Introduction: To evaluate the pharmacological profile of ocular formulations based on cross-linked sodium hyaluronate (CL-SH), taurine (Tau), vitamin B6 (Vit B6) and vitamin B12 (Vit B12) using in vitro and in vivo paradigms.Methods: Rabbit corneal epithelial cells were used to assess wound healing and reactive oxygen species (ROS) formation by scratch assay and oxidative stress , respectively with or without ocular formulations exposure. In vivo studies were carried out on albino rabbits to evaluate corneal nerve regeneration and corneal wound healing with or without treatment with six different formulations. Animals were anesthetized, the corneal epithelium was removed, and formulations were topically administered . Slit-lamp observation was carried out at different time points. After 6 days the animals were killed, and corneas were collected to evaluate corneal re-innervation by immunohistochemistry of selective neuronal marker β-III tubulin.Results: Formulations containing the concentrations 0.16% or 0.32% of cross-linked sodium hyaluronate, taurine, vitamin B6 and vitamin B12 accelerated corneal wound healing. Cells exposed to H2O2 led to significant (p < 0.05) increase of reactive oxygen species concentration that was significantly (p < 0.05) counteract by formulations containing cross-linked sodium hyaluronate (0.32%) and taurine with or without vitamins. The extent of re-innervation, in terms of β-III tubulin staining, was 5-fold greater (p < 0.01) in the eye of rabbits treated with formulation containing 0.32% cross-linked sodium hyaluronate, taurine, vitamins (RenerviX®) compared with the control group (no treatment). Furthermore, re-innervation elicited by RenerviX® was significantly greater (p < 0.01) compared with the group treated with the formulation containing 0.32% cross-linked sodium hyaluronate and taurine without vitamins, and with the group treated with the formulation containing 0.5% linear sodium hyaluronate (SH), taurine, and vitamin B12, respectively.Discussion: In conclusion, among the formulations tested, the new ophthalmic gel RenerviX® was able to contrast oxidative stress, to accelerate corneal re-epithelization and to promote nerve regeneration. SIRCs (P6) were cultured with or without test formulations. All media were filtered with syringe filters, 0.45 µm  to ensure sterile conditions.Statens Seruminstitut rabbit corneal (SIRC) epithelial cells (ATCC CCL-60) were cultured in Eagle’s Minimum Essential Medium  supplemented with 10% of fetal bovine serum , 1X Minimum Essential Medium Non-Essential Amino Acids  and 1X Penicillin/Streptomycin  at 37 °C in 5% CO® Alfa Intes I.T.S. s.r.l); formulation #4 (F4), containing 0.16% SH-CL, 0.5% Tau, Vit B6 and Vit B12; formulation #5 (F5), containing 0.02% SH-CL, 0.5% Tau, Vit B6 and Vit B12; formulation #6 (F6), containing 0.32% SH-CL, 0.5% Tau (no vitamins).Six different ophthalmic formulations were used: formulation #1 (F1), containing 0.5% SH-L, Tau and 0.05% Vit B12; formulation #2 (F2), containing 0.48% cross-linked SH-CL, 0.5% Tau, 0.05% Vit B6 and 0.05% Vit B12; formulation #3 (F3), containing 0.32% SH-CL, 0.5% Tau, Vit B6 and Vit B12 (Renervix4 cells/well). Reached the confluence, cells were washed twice with warm phosphate saline buffer  and then incubated with a serum-free medium for 5 h. Then, the confluent monolayer of cells was scratched with a 200 μL pipette tip. All the wells were washed with fresh medium to remove detached cells before incubation in a serum-free medium containing formulation #1, formulation #2, formulation #3, formulation #4, formulation #5 or formulation #6. To be sure the wounds with the same wound area were compared, a couple of lines were made at two points of the well to link the opposite points in the well with a marking pen, using the lines as a reference for the photographic report at the time of the beginning of the experiment (T0) and for 12 h (T12), 24 h (T24), 36 h (T36), 48 h (T48) and 72 h (T72). Wound area was analyzed from six different wells for each treatment, and all images were acquired with a Leica microscope using a ×20 magnification. The average wound area, expressed in the percentage of control (CTR), was determined using ImageJ Software .The SIRC cells were grown to confluence in six-well dishes – Cellular Reactive Oxygen Species Detection Assay Kit  according to the manufacturer’s protocol, as previously described by Maugeri et al. (Antioxidants 2022. PMID: 35052632). Briefly, SIRC cells were plated into 96-well plates (1 × 104 cells/well). After overnight growth, cells were cultured for 60 min in the control medium (CTR); or in the presence of the formulation #3, containing 0.32% sodium hyaluronate (SH-CL), 0.5% taurine, 0.05% vitamin B6 and 0.05% vitamin B12 (RenerviX®); or in the presence of formulation #6, containing 0.32% sodium hyaluronate (SH-CL) and 0.5% taurine. Then, oxidative stress was induced with 0.3 mM H2O2 treatment for 30 min. Subsequently, cells were washed gently in PBS twice and incubated with 25 μM DCFDA previously dissolved in a buffer solution for 45 min in the dark. ROS concentration was detected by fluorescence spectroscopy with excitation and emission wavelength of 495 nm and 529 nm, respectively, using Varioskan Flash Multimode Reader (Thermo Fisher Scientific). Twelve replicate wells were used for each group.ROS generation was evaluated in SIRC cells, after oxidative stress induction by treatment with Had libitum in a light-controlled room and set temperature and humidity. Animal care and experimental procedures were carried out according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Protocols were approved by the Institutional Animal Care and Use Committee of the University of Catania (project #303). Animals were anesthetized and the corneal epithelium was removed with 0.5 mm corneal rust ring remover , under a dissecting microscope. The eyes were treated as follow: group 1) Formulation #6 containing 0.32% SH-CL and 0.5% Tau; group 2) Formulation #3 containing 0.32% SH-CL, 0.5% Tau, 0.05% Vit B6 and 0.05% Vit B12, RenerviX®); group 3) Formulation #1 containing 0.5% SH-L, 0.5% Tau and 0.5% Vit B12. All formulations were topically administered  starting the same day of corneal epithelial debridement. Slit-lamp observation was carried out at different time points. After 6 days, the animals were killed, and corneas were collected for the immunohistochemical analysis to evaluate corneal re-innervation by immunohistochemical analysis of the selective neuronal marker beta-III tubulin.Male New Zealand albino rabbits (1.8–2.0 kg) were purchased from Envigo . Animals were housed under standard conditions with food and water provided 2O2/methanol to quench endogenous peroxidase activity and then rinsed for 20 min with phosphate-buffered saline. The sections were then heated in a thermoregulated bath (80° for 30 min) with rodent decloaker , to perform antigen retrieval. The blocking step to prevent non-specific binding of the antibody was performed before application of the primary antibody with 1% bovine serum albumin  in PBS for 1 h in a moist chamber. After blocking, the sections were incubated overnight at 4 °C with ß-III Tubulin antibody , work dilution in PBS and 1%BSA 1:100. Immune complexes were then treated with a biotinylated link antibody (HRP-conjugated anti-rabbit was used as secondary antibodies) and then detected with peroxidase labeled streptavin, both incubated for 10 min at room temperature . The immunoreaction was visualized by incubating the sections for 3 min in 3,3′-diaminobenzidine solution . The samples were lightly counterstained with hematoxylin, mounted in vecta mount (Vector Laboratories) and observed with an Axioplan Zeiss light microscope (Carl Zeiss) and photographed with a digital camera . Densitometric analysis was carried with ImageJ. ß-III Tubulin staining in the corneal epithelium was quantified as previously described by The expression and distribution of ß-III tubulin in rabbit cornea were evaluated through immunohistochemical analysis. Briefly, after dewaxing in xylene, the corneal slides were hydrated through graded ethanol and incubated for 30 min in 0.3% Hpost hoc test was used for multiple comparisons. Differences between groups were considered statistically significant for p-values <0.05.Statistical analysis was performed by GraphPad prism 7 . The data generated by all experiments are reported as mean ± SD. One-way analysis of variance (ANOVA) was carried out, and Tukey's p < 0.01) reduction of the average wound area as compared to control. However, starting from 36 h until to 72 h, the corneal cells exposed to F3, F4, and F6 showed the best performance in terms of wound closure compared to control group and the other formulations . No significant differences were observed between F3, F4, and F6. These findings suggest that these formulations exert comparable positive effects on the wound healing rate in SIRC cells. We then analyzed the effect of F3 against oxidative stress induced by treatment with H2O2 (0.3 mM) for 30 min using a DCFDA assay. To assess the role of vitamins contained in F3, we also tested F6, containing similarly to F3, 0.32% SH-CL and 0.5% Tau, but no vitamins. As shown in p < 0.05) increased in SIRC cells after H2O2 treatment compared to control. The treatment with F3 and F6 significantly (p < 0.05) reduced ROS formation after H2O2 stress.We performed wound healing assay to evaluate the impact of the formulations in the wound repair capability of SIRC cells. As shown in 2O2 treatment for 30 min alone or in cells previously treated for 60 min with F3 or F6, using the cytoplasmic probe, DCFDA. *p < 0.05 vs. CTR; #p < 0.05 vs. H2O2 as determined by one-way ANOVA followed by Türkiye’s multiple comparison test. Data are shown as mean ± SD of five independent experiments (n = 5).ROS levels were measured in SIRCs after 0.3 mM Hin vivo study was to evaluate the effects of RenerviX® on corneal wound healing and to evaluate the expression and the localization of regenerated nerve fibers after corneal abrasion in rabbit eye. As showed in ®) containing 0.32% SH-CL, 0.5% Tau, 0.05% Vit B6 and 0.05% Vit B12; Formulation 1 (F1) containing 0.5% SH-L, 0.5% Tau and 0.5% Vit B12] contribute to the corneal wound healing even though no statistical differences were observed between treated groups (The aim of the d groups .p < 0.05 and p < 0.01 vs. CTR). However, the extent of re-innervation was significantly greater in the eye of rabbits treated with RenerviX® compared with the group control and the groups treated with formulations containing 0.32% SH-CL and Tau (F6) and formulation containing 0.5% SH-L, 0.5% taurine and 0.5% vitamin B12 (F1) (p < 0.05 vs. F6 and F1).Corneal re-innervation was examined by immunohistochemical analysis of the selective neuronal marker, beta-III tubulin after mechanical injury. As shown in ®, was able to improve corneal wound healing, to restore functional corneal nerves, and to protect corneal cells from oxidative stress. Treatment with RenerviX® stimulates re-innervation of the injured cornea in rabbit eye with a significant difference when compared to formulation 6 and formulation 1. Finally, no levels of taurine, pyridoxine (vit B6) and cyanocobalamin (vit B12) were detected after 18 h in the cornea samples of rabbit eyes treated with a single instillation of RenerviX®, suggesting that no deposit of these molecules occurred after topical administration. Altogether, these findings suggest that pyridoxine (vitamin B6) present in RenerviX®, significantly contributes to the corneal preservation and recovery after an insult.In the present study we demonstrated that RenerviXPrevious studies demonstrated the importance of vitamins in maintaining ocular surface homeostasis, suggesting the possible protective effects against damages . VitaminMoreover, Vit B6 has been recognized as a potent antioxidant as well as an established cofactor for several metabolic enzymes, including, among others, those involved in protein metabolism, conversion of tryptophan to niacin, and neurotransmitter function .The role of Vit B6 as a therapeutic agent has been demonstrated in several disorders such as diabetes  and cardIn addition, Vit B6 is involved in the immune system regulation and the regulation of neurotransmitters . Being e®), demonstrated to better contrast oxidative stress, to accelerate corneal re-epithelization and to promote nerve regeneration, suggesting an important advantage in clinical practice, ranging from corneal abrasion and/or neuropathy (diabetes or severe dry-eye) to help patients’ recovery after eye surgery.In conclusion, among the formulations tested, the new ophthalmic gel based on 0.32% SH-CL, 0.5% taurine, 0.05% vitamin B6 and 0.05% Vitamin B12 (RenerviX


