The aim of this study was to investigate the association between estimated glucose disposal rate (eGDR), a proxy for insulin resistance, and retinopathy or kidney disease, i.e. micro-, or macroalbuminuria, in young individuals with type 1 diabetes (T1D).Using data from the Swedish pediatric registry for diabetes (SweDiabKids) and the registry for adults (NDR), all individuals with T1D with a duration of diabetes of less than 10 years between 1998 and 2017 were included. We calculated the crude incidence rates with 95% confidence intervals (CIs) and used multivariable Cox regression to estimate crude and adjusted hazard ratios (HRs) for two cohorts: retinopathy cohort or kidney disease cohort, stratified by eGDR categories: < 4, 4 to 5.99, 6 to 7.99, and ≥ 8 mg/kg/min (reference).A total of 22 146  children and adults with T1D  were studied. During a median follow-up of 4.8 years (IQR 2.6–7.7) there were 5040 (24.7%), 1909 (48.1%), 504 (52.3%) and 179 (57.6%) events for retinopathy in individuals with an eGDR ≥ 8, 7.99 to 6, 5.99 to 4, and < 4 mg/kg/min, respectively. Corresponding numbers for kidney disease was 1321 (6.5%), 526 (13.3%), 255 (26.8%) and 145 (46.6%). After multiple adjustments for different covariates, individuals with an eGDR 7.99 to 6, 5.99 to 4 and < 4 mg/kg/min, had an increased risk of retinopathy compared to those with an eGDR ≥ 8 mg/kg/min  1.29 (1.20 to 1.40); 1.50 (1.31 to 1.71) and 1.74 (1.41 to 2.14). Corresponding numbers for kidney disease was  1.30 (1.11 to 1.52); 1.58 (1.25 to 1.99) and 1.33 (0.95 to 1.86), respectively.eGDR, a proxy for insulin resistance, is associated with retinopathy and kidney disease in young adults with T1D. The risk of retinopathy increased with lower eGDR. The risk of kidney disease also increased with lower eGDR; however results show no association between the lowest eGDR and kidney disease. eGDR can be helpful to identify young T1D individuals at risk.The online version contains supplementary material available at 10.1186/s12933-023-01791-x. Individuals with type 1 diabetes (T1D) are at increased risk of macrovascular and cardiac complications, in which microvascular complications may predict the risk , 3. HoweThe term double diabetes, first described in 1991, was initially defined as an individual with T1D with a family history of type 2 diabetes (T2D) . Today, The gold standard for measuring insulin resistance is the euglycemic hyperinsulinemic clamp technique . Since tNot only macrovascular complications are associated with insulin resistance, but also microvascular complications . There iThe study was a nationwide, observational cohort study. The study was approved by the Swedish Ethical Review Authority (Dnr 977-17).st December 2017. Between these time points all children, adolescents and adults in the registries were included if they had a diagnosis of T1D since ten years, or less when first recorded in the registries. The age span in the whole cohort was 0–39 years, i.e., patients were included if they are diagnosed with T1D before 30 years of age and if duration was < 10 years.We used data from the Swedish national diabetes registry for adults (NDR) and the Swedish pediatric registry for diabetes (SweDiabKids), no other registers were used. Participants provided informed consent when entering the registries. NDR defines T1D on the basis of epidemiological data: treatment with insulin and a diagnosis at the age of 30 years or younger, which has shown to be validated in 97% of cases . In chilData extracted from the registers were age, sex, diabetes duration, weight, height, BMI, blood pressure, HbA1c, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, smoking, physical activity, type of diabetes treatment, micro/macroalbuminuria, estimated glomerular filtration rate (eGFR), and result of retinopathy screening. Categorization of the variables from the registers are shown in Tables In the final data set individuals with at least one registration of examination of retinopathy, or kidney disease was included. They also needed at least one registration of eGDR  and at least one registration of each of the covariates that was used in the final model. In total 15 111 individuals were recruited from SweDiabKids and 19 298 individuals were recruited from NDR. 26 786 individuals had T1D with < 10 years duration when entering the registry. After including patients with at least one examination of retinopathy or nephropathy, one eGDR observation and at least one observation for each covariate, 10 289 cases in the retinopathy cohort and 11 857 cases in the nephropathy cohort remained Figure S1  were excluded from retinopathy analysis  and 14 929 individuals (55%) from kidney disease analysis  Figure S1  was calculated as previously described [BMI = 19.02 − (0.22*BMI) − (3.26 * HT) − (0.61*HbA1c) . BMI = body mass index (kg/m2), HT = hypertension (yes = 1/no = 0), and HbA1c = HbA1c (DCCT %).eGDRescribed  based onHypertension was defined as a blood pressure > 140/90 mmHg or current use of any anti-hypertensive agents. For individuals < 18 years old, we used IsoBMI . AnalyseBased on previous studies , 23, we −1, or urinary albumin of 20–200 µg min−1 (20–300 mg/L), or macroalbuminuria defined as an albumin/creatinine ratio > 30 mg mmol−1, or urinary albumin > 200 µg min−1 (> 300 mg L−1) [Endpoints of retinopathy were classified as any retinopathy, preproliferative diabetic retinopathy (PPDR), or proliferative diabetic retinopathy (PDR). Any retinopathy included any signs of retinopathy, i.e. simplex retinopathy, PPDR or PDR. PDR was defined as evidence of current proliferations or any earlier laser photocoagulation. Endpoints of kidney disease were classified as any microalbuminuria defined as two positive test results from three samples taken within one year with an albumin/creatinine ratio of 3–30 mg mmolst of December 2017, whichever came first. Follow up did not end if mild retinopathy, i.e. simplex retinopathy, or microalbuminuria was registered. If more severe forms were registered, milder forms were ignored, and the first diagnosis of the severe form was included.All individuals were followed from the first observation (between 1998 and 2017) when they first appeared in the register, i.e. index date, until the first event of any retinopathy, or any kidney disease , or until the end of the study 31Baseline characteristics  are based on the first available observation (between 1998 and 2017) from index date and after that with no specific time limit, for each individual in the study. Continuous variables are presented as medians with interquartile range (IQR), due to the contribution of data, and categorical variables as proportions. Crude and adjusted hazard ratios (HRs) and confidence intervals (CIs) were estimated using univariable and multivariable Cox regression models with time until endpoints, that is retinopathy or kidney disease, as response variables and time-varying covariates as exposure variables, that is eGDR, and potential confounders. Exposure to risk of retinopathy and kidney disease starts at diabetes onset, but follow-up starts at first eGDR observation. This means that data are left-truncated as well as right-censored handled accordingly.Instead, age at diabetes onset was included as a covariate. All covariates which were adjusted for in the final model were time-varying, but sex. The time-varying variables, i.e. numbers and timespan per patient are shown in Table S2  and 9% were obese (BMI > 30 kg/m2), both cohorts. Finally, 5% of individuals had hypertension. All covariates, multivariable adjusted for in the final model, are shown in Table Baseline characteristics of the study participants in the retinopathy and nephropathy cohorts, respectively, are shown in Table The estimated retinopathy crude cumulative risk curves illustrating the accumulated risk for retinopathy are shown in Fig. We further assigned individuals to different degrees of retinopathy to compare the risk for severe retinopathy with milder degrees: PPDR/PRD/laser photocoagulation vs. non/simplex retinopathy and PRD/laser photocoagulation vs. non/simplex retinopathy/PPDR, respectively, into the same eGDR categories Table .Number of events and event rates was much less for severe retinopathy compared with mild retinopathy. Crude and adjusted for sex and age relative risk for PPDR/PRD/laser photocoagulation vs. non/simplex retinopathy increased in all eGDR categories below 8 mg/kg/min Table . After mThe estimated crude survival curves illustrating the accumulated risk for kidney disease, i.e. microalbuminuria, or macroalbuminuria are shown in Fig. After adjustment for age and sex, and all covariates in Table We further assigned individuals to different degrees of kidney disease, i.e. comparison of microalbuminuria and macroalbuminuria vs. non-albuminuria respectively. Number of events and event rates was much less for macroalbuminuria compared with microalbuminuria Table . Crude aThe reason for using ISO-BMI, instead of BMI, is that this formula “correct” BMI, especially for the youngest children . Since t2 ± SD) for each variable in the eGDR formula for the risk of