. The mixtures were boiled for 10 min. After cooling, 250 µL was transferred to each well in a 96-well microplate and the absorbance was read at 450 nm.The α-amylase inhibitory effect was calculated by the following equation:IC values (concentration of inhibitor required to inhibit 50% of enzyme activity) were determined. Acarbose was used as reference compound.Inhibition of acetylcholinesterase (AChE) activity was determined as described by Ellman et al.  with sli50 values (concentration of inhibitor required to inhibit 50% of enzyme activity) were determined.ICW. saharae extract, three compounds were docked to the α-glucosidase enzymatic activity. The selected compounds were taxifolin, 1,3-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, which are the most abundant in the W. saharae extract. The 3D structures of the considered molecules were downloaded from PubChem database [To support our findings from in vitro inhibition of α-glucosidase activity by the database . All thrdatabase . The crydatabase . Water mdatabase  and BIOVdatabase .p ≤ 0.05. Data were recorded as the mean ± standard deviation (m ± SD) from three independent assays.All of the data were analyzed with MINITAB software  using one-way ANOVA followed by a post hoc honestly significant difference (HSD) Tukey’s test at W. saharae. The extract showed potent antioxidant and α-glucosidase inhibition properties, highlighting that this species provides a promising source for obtaining natural compounds that could be used to develop food additives or pharmaceutical products. Anti-inflammatory properties were also observed with the hydromethanolic extract of W. saharae. Finally, further toxicological and pharmacological studies must be performed for pharmaceutical purposes.Our results provided new information and allowed the characterization of the chemical profile of the hydromethanolic extract of


Extracellular matrix stiffening is characteristic of both aging and glaucoma, and acts as a promoter and perpetuator of pathological fibrotic remodeling. Here, we investigate the role of a mechanosensitive transcriptional coactivator, Yes-associated protein (YAP), a downstream effector of multiple signaling pathways, in lamina cribrosa (LC) cell activation to a profibrotic, glaucomatous state.LC cells isolated from glaucomatous human donor eyes  were compared to LC cells from age-matched nonglaucomatous controls  to determine differential YAP expression, protein levels, and proliferation rates. NLC cells were then cultured on soft (4 kPa), and stiff (100 kPa), collagen-1 coated polyacrylamide hydrogel substrates. Quantitative real-time RT-PCR, immunoblotting, and immunofluorescence microscopy were used to measure the expression, activity, and subcellular location of YAP and its downstream targets, respectively. Proliferation rates were examined in NLC and GLC cells by methyl thiazolyl tetrazolium salt assays, across a range of incrementally increased substrate stiffness. Endpoints were examined in the presence or absence of a YAP inhibitor, verteporfin (2 µM).P < 0.05) increased YAP gene expression and total-YAP protein compared to NLC cells, with significantly increased proliferation. YAP regulation is mechanosensitive, because NLC cells cultured on pathomimetic, stiff substrates (100 kPa) show significantly upregulated YAP gene and protein expression, increased YAP phosphorylation at tyrosine 357, reduced YAP phosphorylation at serine 127, increased nuclear pooling, and increased transcriptional target, connective tissue growth factor. Accordingly, myofibroblastic markers, α-smooth muscle actin (α-SMA) and collagen type I, alpha 1 (Col1A1) are increased. Proliferation rates are elevated on 50 kPa substrates and tissue culture plastic. Verteporfin treatment significantly inhibits YAP-mediated cellular activation and proliferation despite a stiffened microenvironment.GLC cells show significantly (These data demonstrate how YAP plays a pivotal role in LC cells adopting a profibrotic and proliferative phenotype in response to the stiffened LC present in aging and glaucoma. YAP provides an attractive and novel therapeutic target, and its inhibition via verteporfin warrants further clinical investigation. Compounded by an aging population, its prevalence is projected to increase from 76 to 112 million between 2020 and 2040.,–Variation in both susceptibility and progression of glaucomatous optic neuropathy across a range of IOP is intriguing and may in part be explained by biomechanics differences at the optic nerve head. The LC is a fenestrated, meshlike structure across the optic nerve head, composed of collagenous load-bearing beams that support vulnerable, unmyelinated retinal ganglion cell axons as they exit the eye posteriorly forming the optic nerve.,–The resident cells of the LC respond to extracellular mechanical cues via mechanotransduction.