Salmonella is an important pathogen associated with many foodborne disease outbreaks that can cause serious issues regarding public health, economic conditions, and quality of life, among others. Meat is the main human infection route for this bacterium, making food quality control in all production steps paramount. As Salmonella is a mesophilic bacterium, the cold chain is very important during meat processing, so pathogen behavior studies under cool storage, simulating the industry environment, can provide important data to the food industry. In this context, the aim of the present study was to perform a systematic review and meta-analysis of Salmonella behavior in meat during cool storage. Other conditions were also analyzed, such as meat sources , fish, shellfish, media broth, package types, storage time, and bacterial inoculation (concentration and inoculation type).Salmonella behavior in meat stored in cool conditions (between 0 °C and 7.5 °C), by employing a systematic review and meta-analysis. The data were obtained from research articles published in SciELO, PubMed, the Web of Science, and Scopus databases. The results of the retrieved studies were obtained from meat , fish, shellfish, and broth media samples The data were extracted as sample size (n), initial concentration (Xi), final concentration (Xf), standard deviation (SD), standard error (SE), and microbial behavior effects (reduction or growth). A meta-analysis was carried out using the metaphor package from R software. A total of 654 articles were initially retrieved. After applying the exclusion criteria, 83 articles were selected for the systematic review, and 61 of these were used for the meta-analysis. Most studies were conducted at 0 °C to 4.4 °C storage temperatures under normal atmosphere package conditions. Salmonella Typhimurium, S. Enteritidis, and a cocktail (strain mixture) were inoculated at 5.0 and 6.0 log CFU mL−1. Articles both with and without the addition of antimicrobial compounds were found. Salmonella concentration decreases were observed in most studies, estimated for all study combinations as −0.8429 ± 0.0931 log CFU g−1  (p < 0.001), varying for each subgroup analysis. According to this survey, Salmonella concentration decreases are frequent during cool storage, although concentration increases and no bacterial inactivation were observed in some studies.The aim of the present study was to investigate  Salmonella is an important pathogen, responsible for food disease outbreaks and termed salmonellosis. This microorganism can be found in the gastrointestinal tract of farm animals and can contaminate carcasses and meat when slaughter is carried out under inappropriate conditions.This pathogen was responsible for 21.3% of the 2627 foodborne outbreaks that occurred between 2007 and 2019 in Brazil. It is important to highlight that 8998 outbreaks during this period were related, but the causative agents of only 29% were identified [Salmonella [Salmonella Enteritidis being the most common serotype reported [About 1.35 million illnesses, 26,500 hospitalizations, and 420 deaths occur each year in the United States due to foodborne outbreaks caused by Salmonella, such as meat [Salmonella can also be present in processing environments  [Salmonella contamination frequencies in Brazilian cattle carcasses and on their surfaces as 6.7% (6/90) and 2.6% (7/270), respectively [Many food types can be contaminated by  as meat , fish an as meat ,6, and f as meat ,8. Salmoandlers) . One stus 6.7% 6/0 and 2.6It is essential to understand microorganism behavior in food to ensure food quality. Mathematical models have been developed in this regard to predict microbial risks in food products, describing microbial inactivation or growth according to intrinsic and extrinsic factors ,11.S. enterica behavior inoculated in poultry meat stored at 6 °C ± 2 °C for 35 days, reporting a 2.0 log CFU g−1 increase during the first seven days, followed by a 4.0 log CFU g−1 decrease at the end of the experiment. At 2 °C ± 2 °C, the concentration remained constant for two days, decreasing, thereafter, to 1.0 log CFU g−1, until undetectable from 25 to 35 days. Viable Salmonella concentrations have also been observed in frozen ground beef stored under a normal atmosphere for 5 to 75 days, following thawing in a refrigerator at 4 °C for 16 h [Storage temperature is a critical point of control in the food industry, as pathogens can grow at temperatures higher than 5 °C. One study  analyzedfor 16 h .−1 decrease in Salmonella Typhimurium concentrations in ground pork meat stored at 4 °C for 7 days [p < 0.05). Olive extract also significantly affect S. Typhimurium. In another study, a 5.0 to 4.0 log CFU mL−1 decrease in Salmonella concentrations was observed in Muller Hilton broth containing 0.5% malic and acetic acid addition at 4 °C for 21 days [Salmonella concentrations has also been observed in vacuum-packed ground beef stored at 3 °C for 12 days [−1 were observed for S. Typhimurium in vacuum-packed turkey meat treated with rosemary and oregano essential oils stored at 4 °C for 21 days [S. Typhimurium concentrations in poultry meat containing 0.2% rosemary essential oil stored in modified-atmosphere packaging at 4 °C for 7 days.Beyond the cold chain, the food industry has used natural compounds in the control of spoilage and pathogenic microorganisms. The addition of 0.5% cinnamon essential oil, for example, caused a 0.62 log CFU gr 7 days , with hi 21 days . In anot 21 days . A decre 12 days . Decreas 21 days . Kahrama 21 days , on the Salmonella behavior in meat during refrigeration storage. Therefore, a systematic review and meta-analysis of Salmonella behavior in meat and its derivates under cooling storage and several environmental conditions, such as package type and antimicrobial compound addition, was carried out herein.These findings demonstrate discrepant results on https://osf.io/8ayu2, accessed on 29 September 2022) under the doi registration https://doi.org/10.17605/OSF.IO/8AYU2.The systematic review methodology has been registered on OSF (Open Science Framework) platform   in meat , fish, shellfish, and in broth media were the specified populations. The measured outcome was derived from pathogen concentrations detected after cool storage.Initially, three important observations were defined, namely population (sample), intervention or treatment, and measured outcome. Cold storage effects on Salmonella) AND (Meat) AND ((growth) OR  OR (Kinetic)) AND ((cold AND storage) OR (chill) OR (shelf AND life) OR (refrigerat*))”. No restriction filters were applied, and the terms were searched in retrieved paper titles, abstracts, and keywords.The research was performed in the SciELO, PubMed, Web of Science, and Scopus databases, using the following terms as a string: “  was usedSelected articles should be published in English or Spanish. Reviews, book chapters, and articles that did not use bacterial inoculation were excluded.Salmonella concentrations should be reported as log CFU per g, mL, or cm2. Experiments with results in MPN (most probable number) were excluded. As a fourth inclusion criterion for meta-analysis, the primary study must clearly describe the sample size and the standard deviations or errors for means. Several studies that did not report standard deviations or errors were used only for the systematic review. Experiments using ozone, high pressure, irradiation, and combined antimicrobial agents were excluded from the systematic review and meta-analysis. The selected articles were categorized as (1) no use of antimicrobial compounds and (2) use of antimicrobial compounds.A second selection was performed by reading the titles and abstract, followed by full article reading. The third criterion was the use of an approved microbiological method for pathogen enumeration. Salmonella strain (subspecies or cocktail), inoculum concentration , inoculation type (surface and mixture), time (day) and temperature (°C) of storage, sample size (n), initial concentration (Xi), final concentration (Xf), standard deviation (SD), standard error (SE), and effect (reduction or growth) on microorganism behavior.Data of interest described in the articles were organized into spreadsheets by one reviewer. The extracted information included authors, year, sample type (meat or culture medium), package condition , antimicrobial type, antimicrobial concentration, forms of antimicrobial application, Gimp 2.10.8 (GIMP team)  and ImagEffect size was determined by the raw mean difference between the initial and final concentrations, as all the primary studies were reported on a log CFU scale. Each treatment was considered an individual observation.j, the effect size θ is the difference (RawDiff) in the sample means log reduction (R) or growth (G). Equation (1) was used for experiments without the addition of antimicrobial compounds, and Equations (2) and (3) were used for experiments with the addition of antimicrobial compounds.Xi, Xci, and Xti are the means of the initial concentration, initial concentration in the control samples, and initial concentration in the treated samples, respectively, while Xf, Xcf, and Xtf are the means of the final concentration, final concentration in the control samples, and final concentration in the treated samples, respectively.Considering a primary study Sdf and Sdi are the final and initial standard errors, respectively, nf and ni are the numbers of final and initial samples (repetitions), respectively.The variance of mean log reduction or growth (VarRawDiff) was estimated as:SE), this was transformed into SD through Equation (5).When the article only provided the standard error , package conditions , inoculum concentrations, and binomial time versus temperature. Concentration decreases were observed in most studies, but a pathogen concentration increase or no changes were also noted.n = 61 articles/292 treatments) indicated high heterogeneity among studies , and the combined effect size was reduced by −0.8429 ± 0.0931 log CFU g−1 . The combined meta-analysis result (k = 292 treatments) presented a significant effect (p < 0.001), demonstrating that cool temperatures can control and decrease Salmonella concentrations.The first meta-analysis with all the retrieved data , vacuum packages (VC: 34 treatments from 16 articles), and modified atmosphere packages (MAP: 27 treatments from 8 articles)  were more commonly applied compared to temperatures between 5 °C and 7.5 °C (22.03%), probably because the retrieved studies aimed to observe −1 inoculum concentration (25.61%), and Salmonella Typhimurium strains (32.23%) , antimicrobial compound addition (38.84%), normal packaging (53.99%), more than 10 storage days (47.10%), 6 log CFU mL(32.23%) .Beef (24.4%), chicken (18.18%), and pork (12.95%) were the major samples used at 0 °C to 4 °C storage temperatures. Under these conditions, studies were performed using antimicrobial compounds (38.84%), normal atmosphere (53.99%), and storage times of less than 10 days (47.10%).Salmonella Typhimurium was the most inoculated strain (32.23%), and the most frequent inoculum concentrations were 5, 6, and more than 6 log CFU mL−1.In studies conducted at 0 °C to 4 °C, S. Typhimurium strains (7.43%) at a 3 log CFU mL−1 inoculum concentration (7.71%).At 5 °C to 7.5 °C storage temperatures, beef was the most common sample (13.49%), with antimicrobial compound addition (9.36%) and normal atmosphere packing (16.52%), during 11 to 35 storage days using cocktail and −1 in the control (18.73%) and treated (11.29%) groups at 0 °C to 4 °C storage were the most frequent. A reduction of more than 2 log CFU g−1 was also observed at both temperatures. However, about 19.28% of the analyzed treatments presented growth rates of 5.23% and 3.58% at 0 °C to 4 °C, respectively.As a result, decreases lower than 1 log CFU g−1 and 21.48% decreases between 1 log CFU g-1 to 2 log CFU g−1 in pathogen level. From 5 °C to 7.5 °C, the main results were 5.78% reduction <1 log CFU g−1 and 3.03% reductions both >1 log CFU g−1 and >2 log CFU g−1.At temperature intervals from 0 °C to 4 °C, the control group treatments presented 18.73% decreases less than 1 log CFU gSalmonella decreases in most cases, although growth-rate values were noted in all conditions.Most articles analyzed bacterial behavior under a normal atmosphere and demonstrated pathogen reduction. Experiments with vacuum packing and MAP also resulted in Salmonella concentrations, some indicated increased pathogen concentrations even under cool storage. One study, for example, observed a reduction between 1.4 