retinopathy was highest for HbA1c (0.0242 ± 0.0049), followed by BMI (0.0012 ± 0.0011), and hypertension (0.0006 ± 0.0010). Corresponding explained relative risk for kidney disease was for HbA1c (0.0389 ± 0.0103) followed by BMI (0.0163 ± 0.0064), and hypertension (0.0067 ± 0.0059).The estimated explained relative risk (R2) and 9% were obese (BMI ≥ 30 kg/m2) according to the World Health Organization’s definition. The prevalence of obesity in childhood T1D populations is increasing. In the Nordic countries the obesity rate was 18.5% in children with T1D under 15 years of age during the period 2008–2012, which was higher than for the Swedish reference population [This nationwide, observational study shows that eGDR, a proxy for insulin resistance, associates with the risk of retinopathy and kidney disease in young individuals with T1D. The risk of retinopathy increased with lower eGDR. Risks of kidney disease also increased with lower eGDR, however not for the lowest eGDR category (< 4 mg/kg/min).eGDR has been proven a tool for the measurement of insulin resistance in people with T1D  and has pulation . Risk fapulation . We adjupulation , 36.2, in the population in the DCCT/EDIC study increased from 1 to 31% over the course of 12 years [The impact of obesity and microvascular burden in patients with T1D is not fully established . The pre12 years . One pos12 years , 8, 37. 12 years , 38, as 12 years . However12 years .Hypertension, often coexist with insulin resistance, is a well-known risk factor for micro- and macrovascular complications . In the Hyperglycemia is a well-known risk factor for both macrovascular and microvascular complications , 3. HypeThe main strength of this study is the large nationwide population of individuals with T1D with a long follow up. The results show an association between eGDR and risk of retinopathy and kidney disease. We were able to adjust for several important confounders. However, there are limitations to this study. The eGDR formula, including hypertension, has not entirely been validated in youth . DiffereIn conclusion, the current cohort study shows that young people with T1D with low eGDR have higher risk of developing both retinopathy and kidney disease, which indicates that insulin resistance increases the risk of these complications. Since microvascular complications are predictors for cardiovascular disease, and earlier observation demonstrates the association between eGDR and cardiovascular death and mortality, in individuals with T1D, further studies are much needed to explore insulin resistance as a risk factor for micro-, and macrovascular complications in people with T1D.Additional file 1: Table S1. Baseline characteristics of all patients divided in the retinopathy and kidney disease cohort and excluded patients, respectively. Table S2. Number of measurements and timespan per patient, median and (range). Table S3. Baseline characteristics of all patients according to eGDR categories. Table S4. Interaction analysis between the retinopathy cohort  and the kidney disease cohort  with an overlap of 9575 individuals. Number of events, event rate and relative risks for any retinopathy and kidney disease in children and adults with type 1 diabetes. Table S5. eGDR based only on BMI (ISO-BMI excluded). Number of events, event rate and relative risks for any retinopathy and kidney disease in children and adults with type 1 diabetes. Figure S1. Flowchart for the studied group. The overlap between cohorts included 9575 individuals. Proportion of excluded patients between the registers was for the retinopathy cohort 70% vs. 47% (SwedDiabKid vs. NDR) and for the kidney analysis corresponding proportional numbers were 62% vs. 39% (SwedDiabKid vs. NDR), for the two registers. eGDR, estimated glucose disposal rate; NDR, National Diabetes Register.


Carthamus tinctorius L. (CT) oil extract, which has been scientifically proven for its antibacterial and antioxidant activities for the ailment of bacterial skin infections.The current study aimed to develop a topical herbal emulgel containing The CT emulgel was formulated by response surface methodology (RSM) and was evaluated by various parameters like extrudability, spreadability, pH, viscosity, and antibacterial and antioxidant activities. Molecular docking was also performed using AutoDock.Staphylococcus aureus and Escherichia coli at p-value = 0.00, the Emulgel-F8 shows zone of inhibition of 24 mm for E-coli and 19 mm for S-aureus. Emulgel-F9 shows zone of inhibition of 22 mm for E-coli and 15 mm for S-aureus while pure CT- Oil extract shows zone of inhibition of 25 mm for E-coli and 20 mm for S-aureus and ciprofloxacin used as standard shows 36mm zone of inhibition against both E-coli and S-aureus. The comparative investigation through molecular docking binding affinities and interactions of ligands with various target proteins provides insights into the molecular processes behind ligand binding and may have significance for drug discovery and design for the current study.Among all formulated CT emulgels, F9 and F8 were optimized. Optimized formulations had shown good spreadability and extrudability characteristics. Sample F8 had % inhibition of 42.131 ± 0.335, 56.720 ± 0.222, and 72.440 ± 0.335 at different concentrations. Sample F9 had % inhibition of 26.312 ± 0.280, 32.461 ± 0.328, and 42.762 ± 0.398 at concentrations of 250 µg/ml, 500 µg/ml, and 1,000 µg/ml, respectively, which shows that both samples F8 and F9 have significant antioxidant potential. Optimized CT emulgels F8 and F9 had significant antibacterial activity against C. tinctorius L.-based emulgel has good antioxidant and antibacterial activities against E. coli for the treatment of bacterial skin infections.The current study suggests that  Its success overlaps the traditional processes including injectable and oral drug delivery . It incrThe dosage form that is comprised of combined gel and emulsion is referred to as an emulgel, which is formed by adding a gelling agent to either oil and water or water-in-oil emulsions. Emulgel shows diversity, as it has dual control on drug release due to the presence of both aqueous and non-aqueous phases in it . EmulgelEscherichia coli strains have been involved in various soft tissue skin infections that occur in patients of each age. Some infections recover without any treatment, but some require antibacterial treatments , also called safflower or false saffron, is a medicinal plant that belongs to the family Compositae or Asteraceae. This shaft-like species typically grows in dusty Southern Asia, China, India, and Iran climates. It is cultivated mainly for its seeds, which are used as edible oil and contain many useful compounds such as flavonoids, phenolic acids, fatty acids, and alkynes, which contribute to its effect as an analgesic, anti-inflammatory, antioxidant, and antimicrobial , as well as distilled water, Mueller–Hinton agar (OXOID CM0337), and Nutrient broth (OXOID CM0001). All other ingredients were of analytical grade.2.2n-hexane, a hydrocarbon having a boiling point of 68.7°C, was chosen as the preferred solvent. After extraction, oil was obtained by removing the solvent using a rotary evaporator .CT plants were collected from Pakistan’s tropical regions. The oil from the CT plants’ seeds was extracted using a Soxhlet apparatus . For extraction, aporator . After t2.3The experimental permeation enhancer concentration like propylene glycol (PG) and polyethylene glycol (PEG) on emulgel was assessed using Design Expert (version 7.0.3). The quantities of PG and PEG were tested at three altitudes using a central composite rotatable design (CCRD), as indicated in 2.4C. tinctorius L. oil-based emulgel was made using various concentrations of PG and PEG. Initially, the gel phase was made with the necessary Carbopol 940 concentration to water at an appropriate speed with continuous stirring by a mechanical stirrer. Span20 was mixed with liquid paraffin, following the addition of CT oil to form the oil phase. Tween 20 was dissolved in distilled water to form an aqueous phase, and then polyethylene glycol-1000 and methylparaben were added. Then, both phases were heated to 70°C to 80°C, cooled at room temperature, and mixed to make emulsion. To develop emulgel, the emulsion was diluted with gel in a ratio of 1:1 while swirling gently . The following formula was used to calculate the average .M stands for the weight (g) bent to the top slide, L for the length (cm), and T for the time (seconds).where 2.5.32 was applied for 30 seconds by holding the tube with a plunger. Emulgel extruded from each tube was measured (The extrudability test of each CT emulgel determines if it flows freely from the collapsible tubes. The extrudability of CT emulgel was determined using a hardness tester. CT emulgel with a mass of 5 g was placed inside a collapsible tube. A pressure of 1 kg/cmmeasured .2.6By conducting Fourier transform infrared (FTIR) spectroscopy of polymers, drugs, and drug-loaded emulgels, structural factors such as polymer–drug interactions of CT-emulgel formulations were confirmed . FTIR sp2.7The stability compartment was maintained