–To disrupt this maladaptive positive feedback loop, we targeted the potent, mechanosensitive, transcriptional coactivator, Yes-associated protein (YAP), a key player in multiple mechanotransduction pathways, and main downstream effector of the Hippo pathway.–There are many upstream regulators of YAP activity, which include mechanical cues , extracellular ligands , which signal via G protein coupled receptors (GPCR) and receptor tyrosine kinases, inflammation (through TNF-α), hypoxic stress (through SIAH2 ubiquitin E3), and energy stress (Hippo pathway via AMP-activated protein kinase), with mechanical cues setting an overarching responsiveness to GPCR, Wnt, and Hippo signaling pathways.,–The transcriptional activity of YAP predominantly depends on its phosphorylation status. More specifically, the site of phosphorylation subsequently determines its subcellular location. For example, large tumor suppressor (LATS1/2)–mediated YAP phosphorylation at serine 127 (pYAP[s127]) (via the Hippo pathway) leads to direct binding of protein 14-3-3, which favors cytosolic retention through increased nuclear export, thus limiting its transcriptional ability. Whereas YAP phosphorylation at tyrosine 357 (pYAP[y357]) (via SRC family kinases) promotes nuclear pooling, subsequently upregulating its transcriptional activity of target genes through binding to TEAD transcription factors.In this study, we aim to expand our mechanistic understanding of the role of YAP in glaucoma by studying LC cells from nonglaucomatous and glaucomatous eye donors. Furthermore, we interrogate the effect of exposing healthy, nonglaucomatous LC cells to a pathomimetic, stiffened in vitro microenvironment, akin to the glaucomatous milieu. Finally, we use a recently described YAP inhibitor, verteporfin (VP), in an attempt to disrupt LC mechanotransduction, precluding any subsequent profibrotic, positive-feedback cycle.2+ binding adapter molecule 1 .,2 in Dulbecco's modified Eagle medium containing 10% (vol/vol) fetal bovine serum and 1% L-glutamine, and 1% penicillin-streptomycin. Cells were cultured in full media until 80% to 90% confluence was attained and passaged using trypsin/ EDTA for one to two minutes at 37°C. The cells were spun in a centrifuge at 1200g for five minutes, and the supernatant was aspirated and discarded. The pellet was then resuspended in fresh media for subsequent culturing of cells. For all experiments passages 4 to 8 were used, and for comparative experiments, cells from equal passage were used. Unless otherwise stated, reagents and solutions were from Sigma-Aldrich Corp. .Human LC cells  were isolated and cultured after retrieval from donors as previously described.2 on commercially available collagen-1 (rat tail)–coated polyacrylamide hydrogel substrates  of substrate stiffness 4 kPa and 100 kPa. The differential expression of YAP in LC cells grown under these controlled stiffness conditions was examined by Western immunoblotting, qRT-PCR and immunofluorescence. For immunofluorescence, 0.17 mm × 30 mm coverslips placed under the collagen-1–coated polyacrylamide hydrogel substrates  were used. Cell culture on these collagen-coated coverslips enabled easier removal for image capture.LC cells were seeded at a low-density of 2000 cells/cm2 onto substrates for 24 hours, serum-restricted pretreatment for 12 hours, and then subjected to either vehicle only DMSO (control), or VP treatment (VP/DMSO) for a further 24 hours, at which point they were immediately isolated and analyzed. The altered gene expression levels, protein expression, and subcellular location of YAP were assessed as: Untreated control (4 kPa substrate) and treated (100 kPa substrate) groups. Both groups were examined in the absence or presence of VP. A minimum of three independent experiments from different LC cell donors were performed. The duration of culture was intended to be short enough to avoid any potential differences in proliferation confounding results, as cell-cell contact is a negative regulator of YAP and its nuclear translocation and could diminish or reverse any findings specific to substrate stiffness. VP use in the proliferation/ methyl thiazolyl tetrazolium salt (MTS) assay was at a concentration of 2 and 4 µM for a period of 24 hours after a 24-hour period for cell attachment and overnight serum starve in serum-free medium.Dimethyl sulfoxide (DMSO) was used to reconstitute VP  to stock solutions of 2 or 4 µM. NLC cells were seeded at a density of 2000 cells/cmg for five minutes at 4°C to form a pellet. The supernatant was carefully removed without disturbing the pellet and discarded. Crude cell lysate was collected using radio immuno-precipitation assay buffer