and 1.9 log CFU g−1 in Salmonella concentrations in ground beef packed under a vacuum and in a modified atmosphere at 3 °C for 12 days [Salmonella -concentration decreases were detected in modified-atmosphere packed (MAP) beef stored at 7.5 °C for 12 days, although a 1.69 log CFU g−1 pathogen reduction was observed in vacuum-packed beef in the same study [Although most selected studies reported decreases in  12 days . In anotme study .Salmonella concentrations was observed in chicken samples containing 2% acetic acid at 2 °C, 6 °C, and 8 °C for 9 days, with reduction values of 0.7, 0.9, and 0.9 log CFU/g, respectively [Salmonella Typhimurium growth was observed in vacuum-packed minced chicken treated with olive oil and stored at 2 °C for 60 days [A decrease in ectively . However 60 days .Salmonella concentrations in vacuum-packed whole shrimp with potassium sorbate, sodium benzoate, sodium diacetate addition and control were observed at 4 °C for 7 days, ranging from 0.93 to 1.84 log CFU g−1 [−1 in shrimp inoculated with S. Typhimurium, S. Enteritidis, and S. Infantis stored at 4 °C for 2 days. The authors, however, also observed growth of approximately 0.10 and 0.13 of the same Salmonella strains in the same storage conditions.Increasing  CFU g−1 . Edwards CFU g−1  also repSalmonella concentration decreases were observed with malic and acetic acid addition in Mueller Hilton broth stored at 4 °C for 21 days [−1 in Salmonella Typhimurium concentrations were detected in BHI broth stored at 4 °C and 7 °C, respectively, for 7 days [−1 using chitosan coating in inoculated beef stored at 4 °C for 3 days. Salmonella concentrations were also reduced by approximately 1.8 and 1.6 log cycles following the addition of 0.3% carvacrol and thymol essential oils in beef stored at 4 °C for 7 days [Regarding challenge tests with antimicrobial compounds,  21 days . In anotr 7 days . Silva er 7 days  reportedr 7 days .Salmonella cocktail concentrations, although an increase was observed in the control treatment of 0.8 log CFU g−1 bacterium concentrations [Salmonella Typhimurium concentration reduction higher than 3 log CFU g−1 was reported for minced meat stored at 4 °C for 7 days [Salmonella Typhimurium concentrations in modified-atmosphere-packaged beef, with or without marination, stored at 5 °C for 19 days of 0.6 and 2.90 log CFU g−1 in control and treated samples, respectively.A study comprising lactic acid or acidified sodium chlorite addition in minced beef stored at 5 °C for 14 days reported a 0.031 to 0.264 log-cycle reduction in trations . A Salmor 7 days . Nisiotor 7 days  also ind−1, demonstrating result variability and the absence of publication bias.The funnel plot graph  presents-1 , and a reduction of −0.8943 ± 0.1880 log CFU/g  was found for the pork samples. However, studies on chickens indicated a reduction of −0.1639 ± 0.1994 log CFU g−1 , although the pathogen growth rate was observed within a sample confidence interval (p = 0.4111) according to the combined meta-analysis result.The beef sample effect was −0.9951 ± 0.1524 log CFU gp < 0.001) in the meta-analyses results were presented in experiments using beef, pork, turkey, and fish.For turkey and fish samples, reductions of −1.0140 ± 0.1495 log CFU/g  and −1.8090 ± 0.1924 log CFU/g  were observed, respectively. Significant effects (p < 0.001), but antimicrobial addition was reduced by −1.2041 ± 0.1559 log CFU g−1 , while in the control group or in treatments without antimicrobial compounds, a −0.4526 ± log CFU g−1  was observed. This demonstrated the importance of studying antimicrobial compound effects, mainly those of natural compounds, against Salmonella in meat.The second subgroup comprised antimicrobial compound addition, categorized into groups with (152 treatments/37 articles) and without (142 treatments/55 articles) antimicrobial compound addition compounds or controls. Both studies demonstrated a significant effect  (p < 0.001) and −0.8604 ± 0.3273 log CFU g−1  (p < 0.01). No significant effect was observed for vacuum-packaged samples (p = 0.3934), with a combined value of −0.2506 ± 0.2936 log CFU g−1 .The normal atmosphere and modified atmosphere packaging displayed more significant Salmonella concentrations was observed in both temperature intervals, albeit with a difference in p values and higher pathogen concentration decreases at lower temperatures.A decrease in −1   decrease, while Salmonella concentrations at 5 °C to 7.5 °C were reduced by −0.5742 ± 0.1955 log CFU g−1  . Temperatures from 0 °C to 4 °C promoted more significant pathogen concentration decreases, with a statistically significant effect.The general effect at 0 °C to 4.4 °C temperature storage was a −0.9217 ± 0.1063 log CFU gSalmonella concentration reduction was observed for fish, under both normal and MAP atmospheres, with antimicrobial compound addition and stored between 0 °C and 4.4 °C.Concerning all subgroups, a higher Salmonella concentration decreases. At 0 °C to 4 °C, only vacuum-packaged pork samples with up to 4 log CFU mL−1 pathogen concentration treatments presented no significant statistical effect, and, at 5 °C to 7.5 °C, these effects were observed in less than half of the studies.As expected, decreases were noted in all analyses except for treatments using broth samples and MAP packages at 5 °C to 7.5 °C , with treatments analyzed at 0 °C to 4 °C  presenting more significant −1 decreases at intervals 1 and 2, respectively. When considering studies with K > 60, beef samples and antimicrobial compound addition treatments exhibited more significant effects, with −1.24 and −1.25 log CFU g−1 values, respectively.The more significant effects in both temperature intervals were observed for studies employing fish samples, with −1.93 and 1.34 log CFU g−1 (p < 0.001) decrease at interval 1 and a −0.59 log CFU g−1 (p < 0.05) decrease at interval 2. In addition, this same condition was observed in many other results. Only studies on fish presented the same statistical effect at both temperature intervals (p < 0.001). It is important to note that chicken samples did not present significant effects at either temperature interval.Thus, differential statistical effects were observed between treatments according to storage temperature intervals. For example, beef samples presented a −1.2 log CFU gSalmonella growth at interval 2. Decreases in Salmonella concentrations were lower for treatments using the S. Typhimurium and S. Enteritidis mixture inoculation type, up to 4 log CFU g−1 for 10 days, compared to interval 1 treatments.Experiments without antimicrobial compound addition and under vacuum packaging and MAP packaging are less effective in controlling Salmonella concentration decreases were observed in meat under cool storage, which were higher at lower storage temperatures. According to this meta-analysis, other factors also contribute to Salmonella concentration decreases during refrigerated storage time, such as beef and fish samples, normal atmosphere and MAP, antimicrobial compound addition, and storage from 0 °C to 4.4 °C.Salmonella growth control, where decreases in meat between −0.15 and −1.24 log CFU g−1 were observed at colder temperatures, demonstrating the importance of cold chains for both the industry and customers.Cool storage was effective for Salmonella decreases during cool storage, the pathogen was not eliminated in the samples. Thus, the risk of salmonellosis transmitted by meat remains and should be considered.Although most experiments reported


CS-QT-BS3opt1 exhibited significantly (P < .05) high flux, i.e. 4.20-fold more than pure QT dispersion and 1.27-fold higher than QT-BS3opt. CS-QT-BS3opt1 showed significantly greater bio-adhesion (76.43 ± 2.42%) than QT-BS3opt (20.82 ± 1.45%). The antioxidant activity showed that QT from CS-QT-BS3opt1 has more remarkable (P < .05) antioxidant activity at each concentration than pure QT. The CS-QT-BS3opt1 exhibited 1.61-fold higher cytotoxicity against MFC7 and 1.44-fold higher cytotoxicity against MDA-MB-231 than pure QT. The CS-QT-BS3opt1 displayed a significantly greater antimicrobial potential against E. coli than against S. aureus. From all these findings, it could be concluded that surface-modified QT-BS might be an effective approach for increasing the efficacy of QT in the treatment of certain ailments.Quercetin (QT) is a flavonoid that exhibits anti-oxidant and chemo-preventive activity. This research work aimed to develop surface-modified bilosomes (BS) of QT. The BS was prepared by the solvent evaporation method and optimized by the Box-Behnken design. The optimized QT-BS (QT-BS3opt) displayed vesicle size (143.51 nm), PDI (0.256), zeta potential (−15.4 mV), and entrapment efficiency (89.52%). Further, the optimized QT-BS formulation was coated with chitosan (CS). The XRD diffractogram of CS-QT-BS3opt1 did not exhibit extensive peaks of QT, revealing that QT is properly encapsulated in the polymer matrix. The QT-BS3opt and CS-QT-BS3opt1 exhibited sustained-release  up to 24 h with the Korsmeyer-Peppas kinetic model (R Li and his associates formulated QT-SLN with a 571.4% enhanced relative bioavailability as compared to pure QT  is an herbal natural bioactive compound belonging to the polyphenol flavonoid category. It is obtained from various sources like citrus fruit, vegetable, and beverages , chloroform, methanol, and acetonitrile were procured from Sigma Aldrich . Span 60 and sodium deoxycholate (SDC) were acquired from SD fine Chemicals . 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ABTH were obtained from Sigma Chemical Co. . The rest of the chemicals utilized in this experiment are of analytical grade.The quercetin (QT) loaded BS formulations were optimized using 3-factors and 3-levels Box-Behnken experimental design software . The independent factors were lipid, span-60, and SDS, while the dependent variables were vesicle size , entrapment efficiency , and polydispersity index (PDI). The QT-loaded BS (QT-BS) was developed by the solvent evaporation method with a slight modification of the previously reported method  was used to determine the size, PDI, and zeta potential of developed QT-BS vesicles. The QT-BS was diluted twenty times using milli-Q water and measured for VS and PDI at a 90° scattering angle. For zeta potential, the same diluted sample was filled into an electrode cuvette and subjected to the measurement of zeta potential.The ultracentrifugation method was employed for the estimation of EE. The QT-BS was placed in a centrifugation tube and centrifuged at 15,000 rpm  for 30 min. The supernatant was collected and diluted appropriately. Absorbance was measured by UV-Vis-spectrophotometry  at 370 nm. The given The optimized QT-BS formulation was coated with CS. The required quantity of CS (0.20–0.30%) was weighed and dissolved in 0.1% v/v of aqueous acetic acid solution  was determined by the zeta sizer .