at 40.1°C to check the stability of all CT emulgels for 6 months. After 0 months to 6 months, different physical aspects of CT emulgels, such as viscosity, pH, and drug contents, were examined . With Mi2.8in vitro drug release study of CT emulgels utilized the dissolution apparatus [USP type II]. The CT oil extract was administered as a pharmaceutical agent in phosphate-buffered saline (PBS), with a release medium at a pH of 5.5 and a temperature of 37.5°C. This was achieved by encapsulating 1 g of CT emulgel within a cellophane membrane and attaching it to the paddle of a dissolution apparatus using a thread. A volume of 500 ml of PBS was employed, while the paddle rotation speed ranged from 85 to 100 revolutions per minute (RPM). From the release media, 5 ml of the sample was removed after a predetermined amount of time and replaced with the same volume of fresh media  to analyze the physical state of CT oil present in emulgel formulations. The sample sizes used were 14.7 mg and 16.6 mg for F8 and F9, respectively. Under a nitrogen environment, heat stress was applied at a rate of 10°C/min from 20°C to 350°C, and thermogram peaks were recorded .2.10Scanning electron microscopy (SEM) was used to analyze the surface morphology and superficial shape of emulgels. The sampling for SEM was performed by attaching emulgels on metal slabs using adhesive tape and kept in a vacuum chamber for drying. A layer of 10-nm thickness consisting of a dense gold coating was subsequently applied using a sputter coater. Subsequently, a high-resolution SEM analysis was conducted to detect and examine the sample .2.1150). Acarbose was used as a standard antioxidant agent  was calculated to check the antioxidant activity of CT emulgels F8 and F9. At various concentrations , 1 ml from 0.1 mM of DPPH solution was prepared in ethanol and then mixed with 3 ml of the tested compounds in ethanol formulated by successive dilution. The prepared mixture was placed at room temperature of approximately 22°C and shaken for 30 min, and then the reaction mixture’s absorbance was investigated using a UV spectrophotometer at 517 nm. Different concentrations were used to detect the inhibition concentration for 50% of DPPH free radical  × 100.The absorbance of control is the absorbance of pure DPPH solution.2.122.12.1Mueller–Hinton is a commonly used agar medium in microbiology for antimicrobial susceptibility testing. This agar media method was employed for the preparation of the media. To prepare the media, 38 g of agar was added to 1 L of water and then heated to dissolve it completely after autoclaving for 15 min at 121°C. This media was utilized for the agar well diffusion procedure. A nutrient broth media was created to prepare the bacterial solution by adding 13 g of powder to 1,000 ml of distilled water. After thorough mixing, this was sterilized by autoclaving for 15 min at 121°C .2.12.2S. aureus (ATCC-2529) and E. coli (ATCC-27853) were used.Different microbial strains, 2.12.3S. aureus and E. coli was selected. In the next step, nutrient broth media was inoculated with bacterial colonies. To obtain precise results, nutrient media was inoculated with maximum colonies, and for each S. aureus and E. coli, an optical density greater than 1 was found.The freshly prepared culture of 2.12.4Petri dishes were used for plating and solidified by pouring liquefied Mueller–Hinton agar (11–14 ml). Then, the plates were labeled .2.12.5To obtain a precise number of bacteria per 1 ml, serial dilutions were prepared as necessary to attain 1,000 bacteria per 100 µl. For serial dilution, there were five Falcon test tubes for each strain, and a total of 10 test tubes were used. A nutrient broth of 9 ml was added to each test tube and then labeled as 101, 102, 103, 104, and 105 for each strain of bacteria. Ten Falcon tubes were used for each strain of bacterial dilution. Nutrient broth with volume of 9 ml was added to each Falcon tube and then labeled as 101, 102, 103, 104, and 105 for each bacterial strain. From bacterial solution, 1 ml was used for Falcon tube 5, labeled as 105, and mixed and added to Falcon tube 104. The same procedure was repeated until the last one was finished, which was Falcon tube 4 . Dilution 101 with volume of 100 µl was taken via micropipette and poured on plates containing Mueller–Hinton agar, as this contained 1,000 bacteria .2.12.6In the next step, 100 µl of serial dilution was selected and poured into a petri dish labeled as 4, designated for specific bacteria. Following that, with the help of swabs and loops, lawn and wells were made in petri dishes, respectively. A cotton swab was used to spread bacterial solution in multiple directions to prepare equally dispensed bacterial loon. According to the arrangement of sample per plate, 1-ml autoclaved micropipette tips were used to spread wells in the agar media.2.12.7In this step, a micropipette in the range of 10 µl to 200 µl was used to pour 100 µg into the well. The sample was incubated for 24 h at 37°C. CT emulgel with a mass of 100 µg containing 2 µg of the extract was loaded in wells. The standard used was ciprofloxacin 10 μg . The pla2.13E ranges were determined using the microliter broth dilution method. This method filled the wells with 100 µl of nutrient broth to prepare serial dilutions. The ultimate oil extract concentrations in the wells were 10 µg/ml, 5 µg/ml, 2.5 µg/ml, 1.5 µg/ml, 1.25 µg/ml, 0.625 µg/ml, 0.312 µg/ml, 0.156 µg/ml, 0.078 µg/ml, and 0.039 µg/ml. After this, 100 μl of E. coli-based bacterial sample was added to well numbers 1–10, taken from 108 Falcon tube dilution. Then, the last two wells were marked as negative and positive controls. The negative control had only 100 µl of methanol extract solution, while the positive control had 100 µl of bacterial solution. These controls were then incubated for 24 h at 37°C. From this, MIC and MBC values were identified. In order to confirm bacterial absence, 100 µl was taken from the wells marked to have MIC and MBC values, poured into two separate nutrient agar plates, and incubated at 37°C for 18–42 h  determined was the minimum concentration that shows no turbidity, and the minimum bactericidal concentration (MBC) determined was the minimum concentration that produces not even a single colony of bacteria. With the use of MBC and MIC, serial dilutions of oil were primed. To make serial dilutions, MIC and MBC values were prepared using the microliter broth method; with this method, a methanol extract of 10 µg/100 µl was prepared, and MIC and MBC for bacterium  18–42 h .2.14Helicobacter pylori urease crystalline structures for virtual screening. Molecules were docked using MGLTools, AutoDock4, and Autogrid4 binary files. International Union of Pure and Applied Chemistry (IUPAC) designations were used to draw the ligand structures in ChemDraw Ultra.The RCSB protein data repository included urate oxidase and H. pylori urease crystalline structures for virtual screening  was employed to produce a mathematical relationship based on a polynomial equation. The positive and negative significant effects have demonstrated the comparative impacts of each factor on a given response of CT emulgels. Negative numbers represent antagonistic effects on each response, while positive values demonstrate synergistic effects.p-Value (p 0.05) for answer response Y1 indicates linear contributions. A (PG) and B (PEG) both exhibited negligible antagonistic and cross-product effects. According to ANOVA Table 4,According to the equations, it was determined that while “B” had a significant negative antagonistic impact on the release of CT oil in PBS, “A” had a relatively minor negative antagonistic effect at pH 5.5. Both of the variables (AB) demonstrated potent combined effects that demonstrated adverse (antagonistic) consequences. Propylene glycols (A2), which had a high positive (synergistic) effect, had a quadratic relationship with PEG, which is a weak positive (synergistic) relationship. Propylene glycol (A) [−0.38A + 4.74A2] showed that the percentage of drug release increased when PG concentration was higher, while the concentration of PG fell when PG concentration was lower. According to previously published research , the per3.4According to the stability findings, all CT-emulgel formulation preparations were carried out by keeping them all for 6 months at a temperature of 40°C ± 1°C in a stability chamber. The findings demonstrated that all CT emulgels have satisfactory outward appearances, and no significant alterations in drug content, drug release behavior, viscosity, pH, or moisture content were found. All CT-emulgel preparations underwent a 30-day skin irritation test using human volunteers. According to the findings, there were no skin lesions, irritations, or scratches. Similar findings were reported in previously reported articles .3.5In vitro drug release profiles of CT emulgels are shown in 3.6C. tinctorius L. oil, and Carbopol 940 revealed several interesting findings, as shown in C. tinctorius L. oil, and Carbopol 940. This suggests that the formulation process and the addition of excipients did not cause major changes