containing protease inhibitor cocktail, and then the cells were incubated on ice for five minutes and subsequently spun in a centrifuge for 15 minutes at 15,000g at 4°C. The cleared supernatant was collected, and the protein concentration was quantified using the Bradford method. Protein extracts (20 µg) were electrophoresed on 10% polyacrylamide-SDS gels and transferred to a nitrocellulose membrane. For control and treated samples on soft and stiff substrates, equal amounts of protein were loaded in the SDS-PAGE. Membranes were blocked with 5% non-fat milk in Tris-buffered saline solution containing 0.1% Tween-20 at room temperature for one hour before incubation overnight at 4°C with primary antibodies as outlined in On completion of treatments, media was removed and cells incubated in 1X ice-cold PBS. The cells were then trypsinized into ice-cold PBS, spun in a centrifuge at 14,000g for 15 minutes at 4°C to allow phase separation. The upper, clear aqueous phase containing the RNA was carefully removed and placed into a new Eppendorf tube on ice. Isopropanol 500 µL was added to the aqueous phase and kept on ice for five minutes, and then spun in a centrifuge at 12,000 g for eight minutes at 4°C. The supernatant was removed and discarded, and the pellet was washed with 1 mL 75% ethanol and spun in a centrifuge at 7500g for five minutes at 4°C. The supernatant was removed, and the pellet was allowed to air dry. The RNA pellet was resuspended in diethylpyrocarbonate-treated water and stored at −80°C for subsequent conversion to complimentary DNA.Following completion of cell culture in their respective conditions, culture medium was removed and TRIzol Reagent Solution  was added to the flasks/wells. Samples were transferred to a 1.5 mL Eppendorf tube and placed on ice. Chloroform 200 of was added per 1 mL of TRIzol, and the sample was inverted to ensure mixing. Samples were incubated on ice for five minutes and then spun in a centrifuge at 12,000 RNA was reverse transcribed to complimentary DNA as follows; A 0.2 mL PCR Eppendorf was filled with 11 µL diethylpyrocarbonate water (Sigma-Aldrich Corp.), 1 µL oligo dT (Sigma-Aldrich Corp.), 1 µL deoxynucleotides (Sigma-Aldrich Corp.), 2 µL of 10× AMV reverse transcriptase buffer (Sigma-Aldrich Corp.), and 4 µL RNA. This was heated to 70°C for 10 minutes and then left on ice for two minutes. AMV reverse transcriptase 0.5 µL was added and the tube transferred to the thermocycler. The cycle was 45°C for 90 minutes, 90°C for two minutes, and then cooled to 4°C and stored at −20°C.ΔΔCt equation. Primer sequences for genes used are shown in RT-qPCR was performed using 18S as a housekeeping gene to normalize Ct values. A standard qPCR cycle was used as follows: denaturation at 95°C for five minutes, denaturation at 95°C for 10 seconds, annealing at 60°C for 20 seconds, and elongation at 72°C for 20 seconds. This was repeated from the second denaturation step for a total of 45 cycles, followed by a melt curve program of 95°C for five seconds and 65°C for one minute. Fold change in gene expression was assessed using the 2NLC cells were seeded on soft (4 kPa) and stiff (100 kPa) collagen-1, polyacrylamide gels overlying detachable coverslips, cultured for 24 hours, serum starved overnight, before being treated with DMSO (vehicle only) or DMSO/VP (2 µM) for a further 24 hours . They were then fixed in 100% ice-cold methanol for five minutes and blocked in PBS containing 0.1% Triton X-100 and 5% normal goat serum (ThermoFisher Scientific) for 1 hour at room temperature. They were subsequently probed for total-YAP  overnight at 4°C. Coverslips were washed three times in PBS. Species-specific secondary antibodies (A10521) and F-actin  were diluted 1:500 and 1:3000 in PBS respectively, and incubated for two hours at room temperature and protected from light. Coverslips were washed again, and for the third wash we used 4ʹ,6- diamidino-2-phenylindole at a 1:5000 dilution for nuclear localization. Coverslips were washed a final time and were mounted to slides using aqua-polymount . Digital images were captured using a confocal microscope  and processed using imaging software ZEN Lite.3 cells per well in a final volume of 100 µL/well. Cell proliferation was measured at different time points  using the MTS colorimetric cell counting assay according to the manufacturer's protocol (Promega Corporation). MTS is bioreduced by cells into a colored formazan product that reduces absorbance at 490 nm. After incubation, 20 µL of the CellTiter 96 AQueous One Solution Reagent (Promega Corporation) was added to each well, and the plates were incubated in a humidified 5% CO2 atmosphere for one hour. The supernatant was removed, and 150 µL/well of DMSO was added to the plates to solubilize the formazan