○–40○. The instrument was operated at 40.0 kV voltage and 30 mA current, with a scanning speed of 2.0 deg/min.The X-ray diffractogram of QT, CHO, SDC, QT-BS, CS, and CS-QT-BSopt was captured by the X-ray diffraction instrument . Each sample was placed individually into a sample holder, converted into a thin film, and placed into an instrument. The image was captured at 2 theta level after scanning the sample at 5The dialysis bag method was used to evaluate in-vitro drug release of QT from QT-dispersion, QT-BS, and CS-QT-BS-dispersion. To open the pores, the dialysis bag was immersed overnight in distilled water. The release medium  was poured into a beaker and maintained at 37 ± 0.5 ○C using a thermostat magnetic stirrer. The 1 ml of QT-dispersion, QT-BS, and CS-QT-BS dispersion  were filled into a dialysis bag and bound tightly. After that, the dialysis bag was immersed in a release medium and constantly stirred at 100 rpm. At a predetermined time point, 3 ml of the sample was taken from the beaker and added to the same volume of fresh media in the beaker to maintain a constant volume. The release sample was filtered with a membrane filter (0.25 m) and the concentration of QT was determined using the previously reported HPLC method ; Mt & M∞ = amount of drug release at time t and infinitive time; M0 = amount drug at zero time; Mt = amount of drug remaining at time t; k = Korsmeyer-Peppas an dHixon-Crowell constant; n = diffusion exponent for releases mechanism; n = 0.45 indicated Case1 or fickian diffusion; 0.45 < n < 0.89 = anomalous behavior or non-fickian diffusion; n = 0.89 (Case 11 transport) and n > 0.89 = super case II transport  and stood for 4 h at room temperature. After finishing the incubation period, the sample was centrifuged at 15,000 rpm for 30 min, and the supernatant was isolated. Free mucin concentration in the supernatant was observed by UV-spectrophotometry at 251 nm  was taken as a control. The percent antioxidant activity of the tested sample was calculated by the following The study was performed as per the previously described method with a small modification . The various concentrations of QT and optimized CS-QT-BS (10–150 µg/ml) were prepared and 0.1 ml of each dispersion was mixed with 3.9 ml of ABTS solution and vortexed. The mixture was incubated for 30 min in a dark place and analyzed by UV-Vis spectrophotometry at 734 nm. The BTH solution is taken as a blank for comparative analysis. The percentage of scavenging activity was calculated by the following formula 10  and maintained a pH of 7.4  were seeded into a 96-well sterile microplate and allowed to stand overnight. The cells in the 96-well plate were treated with each concentration of QT and CS-QT-BS for 48 h. 0.05 mg/ml of MTT was filled into each well of the 96-well plate and incubated at 37○C for 2 h with a continuous supply of 5% CO2 and 95% O2. After that, DMSO (100 µL) was added to a 96-well plate to dissolve the formazan crystal. Finally, a microplate reader was utilized to evaluate the mixture, with blank DMSO serving as a control. The IC50 and the percentage of cells inhibited were calculated by the given The MTT assay method was used to test the cytotoxicity of pure QT and optimized CS-QT-BS on breast cancer cell lines (MCF-7 and MDA-MB-231). The cell line was obtained from an American culture type . MCF-7 and MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 U/ml streptomycin, and 100 U/ml penicillin. The cell was grown at 37 °C/95% RH with a continuous supply of 5% COStaphylococcus aureus  and Escherichia coli  microbial strains using the cub plate method. The culture was grown in nutrient broth media equivalent to 5 × 106 CFU/ml bacterial load. The nutrient agar medium was prepared and sterilized at 121○C in an autoclave. The 0.5 ml (diluted) of microbial strain was mixed with liquid nutrient agar media and transferred to a sterile Petri plate under aseptic conditions and allowed to stand for solidification. After solidification, the 6 mm cup was made using a sterile stainless-steel borer. The test sample  was filled into each cup and stood for 2 h for absorption of the test sample. Then a Petri plate was placed into the incubator in an inverted position at 37○C for 24 h. The zone of inhibition (ZOI) was then measured using a graduated scale.The antimicrobial evaluation of pure QT-dispersion and CS-QT-BS was done on P < .05.Experimental design software (version 8.0.6) was used for the optimization of the formulation. The value was represented in mean ± SD. GraphPad  was used for statistical analysis. One-way ANOVA and Tukey-Karman multiple comparison tests were used for data analysis. Significant differences were taken at 2, predicted R2, adjusted R2, standard deviation, and PREES) of all applied models were calculated and depicted in 2 of the best-fitted model was greater than that of other models  revealing that the model was well fitted. The response plot (3D and contour) was created for all best-fitted models to explain the effect of the independent variable on the response . The thirteen formulations were obtained in different compositions and the values of their responses were determined and are depicted in r models . The PREresponse  as well response . HoweverThe quadratic model was found to be the best fit model for VS. The polynomial equation was given below:2, and C2 exhibited a significant effect (P < .05) whereas B2 showed an insignificant (P > 0.05) effect on VS. P < .0001) fitted than the other models because it had the highest R2 (0.9996). The PRESS value of the quadratic model was significantly less (44.92) than the other models (P > 0.05), which gave favor to the fitted quadratic model. The adequate precession was > 4 (173.82), representing a satisfactory signal to the fitted model. The predicted R2 (0.9991) was in realistic agreement with the adjusted R2 (0.9992). The response surface plot (3D &contour) was plotted and depicted in In this polynomial equation, the positive and negative signs represented the favorable and unfavorable effects on VS. The model terms A, B, C, AB, AC, BC, Ar models . The ANOr models . The lacThe polynomial equation of EE was constructed, which explained the effect of the formulation variable over the response.P < .05), whereas AC showed an insignificant (P > 0.05) effect on EE. The statistical analysis of EE was applied in the linear, second order, and quadratic, and the results are given in P < .0001) fitted to EE than other models because R2 was found to be high (0.9969). The PRESS value of the quadratic model was significantly less (12.37) than the other models (P > 0.05), which is favorable to the fitted 2nd order (2F1) model. The adequate precession was > 4 (79.86) and represented a satisfactory signal to the fitted model. The predicted R2 (0.9902) was in realistic agreement with the adjusted R2 (0.9950). The response surface plot (3D & contour) of 2nd order (2F1) was plotted and depicted in The model terms A, B, C, AB, and BC exhibited a significant effect .2, B2, and C2 exhibited a significant effect (P < .05), whereas BC showed an insignificant (P > 0.05) effect on PDI. The statistical analysis of PDI was applied in linear, 2nd order, and quadratic, and the results are given in P < .0001) fitted to PDI than other models because R2 was found to be high (0.9992). The PRESS value of the quadratic model was significantly less (0.0001) than the other models , which favors the fitted model. Adequate precession is > 4 (114.77), representing a satisfactory signal to the fitted model. The predicted R2 (0.9902) is in realistic agreement with the adjusted R2 (0.9950). The response surface plot (3D & contour) of the quadratic was plotted and depicted in The model terms A, B, C, AB, AC, Ar models . The ANOr models . The lacThe optimized QT-BS formulation was selected on the basis of small VS, low PDI, and optimum EE. The QT-BS3 formulation was selected as an optimized formulation (QT-BS3opt). The composition of QT-BS3opt was 4% lipid, 7% Span-60, and 8% SDC. The actual value of QT-BS3opt had a VS of 143.51 nm, an EE of 89.52%, and a PDI of 0.256. The predicted value of QT-BS3opt obtained from point prediction software was 145.32 nm of VS, 88.99% of EE, and 0.258 of PDI, respectively. It was observed that a very small percentage of the variation between the actual and predicted value of the responses. The QT-BS3opt formulation was used for further analysis.The optimized QT-BS (QT-BS3opt) has a VS of 143.51 nm , PDI of The QT-BS3opt was successfully coated with natural bio-adhesive chitosan (CS) polymer. The different CS-coated QT-BS formulations are given in The VS and PDI of various CS-coated formulations (CS-QT-BS3opt1-CS-QT-BS3opt3) were analyzed by using a zeta-sizer and the result is depicted in ○, 12.2○, and 27.2○, respectively  and is selected as the best-fitted model. The exponent value is 0.495 and it lies between 0.45<n < 0.89, indicating anomalous behavior or non-fickian diffusion of the drug from the nanoformulation matrix.The release of QT from pure QT-dispersion, QT-BS3opt, and CS-QT-BS3opt1 was determined by using the dialysis bag technique, and the result is depicted in P < .001) than the QT-BS3opt due to the positive charge of CS bound with the negative charge of mucin . The percentage of bio-adhesion represented the amount of mucin bound to the formulation. The bio-adhesion of QT-BS3opt and CS-QT-BS3opt1 was calculated and found to be 20.82 ± 1.45% and 76.43 ± 2.42%, respectively. The CS-QT-BS3opt1 exhibited significantly higher bio-adhesion  high flux (permeation), i.e. 4.20-fold more than pure QT dispersion and 1.27-fold higher than QT-BS3opt. The APC of pure QT-dispersion, QT-BS3opt, and CS-QT-BS3opt1 was found to be 2.2 × 10−3 µg/sec, 6.6 × 10−3 µg/sec, and 8.7 × 10−3 µg/sec respectively. The higher permeation of QT-BS3opt1 than pure QT is due to the presence of bile salt (SDC), which enhanced the fluidity of the membrane  antioxidant potential at all concentrations than pure QT. The maximum activity of CS-QT-BS3opt1 was 93.24 ± 4.65% at 200 µg/ml than pure QT (52.84 ± 4.12% at 200 µg/ml). The high activity of QT in CS-QT-BS3opt1 may be due to the increased solubility of QT because of the presence of bile salt and CS.P < .05) higher activity at each concentration than pure QT. The QT showed maximum activity in pure form was 45.10 ± 3.34% at 200 µg/ml and in the form of CS-QT-BS3opt1 80.76 ± 4.05% at 200 µg/ml. The CS-QT-BS3opt1 showed higher antioxidant activity than pure QT due to an increase in the solubility of QT in the nano-system. The result showed that antioxidant activity by the ABTS method is less than that by the DPPH method.The result of the antioxidant activity of pure QT and CS-QT-BS3opt1 by the ABTS scavenging method is depicted in P < .05) activity against the MDA-MB-231 cell line at different concentrations, i.e. 10 µM (95.65%), 25 µM (88.51%), 50 µM (71.25%), 100 µM (55.02%), and 250 µM (42.12%) in 24 h, and 10 µM (94.23%), 25 µM (81.14%), 50 µM (54.82%), 100 µM (39.43%), and 250 µM (31.65%) in 48 h. The pure QT showed activity against the MFC7 cell line at different concentrations of 10 µM (99.24%), 25 µM (91.35%), 50 µM (77.43%), 100 µM (64.65%) and 250 µM (49.73%) in 24 h and 10 µM (98.23%), 20 µM (87.56%), 50 µM (63.02%), 100 µM (51.43%) and 250 µM (38.76%) in 48 h. However, CS-QT-BS3opt1 exhibited significant activity, i.e. 10 µM (95.34%), 25 µM (81.23%), 50 µM (61.65%), 100 µM (51.58%) and 250 µM (36.76%) in 24 h and 10 µM (93.02%), 25 µM (76.62%), 50 µM (47.7%), 100 µM (33.28%) and 250 µM (19.98%) in 48 h against the MFC7 cell line. In both cell lines, CS-QT-BS3opt1 had significantly higher cell cytotoxicity at each concentration than pure QT and control after 24 and 48 h. It revealed that cytotoxicity depends upon the concentration of QT. The maximum activity was found at 250 µM for both samples (QT and CS-QT-BS3opt1) in 24 and 48 h. As compared to pure QT, CS-QT-BS3opt1 showed 1.61-fold cytotoxicity against MFC7 and 1.44-fold cytotoxicity against MDA-MB-231 in 48 h. The IC50 of pure QT was 244.8 µM and 213.10 µM against MDA-MB-231 in 24 h and 48 h, respectively. In addition, the IC50 value of pure QT against MFC-7 was 221.60 µM and 115.70 µM at 24 h and 48 h, respectively. On the other hand, IC50 of CS-QT-BS3opt1 against MDA-MB-231 was 173.7 µM and 68.54 and against MFC-7 was 61.89 µM and 43.87 µM at 24 h and 48 h, respectively. CS-QT-BS3opt1 showed a lower IC50 concentration than pure QT. The result showed that QT is more cytotoxic against MFC-7 than CS-QT-BS3opt1. The higher activity of QT from CS-QT-BS3opt1 than pure QT might be due to an increase in the solubility owing to the presence of bile salt and Span-60 that were responsible for the accumulation of more QT at the cell site. Further, the coating with CS also helps to improve the anticancer activity by improving the bio-adhesion and opening the tight junction of the cancer cell  high antibacterial activity against both tested bacteria at both tested points (24 and 48 h) in comparison to pure QT. The ZOI of QT from CS-QT-BS3opt1 was found to be 14.65 ± 0.45 mm against S. aureus and 17.25 ± 0.50 mm against E. coli. However, CS-QT-BS3opt1 showed significantly more activity in 48 h, i.e. 17.54 ± 0.48 mm against S. aureus and 20.76 ± 0.42 mm against E. coli. The high activity of QT from CS-QT-BS3opt1 is due to the nano size of the vesicle, which provides a higher surface area for diffusion  and E. coli (Gram-negative) microbial strains using the cup plate method up to 48 h, and the results are expressed in In this work, the QT-loaded bilosome was prepared and optimized by Box-Behnken design. The formulation unveiled a nano-range of vesicle size, high entrapment efficiency, and low PDI. The result of XRD showed the amorphization of QT after incorporation into CH-BLOs. CS-QT-BS3opt1 exhibited sustained drug release up to 24 h with Korsmeyer-Peppas kinetic as a best-fitted model. CS-QT-BS3opt1 displayed better bioadhesion as well as better permeability flux as compared to QT-dispersion. CS-QT-BS3opt1 exhibited a greater antibacterial potential against E. coli than S. aureus than QT-dispersion. As compared to pure QT-dispersion, CS-QT-BS3opt1 showed 1.61-fold cytotoxicity against MFC7 and 1.44-fold cytotoxicity against MDA-MB-231 in 48 h, indicating more QT cytotoxic against MFC-7 than CS-QT-BS3opt1. Finally, all of the findings suggest that CS-QT-BS3opt1 could be a viable technique for the increment of QT efficacy against certain diseases.