in the chemical composition of these components. In The analysis of FTIR spectra of the produced CT emulgels, 3.7The TGA measurement was used to check the thermal stability of emulgel within the 25°C–350°C temperature range. The TGA plot of the emulgel is presented in 3.8The SEM analysis of the emulgel reveals that the surface exhibits an irregularly shaped structure, characterized by the presence of cracks and wrinkles. This observation is depicted in 3.9The antioxidant activity of optimized CT emulgels was also checked using DPPH free radical scavenging assay. In this assay, the protons were accepted as free radicals from the antioxidant substrate, decreasing its absorbance, which was reserved as an amount of radical scavenging.Sample F8 had % inhibition of 42.131 ± 0.335, 56.720 ± 0.222, and 72.440 ± 0.335 at different concentrations. Sample F9 had % inhibition of 26.312 ± 0.280, 32.461 ± 0.328, and 42.762 ± 0.398 at concentrations of 250 µg/ml, 500 µg/ml, and 1,000 µg/ml, respectively. The standard drug used had % inhibition of 24.030 ± 0.578, 26.030 ± 0.619, and 33.607 ± 0.294 at concentrations of 250 µg/ml, 500 µg/ml, and 1,000 µg/ml, respectively, which shows that both samples F8 and F9 have significant antioxidant potential. The radical scavenging activity of optimized CT emulgels F8 and F9 is also shown in 3.10C. tinctorius L. oil-based emulgels F8 and F9 possess antibacterial action against different test strains. The results obtained in terms of the diameter of the zone of inhibition (mm) are summarized in E. coli and S. aureus were inhibited by emulgel formulations.As a result of the study, it was found that E. coli and S. aureus as shown in E. coli with a small zone of inhibition toward S. aureus. However, pure CT oil shows a significant zone of inhibition against both bacterial strains.To determine the antibacterial activity of various formulations of emulgel, the diameter and zone of inhibition (mm) were measured and compared. The specified amount of emulgel was taken in the recent research. It was observed that CT emulgel had significant antibacterial activity against A p-value of 0.00, which is less than 0.05, means that there is a significant difference existing among the means as shown in Here, F8 shows more mean area in the zone of inhibition as compared to F9, but standard drug ciprofloxacin shows a greater zone of inhibition as compared with other formulations.E. coli and 19 mm for S. aureus. Emulgel F9 shows a zone of inhibition of 22 mm for E. coli and 15 mm for S. aureus. Pure CT oil extract shows the zone of inhibition of 25 mm for E. coli and 20 mm for S. aureus. Ciprofloxacin used as standard shows a 36-mm zone of inhibition against both E. coli and S. aureus as shown in Emulgel F8 shows a zone of inhibition of 24 mm for E. coli and 0.25 µg/100 µl and 0.05 µg/100 µl, respectively, for S. aureus as shown in S. aureus and E. coli is also shown in MIC and MBC values were 0.03 µg/100 µl and 0.06 µg/100 µl, respectively, for 4H. pylori urease, recognized for its antibacterial activity, were obtained for virtual screening. Protein Data Bank (PDB) identifiers 1R4U and 4HI0 were utilized to access these structures from the RCSB protein data library. The crystal structures of these proteins were demonstrated in their native state, with a resolution of 2.20 Å. Additionally, the crystal structures of these proteins were examined in their binding state with the investigated ligands 1_KA–10_KA. The results presented in The crystalline structures of urate oxidase, known for its antioxidant properties, and In this comparative analysis, The comparative analysis of the results in The comparative analysis of 5in vitro antibacterial activity. The disc diffusion method was used for this purpose. The diameter and zone of inhibition (mm) were measured and compared against those of bacterial strains E. coli and S. aureus as shown in Myrtus communis, used as an essential oil, correlated to their active constituents. Wazir et al. showed that the results of antibacterial activity were also due to the occurrence of tannins and flavonoids . All of the tested bacteria had a p-value of 0.00, indicating that all the formulations had an antibacterial activity that was statistically significant .E. coli and S. aureus. The MIC and MBC values of pure oil were determined by serial dilution technique, which showed significant results. Similar findings have been reported by previous articles in which Wazir A. et al. documented that different methanolic extracts like Zanthoxylum armatum, Terminalia bellirica, and Swertia chirata show the maximum zone of inhibition against both gram-positive and gram-negative strains  containing in silico results highlighted the diverse interactions between ligands and proteins, such as hydrogen bonding, hydrophobic interactions, and electrostatic interactions. These findings offer insights into the molecular mechanisms underlying ligand binding, which can have implications for future drug discovery and design. This study will focus on identifying and isolating the pure compounds from the emulgel formulations. This will help determine the individual biological effects of these compounds as antibacterial and antioxidant agents. By doing so, the study aims to contribute to developing new and safer treatment options for bacterial infections and conditions related to oxidative stress. The findings have implications for future drug development and highlight the importance of exploring the therapeutic benefits of medicinal plants in a quest for novel treatments with reduced side effects.Furthermore, the study conducted a comparative analysis of molecular docking with antibacterial and antioxidant protein targets. The The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The current research is approved by the ethical committee of the Faculty of Pharmacy, Bahauddin Zakariya University, under 196/PEC/2022. Informed consent was obtained, and ethical guidelines were followed. The approval letter confirms that all necessary precautions were taken to ensure the protection of human subjects and adherence to ethical standards. Notably, all animal experimentations were conducted in accordance with applicable laws, regulations, and guidelines, prioritizing animal welfare and minimizing any potential harm.in silico work, and contributed to manuscript refinement and improvement. GW, YJ, H-AN, and MB performed project administration, review and editing, and data validation. All authors contributed to the article and approved the submitted version.JS and SS conceptualized the study, performed the experiments, and wrote the manuscript. HJ performed review and editing. AsA, AnA, and TZ performed a formal analysis and refined and improved the manuscript. SS supervised, edited, and conceptualized the study. FS supervised the study, performed


Monensin A is a prototypical natural polyether polyketide antibiotic. It acts by binding a metal cation and facilitating its transport across the cell membrane. Biosynthesis of monensin A involves construction of a polyene polyketide backbone, subsequent epoxidation of the alkenes, and, lastly, formation of cyclic ethers via epoxide-opening cyclization. MonCI, a flavin-dependent monooxygenase, is thought to transform all three alkenes in the intermediate polyketide premonensin A into epoxides. Our crystallographic study has revealed that MonCI’s exquisite stereocontrol is due to the preorganization of the active site residues which allows only one specific face of the alkene to approach the reactive C(4a)-hydroperoxyflavin moiety. Furthermore, MonCI has an unusually large substrate-binding cavity that can accommodate premonensin A in an extended or folded conformation which allows any of the three alkenes to be placed next to C(4a)-hydroperoxyflavin. MonCI, with its ability to perform multiple epoxidations on the same substrate in a stereospecific manner, demonstrates the extraordinary versatility of the flavin-dependent monooxygenase family of enzymes. MonCI, a flavin-dependent monooxygenase, transforms all three C = C groups in the polyene substrate into epoxides during monensin A biosynthesis. Here, the authors present the structural basis for this enzyme’s regio- and stereoselective epoxidation activity. The majority of polycyclic polyethers discovered to date are of polyketide origin and are produced by actinomycete bacteria, while others are derived from terpenes and are produced by both terrestrial and marine organisms. Polyether natural products vary in the number, size, and arrangement of the cyclic ethers they contain. Ionophore polyethers  contain multiple five-to-six membered cyclic ethers connected by spiroketals or carbon-carbon bonds, whereas ladder polyethers  contain five-to-nine-membered cyclic ethers that are fused in a trans-syn-trans arrangement. In 1983, Cane, Celmer, and Westley proposed a unified model for ionophore polyether biogenesis which has been shown to be accurate for all natural polyethers studied to date3. According to this hypothesis, all ionophore polyethers are synthesized in the following manner: First, an all-E polyene is constructed