salt crystals. Plates were incubated for 10 minutes at room temperature. Solubilized formazan products were quantified using a Molecular Devices, Spectra Max multiple microplate reader  at a wavelength of 490 nm.Cell proliferation MTS assays were used to compare the proliferation rate in LC cells from glaucomatous patients and age-matched normal donors. Freshly thawed GLC and NLC cells from the same passage were seeded onto “CellTiter 96” 96-well flat-bottom plates  at a density of 106 kPa)  at a fixed timepoint of 96 hours. Additionally, we studied the effect of VP on proliferation using vehicle only (DMSO) or VP/DMSO at a concentration of 2 and 4 µM VP for a period of 24 hours after a 24-hour period for cell attachment and further overnight serum starvation at stiffness values of 4 kPa, 50 kPa, and tissue culture plastic.To ascertain the effect of substrate stiffness on proliferation rates, we repeated the above experiment using NLC cells and a 96 well plate of incrementally increased substrate stiffness values; 0.2/0.5/1/2/4/8/12/25/50 kPa and tissue culture plastic  for comparison between two groups, and by one-way ANOVA with Tukey-Kramer post-test for multiple comparisons. P < 0.05 was taken as the level of significance (P < 0.05). Calculations were performed using the Origin 7.0 software .Data were expressed as means of independent experiments ± SEM. Statistical significance was analyzed by Student's P < 0.05; 3 a.u., P < 0.05; YAP mRNA levels were compared via RT-PCR in GLC cells and age-matched NLC cells and the difference was expressed as a fold change relative to normalized GAPDH. YAP expression was significantly elevated, by approximately 3 fold, in GLC versus NLC cells, 1.138 ± 0.116 versus 0.849 ± 0.095 (P < 0.05) and 120 hours . Accordingly, we found significantly increased proliferation of GLC versus NLC cells at both 96 hours .Real-time PCR displayed enhanced YAP gene expression in NLC cells when grown on a stiffer (100 kPa) substrate compared to soft (4 kPa) substrates, showing 30.1 ± 1. 22 versus 35.8 ± 4.17 fold change  but decreased pYAP(s127) levels on stiff substrates .Not only is total YAP significantly elevated on stiff substrates,  is a transcriptional target gene of YAP.P < 0.05, P < 0.05). Similarly, COL1A1 was reduced on stiff substrates from 0.95 ± 0.08 versus 0.74 ± 0.04 , proliferate at significantly higher rates on substrates having a stiffness of 50 kPa (P < 0.05) and on tissue culture plastic , although its action did not appear to be dose-dependent.  of YAP. Cells on soft substrates show reduced F-actin staining compared to stiff, and YAP protein was localized in the cytoplasm, appearing excluded from the nucleus . In contConsidering the emerging importance of YAP regulation in promoting fibrosis and the significant fibrotic remodeling underpinning glaucomatous progression at the LC, the role of YAP in LC cells represents a potentially novel therapeutic avenue independent of IOP and warrants further delineation. As shown by the in vitro experiments described herein, we have developed a mechanistic understanding of the activity of YAP in LC cells. We compared LC cells from non-glaucomatous and age-matched glaucomatous eye donors, demonstrating enhanced YAP gene expression and upregulated total-YAP protein in glaucoma. Consistent with the proliferative nature of YAP activation, we showed increased proliferation in GLC cells compared to NLC cells. Furthermore, we interrogated the effect of exposing healthy, non-glaucomatous LC cells to a pathomimetic, stiffened microenvironment, and showed the highly mechanosensitive nature of YAP regulation and corresponding differential phosphorylation at serine and tyrosine sites. Additionally, downstream targets CTGF and myofibroblastic markers α-SMA and COL1A1 were increased. Immunofluorescence microscopy demonstrated how substrate stiffness is a critical determinant of YAP nuclear translocation. Finally, we used a YAP inhibitor, VP, and successfully disrupted stiffness-induced LC cell mechanoactivation. We found that treating NLC cells with VP markedly reduces YAP gene expression and transcriptional induction despite culture on a stiffened substrate. Functionally, there was decreased total YAP, decreased pYAP(y357), and increased pYAP (s127) with VP treatment and a concurrent reduction in CTGF, α-SMA and COL1A1, suggesting reduced transcriptional activity, with visibly reduced nuclear localization by immunofluorescence microscopy .Previous work from our group has elucidated differential global and ECM-focused gene expression patterns in LC cells from normal compared to glaucomatous donors.,In attempting to find a cause for this observation, and to further understand what initially drives YAP upregulation at the onset of glaucoma, we investigated