The aim of this study was to compare central and peripheral refraction using an open view Shin-Nippon NVision-K 5001 autorefractor and an open view COAS-HD VR aberrometer in young children.Cycloplegic central and peripheral autorefraction was measured in the right eye of 123 children aged 8 to 16 years. Three measurements each were obtained with both Shin-Nippon NVision-K 5001 autorefractor and COAS-HD VR aberrometer along the horizontal visual field up to 30°  in 10° steps. The refraction from the autorefractor was compared with aberrometer refraction for pupil analysis diameters of 2.5-mm and 5.0-mm.0 and J45 were within 0.15 D and the 95% limits of agreement within 0.90 D across the horizontal visual field.The Shin-Nippon was 0.30 D more hyperopic than COAS-HD VR at 2.5-mm pupil and 0.50 D more hyperopic than COAS-HD VR at 5-mm pupil for central refraction. For both pupil sizes, the 95% limits of agreement were approximately 0.50 D for central refraction, and limits were wider in the nasal visual field compared to the temporal visual field. The mean difference for both J0 and J45 were similar between instruments for both central and peripheral refraction.Defocus components were similar between the Shin-Nippon autorefractor and the COAS-HD VR aberrometer with a 2.5-mm pupil for most visual field angles. However, there was a significant difference in defocus component between the Shin-Nippon autorefractor and the COAS-HD VR aberrometer with a 5.0-mm pupil, wherein the autorefractor measured more hyperopia. The astigmatic components J This technique is considered reliable and accurate in the pediatric age.,,,Currently available instruments that measure objective refraction rely on different measurement principles. A commonly used instrument is the Shin-Nippon autorefractor  that relies on classical measurement principles to measure refractive error.Few studies have compared central refraction values in children obtained with autorefraction and with aberrometry. A study conducted from McCullough et al.There is currently limited information about the validity of peripheral refraction obtained with open view autorefractors or with aberrometers in children. Specifically, there are no studies comparing peripheral refraction in children when using the Shin-Nippon autorefractor and the COAS-HD VR aberrometer. Children are expected to have more variable fixation (e.g. more square wave jerks)Participants were recruited for a longitudinal study investigating the prevalence of refractive errors in Swedish schoolchildren. Details of the study protocol are available in our previous publication.The Shin-Nippon NVision-K 5001 autorefractor uses three ring segments of infrared light (λ= 840 nm) that has a diameter of 2.32 mm. Therefore, the required minimum pupil size for measurement is 2.32 mm. Even though participants may have a larger pupil, the Shin-Nippon autorefractor always computes refraction by analyzing 2.32 mm image of the ring segments. A more detailed description of the Shin-Nippon autorefractor is given in Davies et al.The open-field COAS-HD VR aberrometer  measures lower and higher-order monochromatic aberrations based on the Hartmann-Shack principle.0, and J45 for the COAS-HD VR aberrometer.02 = defocus, C22 = regular astigmatism, C22 = irregular astigmatism, and r2 = radius of pupil diameterC). In addition, to measure the agreement between the Shin-Nippon autorefractor and the COAS- HD VR aberrometer, it is appropriate to match the pupil diameter for analysis.,,,In both instruments, the vertex distance used to calculate refraction was set at the spectacle plane of 12-mm. Both instruments measure refractive error using infra-red light (840 nm) which then were converted to obtain refraction in the visible wavelength. The Shin-Nippon autorefractor converts to a wavelength of 547.06 nm  which was matched in the COAS-HD VR aberrometer.,Prior to cycloplegia, participants underwent a complete eye examination that included visual acuity measurement and subjective refraction. Cycloplegia was induced by instilling two drops of 1.0% cyclopentolate hydrochloride in both eyes. The mean photopic pupil diameter pre-cyclopentolate was 4.3 mm (SD=0.8). Post cyclopentolate, the mean pupil diameter was 7.7 mm (SD=0.6). Three consecutive measurements of the central and peripheral refraction of the right eye were randomly obtained, either with the COAS-HD VR aberrometer or with the Shin-Nippon autorefractor. A single examiner (PD) performed all measurements. During measurements, participants were instructed to look at a series of fixation targets placed at a 3-meter distance . We have0, and J45.,The three measurements obtained from both instruments were converted into vector components M, JOne hundred and twenty-three children aged 8 to 16 years having best-corrected acuity better than 6/7.5 (logMAR 0.1) participated in this study. The mean age of the participants was 12.0 years (SD=2.5 years), 67 were females (54.5%) and 56 were males (45.5%). The mean (SD) central refractive error determined by the Shin-Nippon autorefractor was +0.70 D (SD=1.23 D), range −3.29 D to +5.62 D. The mean central refractive error determined by the COAS-HD VR aberrometer using a 2.5-mm pupil was +0.39 D (SD=1.25 D), range −3.73 D to +5.11 D and when using a 5-mm pupil was +0.19 D (SD=1.20 D), range −3.92 D to +4.51 D. Subjective refraction is reported in Appendix 1.0, and 0.18 D for J45. For the COAS-HD VR and a 2.5-mm pupil, the CR was 0.24 D for defocus, 0.21 D for J0, and 0.14 D for J45. CR can be considered similar between Shin-Nippon autorefractor and COAS-HD VR using a 2.5-mm pupil for all three refraction components. CR was 0.12 D for defocus, 0.05 D for J0, and 0.07 D for J45 with the COAS-HD VR with a 5-mm pupil for all three refraction components. The most repeatable measurements were obtained with the COAS-HD VR for the 5-mm pupil diameter. The CR for central refraction for the Shin-Nippon was 0.29 D for defocus, 0.21 D for J0 (panel B) and J45 (panel C) across the horizontal visual field for the Shin-Nippon autorefractor and the COAS-HD VR aberrometer (for both 2.5 and 5-mm pupil diameter).p < .001. A post-hoc Tukey revealed that the defocus was significantly different in Shin-Nippon autorefractor when compared to COAS-HD VR aberrometer for both the 2.5-mm (p = .028) and 5-mm pupil diameter (p < .001). Defocus also changed significantly across eccentricity with F  = 33.47, MSE= 14.20, p < .001. The interaction between instrument and eccentricity was significant, F  = 3.94, MSE= 1.67, p = .02. We investigated the interaction by performing simple effects pairwise comparisons with Bonferroni adjustments. This confirmed the main effects, that is, defocus from the Shin-Nippon was significantly different across the entire horizontal visual field compared to the COAS-HD VR 5-mm pupil diameter. For 2.5-mm pupil diameter differences were statistically significant only at temporal 30°, nasal 20° and 30°.A mixed-design ANOVA revealed a statistically significant difference for defocus between the two instruments, F  = 8.32, MSE= 78.62, 0) differed significantly between the two instruments with F  = 8.32, MSE= 1.79, p = .019. A post-hoc Tukey revealed that J0 from the Shin-Nippon autorefractor was significantly different from the COAS-HD VR aberrometer for 2.5-mm pupil (p = .028) but not for 5-mm pupil (p = .25). J0 also changed significantly across eccentricity with F  = 1190.45, MSE= 122.44, p < .001 and revealed an expected temporal-nasal asymmetry.,,p < .001. Simple effects analyses with Bonferroni adjustments indicated statistically significant difference between Shin-Nippon and COAS-HD VR (for both pupil diameters) for J0 only for temporal 20° and 30°.Measurements of regular astigmatism (J45) differed significantly between the two instruments. A post-hoc Tukey revealed that J45 obtained with the Shin-Nippon autorefractor was significantly different from J45 obtained with the COAS-HD VR aberrometer for 2.5-mm (p < .001) and for 5-mm pupil (p < .001). J45 also changed significantly across eccentricity with F  = 5.37, MSE= 0.52, p = .006. The interaction between instrument and eccentricity was significant, F  = 5.37, MSE= 0.37, p = .006. Simple effects analyses with Bonferroni adjustments indicated that J45 from the Shin-Nippon was significantly different from the COAS-HD VR 5-mm pupil across the entire horizontal visual field. For the 2.5-mm pupil, differences were statistically significant only at temporal 20° and 30°.Irregular astigmatism . In Appendix 1, we also provide extra analysis of these measurements. Peripheral refraction agreements between the two instruments for the two different pupil sizes are summarized in The mean differences (bias) for defocus between Shin-Nippon and COAS-HD VR were 0.30 D for 2.5-mm pupil (panel A) and 0.50 D for 5-mm pupil (panel D). The bias for both J0 across horizontal visual field were within 0.90 D for 2.5-mm pupil and within 0.67 D for 5-mm pupil. The limits were within 0.50 D across the horizontal visual field except for the 30° nasal visual field for both pupil sizes. The 95% limits of agreement for the J45 across horizontal visual field were within 0.45 D for 2.5-mm pupil and within 0.41 D for 5-mm pupil. The limits were similar across the eccentricity except for the 20° temporal field in both pupil sizes.The 95% limits of agreement for defocus across horizontal visual field were within 1.10 D for 2.5-mm pupil and within 0.98 D for 5-mm pupil. The limits were wider in the nasal visual field compared to the temporal visual field for both pupil sizes. The 95% limits of agreement for the JIn this study, we compared central and peripheral refraction in children given by two open view instruments. We also evaluated the coefficient of repeatability of both instruments for central and peripheral refraction. We found that the best repeatability was obtained with the COAS-HD VR with a 5-mm pupil. Repeatability was worse for the Shin-Nippon autorefractor and the COAS-HD VR with a 2.5-mm pupil for both central and peripheral refraction. For central refraction, the Shin-Nippon autorefractor was 0.30 D more hyperopic than the COAS-HD VR aberrometer when using a 2.5 mm pupil and 0.50 D more hyperopic than the COAS-HD VR when using a 5 mm pupil. As given in Appendix 1, measurements from the COAS-HD VR considering a 5 mm pupil were the most consistent with subjective refraction. For both pupil diameters, the 95% limits of agreement were approximately 0.50 D for central refraction with wider limits in the nasal visual field compared with the temporal visual field for both sizes of pupil tested.The Shin-Nippon autorefractor showed an average variability of 0.29 D for defocus between measurements for central refraction. A previous study that investigated intersession repeatability using the Shin-Nippon NVision-K 5001 autorefractor showed a CR of 0.10 D for defocus in adults.,,,,The central defocus component in our study showed that the Shin-Nippon autorefractor measured more hyperopic central refraction than the COAS-HD VR aberrometer for both the pupil diameters analyzed, as well as the subjective refraction before cycloplegia (see Appendix 1). These results are in line with other authors comparing refractive values from autorefractors and aberrometers.0 and J45 were within 0.50 D for central refraction.,0 and J45. Even though there was a statistically significant difference, from a clinical perspective the difference can be considered negligible.In line with previous studies, the 95% limits of agreement between the compared instruments for the JThe Shin-Nippon autorefractor measured relatively more hyperopic peripheral defocus than the COAS-HD VR aberrometer. This finding is in agreement with Berntsen et al., who also reported that the Grand-Seiko WR-5100 K autorefractor produced more hyperopic refraction than the COAS-HD VR aberrometer in young adults.,Participants in this study were instructed to turn their eyes at the fixation target. The extraocular muscles might distort eye shape and thereby alter peripheral refraction with eye turns. However, previous studies reported no significant difference between peripheral refraction made with the eye turn and the head turn method.In conclusion, clinicians and researchers should be aware of the difference in measurements produced by the Shin-Nippon autorefractor and the COAS-HD VR aberrometer. This study points to the sources of the systematic difference between the two instruments that affect defocus values. Caution must be taken when reporting results from different instruments or when comparing results of studies conducted with instruments that rely on different measurement principles.