from carboxylic acid building blocks by the cooperative action of type I modular polyketide synthases. Next, the polyene is oxygenated repeatedly by a monooxygenase to form the polyepoxide intermediate. Lastly, epoxides are regioselectively ring-opened by one or more epoxide hydrolases that involves an intramolecular nucleophilic attack on the epoxide groups4. In some instances, including monensin biosynthesis, additional tailoring reactions such as hydroxylation and methylation ensue to generate the final product. Recent studies suggest that the biosynthesis of ladder polyethers also follows this general pathway7.Polycyclic polyether natural products have attracted much attention due to their wide-ranging biological activities that include antimicrobial, antifungal, and antitumor propertiesStreptomyces cinnamonensis9. It acts by chelating metal cations, such as Na+ and K+, rendering them membrane permeable and thereby disrupting the physiological ion concentration gradient across the cell membrane. Monensin A is widely used as an anticoccidial agent in poultry and cattle10. Biosynthesis of monensin has been studied extensively for half a century. In 1973, Day et al. used carbon isotope labeling to show that monensin A is synthesized from five acetate, seven propionate, and one butyrate precursors11. In 1982, Cane et al. used oxygen isotope labeling to demonstrate that three of the five ether oxygens  in monensin are derived from molecular oxygen12. In 1983, Ajaz and Robinson showed that the oxygen atom attached to C26 is also derived from molecular oxygen13. In 2003, Oliynyk et al. reported the identification and analysis of the monensin biosynthetic gene cluster from S. cinnamonensis14. Monensin biosynthesis is accomplished by eight type I polyketide synthases, one monooxygenase, two epoxide hydrolases, one hydroxylase, and one methyltransferase. Oliynyk et al. also demonstrated that overexpression of monCI in a heterologous host increases its ability to epoxidize linalool, an unsaturated terpene, by as much as 20-fold, thereby establishing MonCI as the enzyme responsible for the epoxidation step in monensin biosynthesis14. In 2006, Gallimore et al. conducted gene deletion experiments which indicated that MonBI and MonBII have epoxide hydrolase activity15, which was later demonstrated directly by Minami et al. using purified recombinant proteins and a bisepoxide substrate analog16. Also in 2006, Harvey et al. determined that MonCII is a thioesterase responsible for releasing the final product from the acyl carrier protein (ACP)17. In 2014, Hüttel et al. confirmed the hydroxylation and methylation activity of MonD and MonE, respectively18.Monensin A is a prototypical ionophore polyether antibiotic produced by the soil bacterium 19. Each epoxidation reaction is carried out in a highly stereospecific manner, thus exclusively producing the -triepoxypremonensin A. We have conducted biochemical, structural, mutagenesis, and molecular dynamics simulation studies to elucidate the molecular basis of this unique enzymatic transformation. MonCI converted all three C = C groups in the model substrate farnesyl 4-hydroxybenzoate (FHB) into epoxides, providing experimental support for the hypothesis that it performs multiple epoxidations during monensin biosynthesis. Crystallographic analysis revealed that MonCI has an unusually large substrate binding pocket. This allows premonensin A to adopt at least three different poses in the active site cavity, thus enabling three separate epoxidations to take place on the same substrate molecule. Modeling study and molecular dynamics simulation showed that the prearrangement of MonCI’s substrate-binding pocket and the structure of the substrate molecule itself are responsible for the stereo- and regiocontrol exerted by the enzyme.The epoxidation step in monensin A biosynthesis is highly intriguing. A single monooxygenase enzyme, MonCI, is thought to act on premonensin A three times to produce the intermediate product triepoxypremonensin A Fig. 19. Each Escherichia coli BL21-AI and purified using Ni-affinity, anion exchange, and size-exclusion chromatography. MonCI purified as a monomer with an apparent molecular weight of 53.8 kDa  -hydroperoxyflavin29. These monooxygenases are also unique in that they use their own substrate as the electron donor instead of NAD(P)H. We measured the ultraviolet–visible spectrum of MonCI immediately following purification from the E. coli lysate and after a 12-h incubation with substrate and cofactors -hydroperoxyflavin for substrate epoxidation -hydroperoxyflavin species during the oxidative half-reaction, which in turn incorporates one oxygen atom into the substrate molecule while the other oxygen atom is reduced to water31. Reaction mixture containing 160 µM FHB, 15 µM glucose dehydrogenase, 80 mM D-glucose, 4 mM NAD+, 15 µM Fre, 300 mM NaCl, 10% glycerol, and 15 µM MonCI in 50 mM Tris, pH 8.0 was incubated for 6 h at 30 °C and then subjected to LC-MS analysis. This time, four peaks were observed. The largest peak contained mass corresponding to the unreacted FHB, the second largest peak contained mass corresponding to the monoepoxide product, the third largest peak contained mass corresponding to the bisepoxide product, and the smallest peak contained mass corresponding to the triepoxide product 33, human squalene epoxidase (RMSD = 3.0 Å)34, and bacterial p-hydroxybenzoate hydroxylase (RMSD = 3.4 Å)35 H for activity, and lacks a discrete dinucleotide-binding domain, indicating that MonCI binds NAD(P)H transiently. Class A monooxygenases are thought to follow a ping-pong type mechanism, where NAD(P)H only binds in the presence of substrate and NAD(P)+ is released prior to addition of oxygen to the substrate48. This is made possible by an ~8 Å swinging movement of the flavin isoalloxazine ring49. In the “in” conformation, the isoalloxazine ring is located immediately next to the substrate, while in the “out” conformation, the isoalloxazine ring is positioned away from the substrate-binding pocket for interaction with NAD(P)H. Based on the structural similarity of MonCI with other class A monooxygenases that have been experimentally shown to employ flavin movement, especially p-hydroxybenzoate hydroxylase, we predict that the FAD in MonCI also undergoes such movement during catalysis 50 -triepoxypremonensin A product. To elucidate the molecular basis for this exquisite stereoselectivity, we conducted a modeling study based on the MonCI crystal structure. First, we modified the FAD molecule in the crystal structure to the reactive C(4a)-hydroperoxyflavin. Here, we had the option of placing the peroxide group either on the re or si face of C4a of the isoalloxazine ring. In our MonCI crystal structure, the si face is packed against a loop (residues 55–58) and so it is unlikely for molecular oxygen to approach the isoalloxazine ring from this side. In contrast, the re face is fully exposed to the water-filled cavity, and therefore oxygen is likely to approach the flavin’s C4a atom from the re side. Therefore, we generated the structure of MonCI containing the C(4a)-hydroperoxyflavin intermediate in which the peroxide group protrudes out from the re face , and the oxygen atom could be added to either face of the alkene. Therefore, we modeled enzyme-substrate complexes that reflect each of the six possible scenarios. The only restraint we applied was that the C = C group that undergoes epoxidation must lie within 6 Å of the hydroperoxy group of C(4a)-hydroperoxyflavin. We found that steric clash with active site residues prevents the alkene to be placed adjacent to the hydroperoxy group in three of the six possible cases -, -, and -epoxide. However, positioning the substrate in a conformation that is conducive to forming the -, -, and -epoxide resulted in a steric clash. For -epoxide formation, there is a potential clash between 12-Me of premonensin A and the main chain of Ile331. For -epoxide formation, there is a potential clash between 16-Et of premonensin A and the main chain of Ile331. For -epoxide formation, there is a potential clash between 18-Me of premonensin A and the side chain of Leu58. Incidentally, the three favorable stereochemical outcomes we have identified through the modeling exercise match the stereochemistry of the natural intermediate -triepoxypremonensin A. Based on these observations, we propose that the active site pocket shape dictates the binding conformation of premonensin A, which in turn determines the stereochemical outcome of the epoxidation reaction. It is important to note that the structure of the substrate molecule also plays an important role in this type of stereocontrol mechanism. The steric clashes we have identified in the modeling study involve the 12-Me, 16-Et and 18-Me groups of premonensin A . Sequence alignment analysis of MonCI, NanO, SalC, TmnC, and Lsd18 has revealed two highly conserved regions; RKGX1PQX2RHX3HX4LWSX5GA  and AFNPX1X2GHGMSX3X4A  -hydroperoxyflavin to enable the ensuing epoxidation reaction. We conducted a modeling study to understand how premonensin A may reorganize itself inside the