how YAP activity was regulated in healthy NLC cells exposed to a stiffened microenvironment akin to that observed in aging and glaucoma. Age-related ECM remodeling at the LC includes changes to nonenzymatic collagen cross-link pentosidine, total collagen and elastin content, decreased type III to type I collagen ratio, and decreased total sulphated glycosaminoglycans content, all of which infer a stiffer LC with age.Consistent with our hypothesis, we found that mimicking the stiffened “in vivo” glaucomatous LC for healthy nonglaucomatous LC cell culture led to a comparable upregulation of YAP “in vitro” . Increas,––,Linking ECM stiffness to YAP dysregulation, we note that several gene variants implicated in glaucoma such as caveolin 1 and 2 , lysyl oxidase–like 1 (LOXL1), and myocilin have separately been shown to modulate YAP regulation in a highly mechanosensitive manner, through actin-cytoskeleton–dependent, MST1/2 kinase, and Wnt signaling pathways, respectively.–The specific phosphorylation site of YAP is of critical importance in determining its activity as a transcriptional co-activator, but is becoming recognized as increasingly complex. Earlier publications focused largely on the well characterized Hippo-mediated YAP phosphorylation at serine 127, resulting in a “Hippo-on” state, which results in cytosolic retention via binding with 14-3-3 proteins, reduced transcriptional activity due to exclusion from the nucleus, and proteasomal degradation.,–71 who suggested a 20-fold increase in stiffness in glaucoma through atomic force microscopy, although more recent studies suggest qualitative rather than quantitative agreement with glaucomatous TM, Schlemm's canal, and peripapillary scleral stiffening, with ranges as low as 2 fold increased stiffness in glaucoma.–In light of the important role of YAP in cellular proliferation, together with our findings of YAP upregulation in glaucomatous LC cells, we demonstrated increased proliferation of NLC cells on stiffened substrates C.31,67–6,Given that there are several formally discreet mechanisms for YAP regulation , the inhibition of the convergent endpoint of YAP-TEAD binding represents an attractive and potentially highly effective prospect. In this study, we used VP without light activation. Verteporfin is a small-molecule benzoporphyrin derivative, commonly used as a photosensitizer for photodynamic therapy, with Food and Drug Administration approval for treatment of neovascular age-related macular degeneration, presumed ocular histoplasmosis, and pathological myopia, and off-label use, with laser, for central serous retinopathy.Exponential research output within the realm of cancer biology has demonstrated successful YAP inhibition with VP in several cell lines. VP treatment has demonstrated decreased cell growth, proliferation, migration, and invasion, with reported molecular mechanisms observed including reduced YAP, vimentin, STAT3, FAK, COX2, SOX2, TEAD, KRAS, mTOR, p-mTOR, ROCK2, MEK, p-MEK, p-ERK, GLUT1, Cyclin-D1, c-myc, Survivin, VEGFA, MMP2, alpha-SMA, IL-11, IL-6, FOXM1, CTGF, CYR61, bcl-2, and increased pYAP(s127), BAX, cleaved PARP, cleaved Caspase 3 and 9.80 who examined human TM cells in vitro through culture on collagen gels and used VP without light activation to establish a means for YAP inhibition. They reported that after 24 hours treatment, VP abolishes TM cell–mediated collagen gel contraction in a dose-dependent manner. Additionally, it attenuates expression of YAP and CTGF (a direct YAP target gene) in a dose-dependent manner, and last, it has no significant cytotoxicity below 2 µM. Yemanyi et al.81 similarly used hTM cells to uncover the crosstalk between the LPA, Interleukin 6 (IL-6), YAP/TAZ, and Signal transducer and activator of transcription 3. LPA or IL6 trans-signaling were shown to activate YAP, TAZ, and STAT3 overexpressed key components of the actomyosin machinery, in conjunction with increased expression of specific receptors and ligands, α-SMA, and fibrotic ECM genes/proteins. VP abrogated LPA or IL6 trans-signaling–mediated activation of YAP, TAZ, and STAT3. Focusing on conjunctival fibroblasts, Futakuchi et al probed the relationship between TGF-β2 and YAP.Several recent reports have outlined YAP inhibition via VP without light activation in cells of ocular origin, namely TM cells and conjunctival fibroblasts. The first of such studies was by Chen et al.Our in vitro findings presented herein implicate dysregulated YAP activity at the lamina cribrosa in the pathogenesis of glaucoma. In confirming the mechanosensitive nature of this dysregulation, and its potential inhibition with VP, we highlight the therapeutic potential in repurposing VP without light activation and propose it to be a worthy candidate for further research.