The soil hydro-physico-chemical properties, morpho-physiological responses, yield, and quality were measured. HA addition amended the soil structure by allowing rapid macroaggregate formation, decreasing bulk density and pH, and increasing porosity and electrical conductivity, thereby improving soil hydraulic properties. HA0.2 and HA0.4 additions improved growth, yield components, and grain minerals, resulting in higher grain yield by 28.3–54.4%, grain protein by 10.2–13.4%, wet gluten by 18.2–23.3%, and dry gluten by 23.5–29.5%, respectively, than HA0. Foliar application of ZnONPs or L-TRP, especially at higher concentrations compared to the control, noticeably recorded the same positive results as HA treatments. The best results were achieved through the integration of HA0.4 + ZnONPs100 or L-TRP0.5 to the tested nutrient-poor sandy soil. The interactive application of HA0.4 + ZnONPs100 or L-TRP0.5 and the use of mineral fertilizer, which is considered a surplus point in permaculture, can be recommended for sustainable wheat production in nutrient-poor sandy soil.Sandy soils (containing > 50% sand) are widely distributed worldwide and are characterized by their poor structure, low organic matter, weak hydraulic and nutritional properties, and low crop productivity. Using a 2-year pot experiment, in this study, we investigated the effects of humic acid (HA) as a soil amendment and study two plant growth stimulants (PGSs), zinc oxide nanoparticles (ZnONPs), and L-tryptophan (L-TRP), as a foliar application on wheat grown in nutrient-poor sandy soil. Three HA rates (0 (HA Triticum aestivum L.) is the world’s second most cultivated cereal crop, which supplies approximately 20% of the total world cereal requirements  form. Zn is an activator of ~ 300 photosynthetic and metabolic enzymes, such as DNA- and RNA-polymerase, superoxide dismutase, and carbonic anhydrase (CA), which are involved in the photosynthesis and metabolism of carbohydrates, lipids, and nucleic acids , such as zinc oxide nanoparticles (ZnONPs) and L-tryptophan (L-TRP), to boost crop yield in normal and defective nutrient-deficient soils has recently received much attention , chemically known as L-d et al.  found th To the best of our knowledge, no study has yet assessed the collaborative effect of soil-applied HA with exogenous foliar L-TRP or ZnONP application on bread wheat. We hypothesized that amending nutrient-poor sandy soil integrated with foliar application of ZnONPs or L-TRP would potentially improve soil hydro-physico-chemical properties and wheat yield and quality. Therefore, the main objective was to investigate the combined effect of HA and foliar L-TRP or ZnONP application on the soil hydro-physico-chemical properties, morpho-physiological parameters, grain yield, and quality of wheat grown under nutrient-poor sandy soil conditions.−1, and precipitation of 1.0 ± 0.6 mm across the two-winter wheat growing seasons of this study. The soil used in this study was collected close to the experimental site at 0–0.3 m depth, and unwanted materials  were excluded. Before filling the pots, the soil was air-dried, ground, homogenized, and sieved through a 2-mm stainless steel sieve. The physicochemical characteristics of the tested soil were determined following standardized methods , permanent wilting point (PWP), and available water (AW) were 12.25%, 3.54%, and 8.71%, respectively. Chemically, this nutrient-deficient soil has pH (7.82), electrical conductivity for soil paste extract , OM (0.38%), SOC (0.22%), available N (20.42 mg kg−1), available P (5.03 mg kg−1), and available K (8.67 mg kg−1). This soil is also poor in available micronutrient concentrations, such as iron , Zn (4.01 mg kg−1), manganese , and copper .A 2-year (2016/17 and 2017/18) pot experiment was conducted in an open greenhouse of the Agronomy Department (latitude: 30° 3′ 16.83′′ N and longitude: 31° 19′ 11.83′′ E), Faculty of Agriculture, Al-Azhar University, Cairo, Egypt. This experiment investigated the beneficial effects of soil-applied humic acid (HA) and foliar applications of plant growth stimulants (PGSs) on the soil hydro-physico-chemical properties, morpho-physiological parameters, grain yield, and its components, and grain quality traits of wheat crops grown under nutrient-poor sandy loam soil conditions. The climate conditions of the experimental area during the experimental period of the 2016/2017 and 2017/2018 growing wheat seasons are displayed in Fig. Triticum aestivum L.) cultivar, was kindly supplied by the Wheat Research division, Agriculture Research Center, Giza, Egypt, to be used as plant material for this experiment. The major constituents of the water dissoluble HA substance  are shown in Table Sakha 93, local high-yielding wheat (0), 0.2 (HA0.2), and 0.4 (HA0.4) g kg−1 soil) and five levels of foliar spraying using two PGSs  with three replicates. Each replicate consisted of three pots, totaling 135 pots for the whole experiment. Experimental pots were divided into three equal main sets, each with 45 pots, based on soil-applied HA treatments. Then, each of the former main sets was divided into five subsets, each with 15 pots, to include foliar application with different PGS treatments. Before filling the experimental pots, the used soil was thoroughly mixed well with HA treatments, urea (46% N) to fulfill the N requirements, calcium superphosphate (15.5% P2O5) to fulfill the P requirement, and K sulfate (48% K2O) to fulfill the K requirement. All experimental treatments were supplied with 74 kg P2O5 ha−1, 167 kg N ha−1, and 84 kg K2O ha−1 as recommended doses. The full amounts of P and K fertilizers and the first (1/3 N) dose were applied basely at planting time when filling the experimental pots, while the other second (1/3 N) and third (1/3 N) doses were applied after 25 and 50 days from planting (DFP), respectively. In each pot, eight homogenous grains were sown on November 15th and 18th in the 2016/17 and 2017/18 seasons, respectively. After 2 weeks (full emergence), five uniform seedlings were maintained per pot. The pots were irrigated regularly to maintain a proper moisture level of approximately at field capacity. Before spraying, the ZnONPs were resuspended well in DDW to increase the dispersion and solubility of their particles using an ultrasonicator  instrument for 20 min. A few drops of Tween-20 were added to the foliar treatments (ZnONPs and L-TRP solutions) as an adhesive agent to increase the solution adhesion to plant leaves. A portable hand sprayer was used for spraying the different concentrations of ZnONPs and L-TRP on the upper surface of wheat leaves until there was run-off (~ 120 ml pot−1). Foliar applications of the different concentrations of the two PGSs (ZnONPs and L-TRP) were performed three times at three distinguished wheat growth stages . These phenological stages correspond with the BBCH 20/21, BBCH 30/31, and BBCH 50/51 stages, respectively, according to the BBCH  scale  comprising three soil-applied HA levels  was calculated by summing the chlorophyll a and b contents  stage, two plants from each pot  were carefully uprooted to avoid damaging the root systems of the remaining plants. These plants were accurately separated into roots and shoots , and the roots were then washed gently with tap water to remove any adhering soil particles. Root length (cm) was measured up to the tip of the longest seminal root length as described by Farrell et al.  using a −1. All spikes were oven-dried at 70 ± 2 °C until a constant weight was reached to obtain the spike dry weight pot−1 (g). Then, wheat grains were threshed, cleaned, and weighed to record grain weight pot−1 (g) (corrected to 12% grain moisture content) and 1000-grain weight (g).The remaining three plants in each pot were harvested at the full ripening (BBCH 88/89) stage. The harvested plants were used to determine plant height (cm), spike length (cm), and spike no. plant−1 and Zn in μg g−1) contents based on dry weight (DW), ground and homogenized grain samples were acid-digested in a mixture of HClO4 and H2SO4  and diluted with DDW , Wi = weight percentage of the dry aggregate size class with respect to the total sample, and n = number of size classes. The pressure plate procedure outlined by Klute (−3) were determined according to Klute (e (dS m−1) was measured using a Metler EC meter according to US Salinity Laboratory Staff  in the laboratory and separated into seven different aggregate sizes  by dry-sieving and then directly weighed. The dry mean weight-diameter (MWD) was calculated according to the method described by Six et al.  as folloby Klute  and Jameby Klute  was gravto Klute . Soil pHto Klute . The ECery Staff .p ≤ 0.05 probability level.Before running the analysis of variance (ANOVA) for the two experimental seasons, the homogeneity of error variance Levene  for all 0.2 and HA0.4 to the experimental sandy loam soil significantly improved the aggregate size distributions at different diameters and MWDs. Compared with HA0, the addition of HA0.2 and HA0.4 significantly increased the dry macroaggregates  and MWD by 7.2 and 9.4%; however, they decreased the dry microaggregates . Soil hydro-physico-chemical properties were significantly affected by the soil-applied HA treatments , TP by 3.9 and 7.2%, and ECe by 10.9 and 16.0%, respectively. However, as the HA level increased from 0.2 to 0.4 g kg−1 soil, the BD and pH of the experimental soil gradually