binding pocket of MonCI while it is tethered to MonACPX. First, we attached the 4´-phosphopantetheine prosthetic group to the conserved Ser60 of MonACPX in our MonCI-MonACPX complex model ,  and  epoxides were lower than RMSDs of non-native poses that would generate the opposite configurations, although the differences are within standard deviations.We performed atomistic molecular dynamics simulations to investigate the dynamics of the ligand-free MonCI, premonensin A (free acid) bound MonCI, and C12-C13 epoxidized substrate (free acid) bound MonCI. Premonensin A, when placed inside MonCI, is expected to take on a short and wide form as it folds into a U-shape, a longer and narrower J-shape, or a P-shape, depending on which C = C group is positioned at the active site. To examine if premonensin A in different shapes and orientations can fit stably within MonCI’s active site cavity, RMSDs of substrate-binding channel residues and RMSDs of substrate-FAD complexes were evaluated -hydroperoxy group -hydroperoxy moiety. In the systems where C16 = C17 and C20 = C21 are initially near FAD, substrates change shapes, and the C = C bonds move away from the flavin group. Overall, C12 = C13 moves away from the reaction site less than C16 = C17 and C20 = C21, and hence C12 = C13 is the more likely double bond to be epoxidized first. Tracking the stability of C12-C13 epoxidized substrate in MonCI active site further suggests that epoxidation proceeds at C16 = C17 in  configuration and then C20 = C21 in  configuration, since C16 = C17 and reactive flavin moiety stay closer to each other within the active site ( ~ 3.5 Å) than C20 = C21 and the reactive flavin moiety (~4.0 Å).Epoxidation reactions of premonensin A in MonCI could occur in a predetermined order. In some cases, probability of a reaction taking place has been shown to depend on the stability of the substrate within the enzyme’s active site pocketoup Fig. . In the The class A monooxygenases characterized to date utilize a similar mechanism for installing an oxygen atom in their respective substrate molecule. The stereochemical outcome and the position of the carbon atom(s) of the substrate that is oxygenated depends on the orientation of the substrate with respect to the reactive flavin in the enzyme’s active site. Generally, the substrate orientation is dictated by specific interactions formed between the substrate molecule and the protein residues lining the active site pocket. However, MonCI employs a different strategy in which careful coordination of the hydrophobic packing interaction between the aliphatic premonensin A and binding cavity residues controls the substrate orientation. Furthermore, MonCI’s oversized substrate-binding pocket permits premonensin A to move and reorient while it is bound to the enzyme, thereby facilitating three epoxidations at three different positions of the same substrate molecule. Another unique aspect of MonCI is that it acts on a substrate that is covalently tethered to a carrier protein. This modality is expected to confer the needed substrate specificity, presumably compensating for the fact that premonensin A is unable to establish extensive specific interactions with MonCI.33. MonCI and Bmp2, which use carrier-bound substrates, appear to have evolved separately from those that act on a free substrate such as the extensively studied 4-hydroxybenzoate hydroxylase. Our work adds another unique chemical transformation to the long list of enzymological functions performed by the superfamily of flavin-dependent monooxygenases.Phylogenetic analysis placed MonCI in a distinct clade from the other class A flavin-dependent monooxygenases  and transformed into E. coli BL21-AI. Gene fragments of MonACPX were synthesized and inserted into pET-28a (+) vector. Then PCR experiments were performed, and the genes of the control ACP were inserted into a plasmid containing an N-terminal SUMO-tag. All the mutagenesis experiments were done using overlap PCR. Sequencing was done for all the MonCI and MonACPX to confirm the accuracy of the cloning.MonCI gene from −1l-arabinose and incubated for 20 h at 15 °C. Cells were harvested by centrifugation, resuspended in lysis buffer  and lysed by sonication. After centrifugation at 18,000 × g for 40 min, the cleared supernatant was loaded onto a HisTrap column  and washed with wash buffer . MonCI was eluted with a wash buffer supplemented with 200 mM imidazole. Fractions containing MonCI were diluted with loading buffer  and subjected to anion exchange and size-exclusion chromatography using a HiTrapQ  and a Superdex 75 10/300 GL column , respectively. The protein solution was concentrated to 6.2 mg ml−1 and stored at −80 °C. The overall yield was about 1.2 mg of MonCI per liter of culture and the protein purity was >95% as judged by polyacrylamide gel electrophoresis.Protein expression was induced using 100 µM isopropyl-ß-D-galactoside and 2 mg mld-galactoside and incubated for 16–18 h at 18 °C. Cells were harvested by centrifugation, resuspended in lysis buffer  and lysed by sonication. After centrifugation at 13,000 × g for 45 min, the cleared supernatant was loaded onto a HisTrap column  and washed with wash buffer . MonACPX was eluted with a wash buffer supplemented with 250 mM imidazole. Purified MonACPX were incubated with buffer containing 20 mM Tris, pH 8.5, 150 mM sodium chloride, 2.5 mM calcium chloride and 100 U/ml thrombin for 20 h at room temperature to cleave the N-terminal His tag. The resulted MonACPX without the His-tag was further purified by size-exclusion chromatography was performed using a Superdex 75 Increase 10/300 GL column with the buffer containing 20 mM Tris, pH 7.5, 150 mM sodium chloride and 10% glycerol. The protein solution was concentrated to 11.26 mg ml−1 and stored at −80 °C.The wild-type MonACPX was induced using 100 µM isopropyl-ß-d-galactoside and incubated for 16–18 h at 18 °C. Cells were harvested by centrifugation, resuspended in lysis buffer  and lysed by sonication. After centrifugation at 13,000×g for 45 min, the cleared supernatant was loaded onto a HisTrap column  and washed with wash buffer . MonACPX mutants were eluted with a wash buffer supplemented with 250 mM imidazole. The SUMO-tag in MonACPX and DEBS ACP2 were cleaved by ulp1 and then the proteins were purified by passing through the HisTrap column  again. The proteins were further purified by anion exchange using an HiTrap Q-HP column  with buffer A as 20 mM Tris, pH 7.5, 150 mM sodium chloride, 10% glycerol and bufffer B as buffer A supplemented 1 M sodium chloride. The proteins were finally purified by size-exclusion chromatography using a Superdex 75 Increase 10/300 GL column with the buffer containing 20 mM Tris, pH 7.5, 150 mM sodium chloride and 10% glycerol. MonACPX solutions was concentrated to 11.3 mg ml−1 and stored at −80 °C. DEBS ACP2 solution was concentrated to 8.9 mg ml−1 and stored at −80 °C.MonACPX mutant and DEBS ACP2 expression was induced using 100 µM isopropyl-ß-dhg gene of Priestia megaterium was introduced into the Escherichia coli BL21(DE3) strain. The culture was grown in Luria Broth medium to OD600nm of 0.6 and expression of N-terminal His-tagged glucose dehydrogenase was induced by 100 µM isopropyl-ß-d-galactoside. The culture was incubated for another 20 h at 18 °C.pET-28a (+) vector carrying the g for 40 min, the cleared supernatant was loaded onto a HisTrap column  and washed with wash buffer . Glucose dehydrogenase was eluted with a wash buffer supplemented with 250 mM imidazole. Fractions containing glucose dehydrogenase pooled and exchanged into a buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol. The sample was concentrated to 10 mg ml−1 for storage.Cells were harvested by centrifugation, resuspended in lysis buffer  and lysed by sonication. After centrifugation at 13,000×−1 in 20 mM Tris pH 7.5, 150 mM sodium chloride, 10% glycerol) and reservoir solution  using the sitting drop vapor diffusion method. Protein crystals were transferred to a cryoprotectant solution comprised of the crystallization buffer supplemented with 20% glycerol prior to freezing. X-ray diffraction data from a native MonCI crystal and bromine soaked MonCI crystal were collected at beamline 12-2 of the Stanford Synchrotron Radiation Lightsource (SSRL). X-ray diffraction data were indexed, integrated, and scaled using the program autoXDS63, and the phase was solved by multi-wavelength anomalous diffraction using the program SHELX64. Initial MonCI model was generated using the program Buccaneer65. Iterative structure refinement and model building were carried out using the programs REFMAC66, Phenix_Refine67, and Coot54.MonCI crystals were obtained from a 1:1 mixture of protein solution (6.2 mg ml2. 4-dimethylaminopyridine (DMAP)  and N,N’-dicyclohexylcarbodiimide (DCC)  were slowly added at ice temperature. The mixture was stirred vigorously overnight at room temperature. Reaction was quenched using NaCl (3 ml std. sln). The organic layer was separated, and the aqueous phase was extracted with CHCl2 (10 mL x3). The combined organic layers were washed with NaHCO3 (80 mL), dried over Na2SO4 and the solvent was removed under reduced pressure. Flash chromatography  provided the product (0.48 g 31% yield). The final product was evaluated by nuclear magnetic resonance (NMR) (JOEL-ZETA 400 MHz and Bruker 400 MHz spectrometers). For mass spectrometry, 50 µg mL-1silver nitrate was added to the sample for better ionization. 