decreased from 2.6 to 5.2% and from 0.4 to 0.8%, respectively, compared with HA0  treatment, HA0.2 and HA0.4 gradually increased wheat morphophysiological parameters, namely, shoot length by 9.4 and 12.9%, root length by 25.8 and 34.3%, shoot dry weight by 26.2 and 46.8%, root dry weight by 38.7 and 54.8%, and chlorophyll content by 18.8 and 34.0%, respectively. Among the HA × PGS interaction levels in Table 0.4 × ZnONPs100 followed by HA0.4 × L-TRP0.50 resulted in the highest shoot length (55.0 and 54.7 cm), root length (16.15 and 16.02 cm), shoot dry weight (6.83 and 6.62 g), root dry weight (0.60 and 0.56 g), and chlorophyll contents (59.5 and 58.9 mg cm−2), respectively, over both growing seasons.Compared with the unamended , spike length (12.10 and 12.18 cm), spike dry weight pot−1 (22.58 and 23.00 g), grain yield pot−1 (17.25 and 18.15 g), 1000-grain weight (48.0 and 48.9 g), and spike no. plant−1 (6.33 and 7.00), respectively , total protein in grains by 10.2 and 13.4%, wet gluten by 18.2 and 23.3%, and dry gluten by 23.5 and 29.5%, respectively, compared with the unamended (HA0) treatment. The uptake and accumulation of grain nutrients and grain quality-related traits gradually increased with increasing ZnONP or L-TRP supplementation compared with the unsprayed control. L-TRP0.50 markedly increased grain nutrients , total protein in grains by 21.6%, wet gluten by 15.8%, and dry gluten by 22.6%, while Zn content in wheat grains increased by 157.1% due to ZnONPs100 supplementation over the unsprayed control plants. Regarding the HA × PGS interactions, the highest contents of N (20.0 mg g−1 DW), P (4.72 mg g−1 DW), K (23.6 mg g−1 DW), total protein (11.38%), and wet (32.3%) and dry (12.34%) gluten in grains were observed in wheat plants treated with the HA0.4 × ZnONPs100 interaction, closely followed by those treated with the HA0.4 × L-TRP0.50 interaction (Table −1DW) in grains was obtained when applying the HA0.4 × ZnONPs100 interaction.Data presented in Table p ≤ 0.01) negatively correlated with MWD (− 0.92**), TP (1.0), and moisture content at FC (− 0.95**), and AW (− 0.95**), whereas TP was significantly (p ≤ 0.01) positively correlated with MWD (0.92), moisture content at FC (0.95**), and AW (0.95**). For the wheat plant measurements from the data obtained under the HA × PGS interaction over both seasons, Fig. 1 and 1000-grain weight were significantly (p ≤ 0.01) positively correlated with shoot length (0.91** and 0.95**), root length (0.90** and 0.67**), shoot dry weight (0.90** and 0.86**), root dry weight (0.91** and 0.82**), chlorophyll content (0.92** and 0.94**), plant height (0.87** and 0.88**), spike length (0.70** and 0.94**), spike dry weight pot − 1 (0.93** and 0.89**), and spike no. plant − 1 (0.85** and 90**), respectively.To explore the interrelationship among the measured traits at the soil and plant levels, correlation analysis was performed on the data obtained under our experimental conditions. For the soil measurements from the data obtained under HA treatments over both seasons, Fig.  FC − 0.9**, and Ae, low OM content, OC content, and nutrient deficiency. These unfavorable characteristics make it defective and less productive because it is classified as having poor hydro-physico-chemical properties. Osman , total grain protein, and wet and dry gluten content of grains were significantly improved with HA100 and L-TRP0.50 markedly increased the grain nutrient content, yield-related traits, and grain quality of bread wheat compared to the control. These positive effects in our study are in accordance with those of Hussain et al.  of soil-applied HA with foliar application of any of the PGSs at a high concentration . These positive results for the soil and wheat crop were obtained with the benefits of the combined application of HA to the soil with exogenous plant growth promoter supplementation under nutrient-deficient sandy loam soil conditions.Foliar spraying with ZnONPsn et al.  and Adren et al.  on wheatn et al. . Furthern et al. , Mohamedn et al. , and Al-n et al. , who fouThe addition of humic acid improved the soil hydro-physico-chemical properties of the degraded nutrient-poor sandy soil. As an excellent soil amendment, humic acid improved the soil structure by allowing rapid macroaggregate formation, decreasing bulk density and pH, and increasing total soil porosity, electrical conductivity, dry mean weight-diameter, and soil water retention capacity. Improving the hydro-physico-chemical properties of degraded nutrient-poor sandy soil positively reflected morphophysiological responses, grain yield, and grain quality. Exogenous foliar application of zinc oxide nanoparticles or L-tryptophan, especially at higher concentrations compared to the control, had a positive effect on wheat morpho-physiological responses, which consequently boosted grain yield and quality. In


Staphylococcus aureus, a major pathogen of community-acquired and nosocomial-associated infections, forms biofilms consisting of extracellular matrix-embedded cell aggregates. S. aureus biofilm formation on implanted medical devices can cause local and systemic infections due to the dispersion of cells from the biofilms. Usually, conventional antibiotic treatments are not effective against biofilm-related infections, and there is no effective treatment other than removing the contaminated devices. Therefore, the development of new therapeutic agents to combat biofilm-related infections is urgently needed. We conducted high-throughput screening of S. aureus biofilm inhibitors and obtained a small compound, JBD1. JBD1 strongly inhibits biofilm formation of S. aureus, including methicillin-resistant strains. In addition, JBD1 activated the respiratory activity of S. aureus cells and increased the sensitivity to aminoglycosides. Furthermore, it was shown that the metabolic profile of S. aureus was significantly altered in the presence of JBD1 and that metabolic remodeling was induced. Surprisingly, these JBD1-induced phenotypes were blocked by adding an excess amount of the electron carrier menaquinone to suppress respiratory activation. These results indicate that JBD1 induces biofilm inhibition and metabolic remodeling through respiratory activation. This study demonstrates that compounds that enhance the respiratory activity of S. aureus may be potential leads in the development of therapeutic agents for chronic S. aureus-biofilm-related infections. Biofilms are clusters of microbial cells covered with an extracellular matrix (ECM) comprising biomolecules such as DNA, polysaccharides, and proteins –3. Dispe–Staphylococcus aureus, a major human pathogen (S. aureus produces extracellular DNA (eDNA), proteins, and polysaccharide intercellular adhesins (PIAs) as ECM components, with the components important for biofilm formation varying among strains , by inhibiting the production of ECM components and resensitizing MRSA to β-lactam antibiotics  strains, four methicillin-resistant S. aureus (MRSA) strains, and two strains of S. epidermidis , both possessed a propylidene group as well as JBD1. ANG1, a JBD1 analog that harbors an ethylidene group instead of the propylidene group, did not inhibit biofilm formation by SH1000 (50 > 100 μM), except for weakly inhibiting biofilm formation by S. aureus MR2 (IC50 = 56.6 μM) -3-cyclohexyl-5-[1-(hexylamino)propylidene]-6-hydroxypyrimidine-2,4-dione, named JBD1 , dose-des SH1000 . The CFUs SH1000 . JBD1 dis SH1000 . Measures SH1000 . JBD1 indermidis  with IC5 38.5 μM . To clar 38.5 μM . Among ty SH1000 . ANG1 di56.6 μM) . These r10.1128/mbio.00845-22.1FIG S1S. aureus SH1000 were formed in the presence and absence of JBD1 and MK-7, and the CFUs were measured. Mean values were compared via one-way ANOVA. *, P < 0.05 (Tukey’s multiple comparison test). (B) Mature biofilms of S. aureus SH1000 were treated with JBD1. The quantitative values of the crystal violet-stained biofilms on 96-well plates are shown. Data represent the means with standard error from three independent experiments. Mean values were compared via one-way ANOVA. **, P < 0.01, ****, P < 0.0001 (Tukey’s multiple comparison test). Download FIG S1, PDF file, 0.4 MB.Effects of JBD1 on colony-forming units (CFUs) of biofilms (A) and stability of mature biofilms (B). (A) Biofilms of Copyright © 2022 Okuda et al.2022Okuda et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00845-22.2FIG S2S. aureus SH1000 after culturing for 24 h in the presence or absence of JBD1 (magenta) or ANG1 (green). Data represent the means of three independent experiments with standard error. Mean values were compared against 0 μM via one-way ANOVA. (B) Growth curves of S. aureus SH1000 in the presence of 5% DMSO (blue), 20 μM JBD1 (magenta), 20 μM ANG1 (green), 20 μM JBD1 and 100 μM MK-7 (orange), and 100 μM MK-7 (gray). Data represent the means of three independent experiments with standard error . Download FIG S2, PDF file, 0.1 MB.Effects of JBD1 and ANG1 on cell growth. (A) Absorbance of Copyright © 2022 Okuda et al.2022Okuda et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00845-22.3FIG S3S. aureus SH1000 . Download FIG S3, PDF file, 1.5 MB.Structure-activity relationships of biofilm inhibitors. Chemical structures of JBD1 and 24 structural analogs. The values in parentheses indicate biofilm-inhibitory activity against Copyright © 2022 Okuda et al.2022Okuda et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the N-acetylglucosaminidase  after staining live and dead cells with fluorescence probes SYTO9 and PI, respectively . Under c10.1128/mbio.00845-22.5FIG S5S. aureus SH1000 formed in the absence of a compound (the control) and the presence of 50 μM JBD1 (JBD1) or 50 μM JBD1 and 100 μM MK-7 (JBD1MK-7) were stained with SYTO9 (green) and PI (red), which stain live and dead cells, respectively. Three-dimensional structures of the biofilms were observed using CLSM. Download FIG S5, PDF file, 2.5 MB.Confocal laser scanning microscopy (CLSM) images of biofilms. Biofilms of Copyright © 2022 Okuda et al.2022Okuda et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. aureus, experiments using the clinically isolated MSSA strain (MS9) and the MRSA strains  were performed. JBD1, but not ANG, increased susceptibility to the aminoglycosides   . Taken t10.1128/mbio.00845-22.7TABLE S1S. aureus strains in the presence or absence of compounds. Download Table S1, PDF file, 0.10 MB.MICs (μg/mL) of antibiotics against Copyright © 2022 Okuda et al.2022Okuda et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bacterial uptake of aminoglycosides is dependent on a proton motive force (PMF) acting across the cell membrane . The PMF10.1128/mbio.00845-22.6FIG S6S. aureus SH1000 biofilms were formed for 4 h in the presence of 5% DMSO (the control), 20 μM JBD1 (JBD1), 20 μM ANG1 (ANG1), 20 μM JBD1 and 100 μM MK-7 (JBD1MK-7), and 100 μM MK-7 (MK-7). The quantitative values of the crystal violet-stained S. aureus SH1000 biofilms formed on 96-well plates are shown. Mean values were compared via one-way ANOVA. *, P < 0.05, ****, P < 0.0001 (Tukey’s multiple