1H NMR spectra were referenced internally to the residual proton resonance in CDCl3 (δ 1H = 7.26 ppm) as the internal standard. Chemical shifts (δ) were reported as part per million (ppm) in δ scale downfield from TMS. 1H NMR data were recorded as follows: multiplicity .Farnesol  and p-hydroxybenzoic acid  were dissolved in 60 ml of CHCl1H NMR  δ 7.95 , 6.85 , 5.44 , 5.10 , 4.81 , 2.06 , 1.68 , 1.59 . 13C NMR  δ 167.29, 160.66, 142.73, 140.51, 135.66, 131.91, 124.32, 123.84, 123.12, 118.67, 115.26, 61.98, 44.30, 39.62, 31.99, 26.85, 25.65, 23.44, 17.55, 16.04. HRMS [M+Ag]+ = 449.1238 m/z. 2Cl2 and added to FHB (0.3 g 0.88 mmol) in batches at 0 °C and left for 1 h. The reaction was quenched with 1 M NaOH and then extracted with ethyl acetate and washed with saturated NaHCO3. After drying with Na2SO4, the mixture was evaporated and separated by flash chromatography , which provided the product as a clear, colorless liquid . The final product was evaluated by NMR (JOEL-ZETA 400 MHz and Bruker 400 MHz spectrometers) and LC-HRMS .Meta-chloroperoxybenzoic acid  was dissolved in CH1H NMR  δ 7.95 , 6.85 , 5.49 , 5.24–5.05 , 4.80 , 2.70–2.75 , 1.66 , 1.33–1.20 , 0.84 . 13C NMR  δ 167.29, 160.66, 142.73, 140.51, 135.66, 124.32, 123.84, 118.67, 115.26, 64.76, 61.98, 58.90, 44.30, 39.62, 31.99, 26.85, 25.65, 23.44, 17.55, 16.04. HRMS [M + H]+ = 359.2223 m/z .+, and 10% glycerol. The reaction mixture was incubated for 6 h at 30 °C. Ethyl acetate was used to extract the unreacted substrate and product, and then vacuum evaporated to obtain samples for LC-MS analysis. Samples were dissolved in methanol and centrifuged at high speed for 10 min prior to LC-MS analysis. Large scale enzymatic assay was performed in 20 ml reaction system with 50 mM Tris, pH 8.0 buffer, 160 µM FHB, 15 µM MonCI, 4 mM NADH, 15 µM Fre, 300 mM NaCl, and 10% glycerol for 6 h at 30 °C. After the reaction, the enzymatic catalytic product was extracted by ethyl acetate, evaporated, dissolved by methanol and purified by HPLC .The reaction mixture contained either (1) 50 mM Tris, pH 8.0, 160 µM FHB, 15 µM MonCI, 4 mM NADH, 80 µM FAD, 15 µM Fre, 300 mM NaCl, and 10% glycerol, or (2) 50 mM Tris, pH 8.0, 160 µM FHB, 15 µM MonCI, 15 µM MonCI, 80 µM FAD, 300 mM NaCl, 15 µM GDH, 80 mM D-glucose, 4 mM NADm/z) was applied with an Agilent ZORBAX Eclipse XDB-C18 column at a flow rate of 0.2 mL/min and column temperature of 30 °C. Buffer A was 99.9% H2O and 0.1% formic acid, and buffer B was 99.9% methanol and 0.1% formic acid. Mass spectra were collected in positive ion mode 100–1000 m/z. The program was set as: 60% B at time 0–0.5 min, 60% B and 60–95%B from 0.5 to 6 min, 95–60% B from 6 to 8 min, 60% B from 8 to 9 min. For FHB epoxidation products, peak 365.2093+ m/z, 381.2042+ m/z, 397.1991+ m/z were selected for analysis.The control experiments of the enzymatic assay without MonCI or cofactor NADH were performed with other conditions the same for the reactions. The products were extracted by ethyl acetate, vacuum evaporated, dissolved in methanol and and centrifuged at high speed for 10 min prior to LC-MS analysis. LC-MS  in a negative mode. The observed m/z was 357.2072 matching the theoretical Monoepoxide [M-H]- = 357.2060 m/z.To quantify the amount of the enzymatic catalytic product, HPLC (Shimadzu LC-2010A HT) analysis was performed. Buffer A was Hm/z) was employed to determine the mass of the substrate and products from the MonCI catalytic reactions. The products of reaction (1) and (2) were processed the same as the above methods and applied to column Kinetex 2.6um C18 100 with a flow rate of 0.3 ml/min and column temperature of 30 °C.The program was set as: 5% B at time 0–1 min 5–95% B from 1 to 3 min, 95% B from 3 to 8 min, and 95% B–5% B from 8 to 8.1 min. For the enzymatic reaction carried out under condition (1), the detected masses were 341.2119 m/z for FHB, 357.2072 m/z for monoepoxide, and 373.2019 m/z for bisepoxide. For the enzymatic reaction carried out under condition (2), the detected masses were 343.2269 m/z for FHB, 359.2221 m/z for monoepoxide, 375.2177 m/z for bisepoxide, and 391.2128 m/z for triepoxide. These mass spectra were collected in positive ion mode.High-resolution LC-MS  was added to 100 µL 160 µM purified enzymatic catalytic product, 160 µM synthetic 17,18-epoxy-4-hydroxybenzoate standard in methanol, or methanol. Then the mixture was transferred to three centrifuge tubes and heated in an 80 °C oven for 2.5 h. Then the tubes were taken out and put on ice for 10 min and 66 µL triethylamine acetone solution (1:1 mixture) was added immediately to the mixture. After high-speed centrifugation, the supernatants were scanned with an ultraviolet spectrophotometer (UV1800 PC) for an absorption peak at a wavelength between 200 and 700 nm, and a spectrogram was drawn.68 and ESPript server69, which identified the ACP domain from module 5 of MLSA1 (PDB ID: 6H0J) as the best template structure to use in homology modeling of MonACPX. The homology structure of MonCI was generated using the program MODELLER70. Evaluation of structural models was performed by calculation of z-DOPE score with MODELLER program and the best MonACPX model was selected with comparing the score for all residues. Visualization and image processing were performed by Pymol and Maestro 12.2  program.Sequence alignment of ACPs was performed by CLUSTALW71. Docking was performed using the MonCl crystal structure and the homology model of apo-MonACPX. The HADDOCK2.2 webserver’s default input settings were used, and passive residues were assigned automatically around the active residues. Active residues for MonCl were those located near the substrate-binding pocket entrance (side entrance): Asp377, Asp378, Ile381, Thr398, Asp399, Pro400, Arg401, Leu402, Ile403, Gly404, Val405, Asp406, Gln409, Arg412, Phe413, Pro440, Gln441, Ala442, Glu443, Gly445, Ser446, Asn447, Arg448, and Leu450. Two docking runs were performed using different active residues for ACP. First run was performed defining a narrower  and second run a wider  selection of active residues surrounding Ser60 of MonACPX, the attachment site for the 4´-phosphopantetheine group. Among the docked structures that were generated, we eliminated those in which Ser60 was not aligned with the side entrance and then chose the solution that had the best HADDOCK score (−86.2 ± 3.6) as the final model MonACPX-MonCI.Structures of the MonACPX-MonCI complex were predicted using the HADDOCK2.2 webserverpsfgen plugin72. For simulations which included premonensin A, the substrate was manually positioned within the channel with the substrate orientation varying by system. The initial substrate position was an approximate binding pose with the reactive double bond placed ~5 Å from and perpendicular to the O-H bond of the reactive −OOH group of FAD. The systems were solvated and ionized using the VMD solvate and ionize plugins72. Final systems contained approximately 78,000 atoms, with a salt concentration of 150 mM NaCl to mimic physiological conditions.Each simulated system contained MonCI, FAD, and except for one system, premonensin A. The MonCI and FAD coordinates were obtained from chain A of the crystal structure (PDB ID: 8T3P). 481 water molecules that are in the crystal structure and are associated with chain A were also included in simulations. In all cases, hydrogen atoms were added using the VMD 73 and the TIP3P water model, with the CHARMM general force field (Cgenff)77 used to model the substrate. MD simulations were performed in the NpT ensemble using the NAMD2.13 package78. Temperature and pressure remained constant at 310 K and 1.01325 bar, respectively. The Langevin constant was γLang = 1 ps−1. A simulation timestep of 2 fs was used, with evaluation of van der Waals interactions at every timestep and evaluation of long-range Coulomb interactions performed every two timesteps using the particle-mesh Ewald method79.Interactions for simulated molecules were defined using the CHARMM36 force-field parameters2). A further 1 ns of equilibration was performed with only the heavy atoms of the protein-FAD-substrate complex restrained, followed by 20 ns of equilibration in which disordered coil protein residues were not restrained. A final 20 ns equilibration run was performed with harmonic restraints present only on the substrate atoms constituting the double bond nearest FAD. Production runs, with all restraints released, were performed for 200 ns. The analyses were performed during the last 100 ns of the production runs.All systems were initially minimized for 2,000 steps, after which water and ions were equilibrated for 1 ns while the protein-FAD-substrate complex was restrained using harmonic forces with a spring constant of 1 kcal/. The ITC data was integrated with a “one site” model. All titration experiments were performed twice. The software Origin 7.0 (OriginLab) was used for data analysis.All protein samples were buffer exchanged into buffer 100 mM phosphate, pH 8.0. All the samples were degassed by centrifugation at 15000 rpm for 10 min. When using wild-type MonACPX to titrate wild-type MonCI, wild-type MonCI (60 μM) was in the cell while wild-type MonACPX (1.2 mM) was in the syringe. When using control ACP to titrate wild-type MonCI, wild-type MonCI (100 μM) was in the cell while control ACP (1.0 mM) was in the syringe. The concentrations of MonCI were determined by UVE. coli) was scanned to obtain the MonCI-Flox[O] spectrum. After incubating MonCI with FHB for 12 h, the sample was buffer exchanged to the enzymatic reaction buffer to remove the cofactors as well as the substrate and product and was scanned to obtain the spectrum of MonCI-Flox.The flavin absorption spectra of purified MonCI were analysed with a Jinghua UV1800 PC spectrophotometry. Untreated MonCI .The phylogenetic tree was calculated based on the amino acid sequence alignment of biochemically characterized enzymes’ sequences by using the ClustalW method in MEGA11 as reported by Adrie H. Westphal et al.Further information on research design is available in the Supplementary InformationReporting SummarySource data file