comparison test). Download FIG S6, PDF file, 0.1 MB.Effects of compounds on biofilm formation at 4 h of growth. Copyright © 2022 Okuda et al.2022Okuda et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. aureus, is a menaquinone (MK), a common electron carrier in the respiratory chains of Gram-positive bacteria   . We inve chains) . MK-7 ad chains) . These r chains) . These rS. aureus  encoding an amino acid permease, ilvA1 (SAOUHSC_01451) encoding a threonine dehydratase, and ald1 (SAOUHSC_01452) encoding an alanine dehydrogenase, suggesting that JBD1 affects the amino acid profile of S. aureus. Expression of pflB (SAOUHSC_00187), which encodes an enzyme that converts pyruvate to formic acid and acetyl-CoA, was also decreased in JBD1-treated cells compared to that in JBD1MK-7-treated cells (pfl expression is enhanced in biofilms (compared to planktonic cells) (pflA (SAOUHSC_00188), which encodes a PflB-activating enzyme, was also determined using real-time PCR because pflA is located in the same operon as pflB. Real-time PCR using total RNA extracted from the cells after 4 h of culture  , and ourc cells) . The exp10.1128/mbio.00845-22.8TABLE S2Table S2, XLSX file, 0.01 MB.RNA sequencing data related to Copyright © 2022 Okuda et al.2022Okuda et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the srrAB-deficient strains , amino acid fermentation , and amino acid transport (steT) , whose expression was downregulated by JBD1, also had no effect on biofilm formation. S. aureus not only synthesizes amino acids but also transports amino acids into the cell from the external environment  . Additioormation , 40. Simormation . These fironment . This maS. aureus, and MK-7 significantly suppressed the effect. This result indicates that respiratory stimulation by JBD1 triggers metabolic remodeling as well as inhibition of biofilm formation. In addition, the PC1 in PCA correlates well with the extent of biofilm formed in each group, suggesting that biofilm control becomes possible by controlling the metabolic profile.JBD1 induced a large variation in the metabolic profile of S. aureus. By clarifying the target molecule and mechanism of action of JBD1, it may be possible to develop a new method for controlling intractable biofilm-related infections.In summary, the compound JBD1, obtained by HTS, inhibited biofilm formation and induced metabolic remodeling by promoting increased respiratory activity of S. aureus and S. epidermidis strains were grown overnight in Brain Heart Infusion (BHI) broth  unless otherwise mentioned. All mutants were constructed from S. aureus SH1000  broth, 5% DMSO, with or without test compounds. Aliquots of the cell suspensions (200 μL) were transferred to the wells of a 96-well microtiter plate  and incubated for 4 to 24 h at 37°C under static conditions. Bacterial growth was assessed by measuring the absorbance of cultures at 595 nm using a microplate reader . After removing culture supernatants and planktonic cells, biofilms that formed at the bottom of the wells were washed twice with 200 μL phosphate-buffered saline (PBS), to remove loosely adherent cells, dried, stained with 100 μL of 0.05% crystal violet for 1 min, and subsequently washed twice with PBS. Finally, biofilm formation was quantified by measuring the absorbance at 595 nm using a microplate reader (Infinite 200 PRO). As the biofilms can form at the bottom of the wells in a patchy manner, multiple reads (4 × 4) per well, using the Infinite 200 Pro, were used to obtain the average value in each well. The IC50 was calculated using the following formula: IC50 = 10[log (A/B) × (50 − C)/(D − C) + log (B)], where A corresponds to concentrations of test compound directly above 50% inhibition; B corresponds concentrations of test compound directly below 50% inhibition; C is percent inhibition directly below 50% inhibition; and D is percent inhibition directly above 50% inhibition.Overnight cultures of S. aureus SH1000, we conducted HTS of 9,600 compounds stocked at the Drug Discovery Initiative of The University of Tokyo. Biofilm formation and quantification under the conditions detailed above were conducted automatically using a custom-built FreedomEVO System (Tecan). The final concentration of each compound for screening was 20 μM. Compounds that inhibited bacterial growth by 20% or more were excluded from the hits.To identify compounds that inhibit biofilm formation in S. aureus SH1000 was diluted 500-fold into BHIG broth, and 200-μL aliquots were cultured in 96-well flat-bottomed polystyrene plates (Corning) at 37°C for 24 h under static conditions. Then, JBD1 was added to the wells at a final concentration of 20 or 100 μM . After incubation at 37°C for 2 h, the biofilms were quantified as described above.An overnight culture of S. aureus SH1000 was diluted 500-fold into BHIG broth containing 5% DMSO, with or without test compounds, and 2-mL aliquots were cultured in a 40-mm dish at 37°C for 24 h under static conditions. After removing the supernatants, the biofilms were washed twice with 2 mL PBS. The CFU was determined by plating each dilution on BHI agar.An overnight culture of S. aureus SH1000 was diluted 500-fold into fresh BHIG broth 5% DMSO, with or without test compounds, and 200-μL aliquots were cultured in 96-well flat -bottomed polystyrene plates (Corning) at 37°C for 24 h under static conditions. The absorbance of each culture at 600 nm was measured every 30 min for 24 h using a Bio Microplate Reader HiTS .An overnight culture of S. aureus cells were stained using WGA-Alexa 488  according to a previously published method , and 2-mL aliquots were cultured in 12-well flat-bottomed polystyrene plates (Corning) at 37°C for 24 h under static conditions. After removing the supernatants, the sedimented cells were resuspended in PBS, spotted on glass slides, and left to dry at room temperature. Then, the cells were stained with 5 μg/mL WGA-Alexa 488 (Invitrogen) in PBS for 20 min at 37°C, washed twice with PBS, and subsequently stained with DAPI  for 5 min at room temperature. Stained cells were mounted with the Vectashield mounting medium  and observed using a fluorescence microscope .For fluorescent imaging of extracellular PIA, d method , with moS. aureus SH1000 were cultured statically in 2 mL of BHIG broth containing 5% DMSO with or without JBD1 (50 μM) or JBD1 (50 μM) and MK-7 (100 μM) in a 35-mm glass-bottom dish  at 37°C for 24 h. After removing the supernatants, the biofilms formed on the glass were washed three times with 300 μL of PBS and stained with 200 μL of Filmtracer LIVE/DEAD Biofilm Viability Kit (Invitrogen) according to the manufacturer’s protocol. The stained biofilm was washed twice with 200 μL of double-distilled water (DDW), mounted with ProLong Glass Antifade Mountant (Invitrogen), and observed using an LSM880 microscope .S. aureus strains were diluted 500-fold in BHIG broth containing 5% DMSO, with or without test compounds . Aliquots of the cell suspensions (195 μL) were transferred to the wells of a 96-well microtiter plate (Corning) containing antimicrobial agents of appropriate concentrations (5 μL) and incubated for 24 h at 37°C under static conditions. Bacterial growth was assessed by measuring the optical density at 595 nm (OD595) using a microplate reader (Infinite 200 PRO). The minimum concentration of the antibiotic that suppresses the increase of OD595 to less than 20% from the start of culture was defined as the MIC.Overnight cultures of S. aureus SH1000 was diluted 500-fold into BHIG broth 5% DMSO, with or without test compounds , and 2-mL aliquots were cultured in 12-well flat-bottomed polystyrene plates (Corning) at 37°C for 4 or 24 h under static conditions. Cells from 1 mL of the culture were harvested by centrifugation at 5,000 × g and 25°C for 10 min and resuspended in 1 mL of PBS. Then, 1 μL of component A (preliminarily diluted 10-fold with DMSO) included in the BacLight RedoxSensor Green Vitality Kit (Invitrogen) was added to the cells and incubated for 10 min at room temperature. Cells were collected by centrifugation, fixed in 4% PFA for 5 min at room temperature, washed twice, and resuspended in 1 mL of PBS. The cell suspensions were diluted 10-fold with DDW, and stained cells were analyzed using a flow cytometer  at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Cells were flowed at 10 μL/min, and 10,000 events were recorded for each sample.An overnight culture of S. aureus SH1000 cells cultured in BHIG broth containing 5% DMSO for 24 h in the presence or absence of the test compound . After harvesting cells by centrifugation, ECMs were extracted based on previously described methods ( methods . Dot blo methods . ECM samS. aureus SH1000 was grown under biofilm conditions for 4 h in the presence or absence of the test compounds (20 μM JBD1 or 20 μM JBD1 and 100 μM MK-7). Cells were harvested by centrifugation at 5,000 × g at 25°C for 10 min and immediately incubated with an appropriate volume of RNAprotect Bacteria Reagent  for 5 min. Cells were collected by centrifugation at 5,000 × g at 25°C for 10 min and stored at −80°C. Total RNA was extracted using an RNeasy Midi Kit (Qiagen) according to the manufacturer’s instructions, except that cells were treated with 0.2 mg/mL lysostaphin (Wako) at 37°C for 30 min before extraction. The extracted total RNA samples were stored at −80°C until use. GEBEWIZ (https://www.genewiz.com/) performed RNA-seq including a quality check of RNA samples, stranded library preparation with rRNA depletion, sequencing using Illumina HiSeq (2 × 150 bp), and data analysis. Data were aligned to S. aureus NCTC 8325 reference genome (NC_007795.1) via software Bowtie2 . Real-time PCR was performed using a Step One Plus (Applied Biosystems) and Power SYBR green PCR master mix (Applied Biosystems). Reactions (20 μL per sample) were performed in triplicate using 96-well plates. All reactions contained 5 ng of cDNA, 10 μL of Power SYBR green Master Mix, 0.2 μM of each primer, and nuclease-free water. According to a previous report, transcript levels of hu were used to normalize the transcript levels (TC) of all samples , with modifications. Briefly, S. aureus SH1000 was cultured in 50 mL of BHIG broth containing 5% DMSO for 8 h in the presence or absence of compounds . Cells were harvested by centrifugation at 5,800 × g at 4°C for 5 min. To remove ECM molecules that cause clogging during subsequent filtration , the cells were washed with 10 mL of 1.5 M NaCl as described previously (595 with the sample volume in mL (OD·mL) of 20 was collected and resuspended in 1.6 mL of methanol by sonication. After addition of an internal standard solution (HMT), cell suspensions were centrifuged at 2,300 × g at 4°C for 5 min. The supernatants were then filtered through a cellulose membrane filter . The extracted metabolite samples were stored at −80°C until use. Targeted quantitative analysis of metabolites using capillary electrophoresis time of flight mass spectrometer was performed by HMT as described previously . Statistical analysis with one-way ANOVA or Student's