At present, memristive neural networks with various topological structures have been widely studied. However, the memristive neural network with a star structure has not been investigated yet. In order to investigate the dynamic characteristics of neural networks with a star structure, a star memristive neural network (SMNN) model is proposed in this paper. Firstly, an SMNN model is proposed based on a Hopfield neural network and a flux-controlled memristor. Then, its chaotic dynamics are analyzed by using numerical analysis methods including bifurcation diagrams, Lyapunov exponents, phase plots, Poincaré maps, and basins of attraction. The results show that the SMNN can generate complex dynamical behaviors such as chaos, multi-scroll attractors, and initial boosting behavior. The number of multi-scroll attractors can be changed by adjusting the memristor’s control parameters. And the position of the coexisting chaotic attractors can be changed by switching the memristor’s initial values. Meanwhile, the analog circuit of the SMNN is designed and implemented. The theoretical and numerical results are verified through MULTISIM simulation results. Finally, a color image encryption scheme is designed based on the SMNN. Security performance analysis shows that the designed cryptosystem has good security. Because of its rich chaotic dynamics, Hopfield neural networks (HNNs) have attracted much attention from chaos scholars ,2,3. It Since the HNN was proposed, various HNNs have been proposed and their chaotic behaviors studied. For example, normal chaos ,10,11 anThe multi-scroll attractor is a complex chaos phenomenon ,25,26, wInitial boosting behavior is a kind of complex behavior that shows extreme multistability ,34. GeneThis paper investigates the chaotic dynamics of the star memristive neural network and its application in color image encryption. To the best of our knowledge, this is the first time that the chaotic dynamics of the star neural network has been investigated. The main novelty and contributions of this study are summarized as follows. (1) We propose a star memristive neural network model based on one Hopfield neural network with four neurons and one memristor. (2) The star memristive neural network exhibits rich and complex multi-scroll attractors and initial boosting dynamics. (3) A star memristive neural network circuit is realized and numerical simulation results are further demonstrated. (4) We design a color image encryption cryptosystem by using the multi-scroll sequences to show the practical application of the presented star memristive neural network.The rest of this paper is organized as follows. A star memristive neural network model is constructed in i, v, and φ are the current, voltage, and state variables of the memristor, respectively. The function h(φ) is the state function of the memristor, where N and M are two control parameters. The memristor model can exhibit typical memristive characteristics, as shown in A = 1, 2, 3). F = 0.05, 0.1, 0.5), the area of the tight hysteresis loop of the memristor decreases gradually.Memristors are usually used to describe electromagnetic induction in the nervous system caused by electromagnetic radiation. Here, a flux-controlled multi-segment nonlinear memristor is introduced . Its matiC, iR, and ix are the membrane capacitance, membrane resistance, and membrane potential of the neuron i, respectively. ijw is the synaptic weight coefficient between neuron j and neuron i. In addition, tanh(.) denotes the neuron activation function, and iI is an external input current. Based on the original HNN (3), setting iC = 1, iR = 1, and iI = 0, a new HNN with four neurons is proposed as follows:ix is the membrane potentials of neuron i. As shown in φ is the magnetic flux across the membrane of neuron 1, µ is the feedback intensity of external stimuli induced by electromagnetic radiation, and µw(φ)x1 is the electromagnetic induction current.A Hopfield neural network with a brain-like structure can be used to emulate chaotic behaviors of the biological nervous system. A Hopfield neural network with n neurons can be described by (3)Cix˙iThe equilibrium points of the star memristive neural network and their stabilities are analyzed in this subsection. Setting the left side of Equation (5) to 0, the equilibrium points are solved bySolving Equation (6), one can obtainis = sech2(ix). When setting a = 1, b = 0.01, c = 0.7, and d = 1.3, the corresponding eigenvalues and stabilities are given in Obviously, there are infinite discrete equilibrium points. The Jacobian matrix of each equilibrium point can be calculated byIn this section, chaotic dynamics of the proposed star memristive neural network are revealed by using nonlinear dynamics methods including bifurcation diagrams, Lyapunov exponents, phase plots, Poincaré maps, and basins of attraction. It should be noted that the numerical simulations are completed in MATLAB R2017a with the ODE45 algorithm. Meanwhile, the start time, the time step, and the time length are set as 500, 0.01, and 4000, respectively.a = 1, b = 0.01, c = 0.7, d = 1.3, N = 0, and initial conditions  = , the parameter µ increases from −0.2 to 0, and the µ-based bifurcation diagram is plotted in x1max is the maxima of the membrane potential x1. Moreover, the corresponding Lyapunov exponents are shown in µ.In this subsection, the chaotic dynamics related to the memristive coupling strength µ is analyzed. Setting the parameters a = 1, b = 0.01, d = 1.3, µ = −0.01, N = 3, and initial states are kept unchanged, c is selected as the control parameter. A bifurcation diagram and the corresponding Lyapunov exponents related to parameter c∈ are shown in N + 1 and 2M + 2.In this subsection, the chaotic dynamics related to memristor control parameters N/M are analyzed. The numerical simulation shows that the SMNN can generate an arbitrary number of multi-scroll chaotic attractors related to N/M. When the parameters φ-x3 and φ-x4 phase planes for the 7-scroll attractor with x1 = 0 are depicted in In addition, the Poincaré maps on 0 under a = 1, b = 0.01, c = 0.5, d = 1.3, µ = −0.01, M = 4, and x10 = x20 = x30 = x40 = 0.1, as shown in φ0 variation are given in φ0-axis. That is to say, the SMNN enjoys complex dynamics of initial boosting coexisting chaos, which means that it has excellent robustness. Moreover, to further verify initial boosting dynamics, the local attraction basin in the φ0-x10 plane is given in In this subsection, the initial boosting chaotic phenomenon is analyzed. It is wonderful that the presented SMNN can generate initial boosting coexisting chaos. For instance, we plot the bifurcation diagram of the φWith the rapid development of artificial intelligence, the physical realization of neural network models is very important for developing neuromorphic hardware systems ,43. It ix1, x2, x3, and x4 are emulated by four out voltages v1, v2, v3, and v4, respectively. All synaptic weight coefficients are simulated by the resistors R1-R10. Based on the circuit in Before implementing the neural network circuit, we first introduce a hyperbolic tangent excitation function circuit  and a meC1 = C2 = C3 = C4 = C = 1 nF, RC = 10 us, and resistance R = 10 kΩ, then C can be chosen as 1 nF. Considering the fixed synaptic weight coefficients, part resistors can be calculated as R1 = 8.3 kΩ, R2 = 2.7 kΩ, R3 = 2.7 kΩ, R4 = 0.9 kΩ, R5 = 10 kΩ, R6 = 100 kΩ, R7 = 12.5 kΩ, R8 = 5.5 kΩ, R9 = 16 kΩ, and R10 = 5 kΩ. In addition, Ra = R/aµ, Rb = Rg/bµ, Rc = R/c, and Rd = R/d are adjustable resistors. In the above formula, g represents the factor of the multiplier M. In this circuit, g is equal to 0.1.Assuming that a = 1, b = 0.01, µ = 0.01, c = 0.7, d = 1.3, and initial values , and setting Ra = 1 MΩ, Rb = 10 MΩ, Rc = 13.9 kΩ, Rd = 8 kΩ, and initial capacitor voltages to 0.1 V, multi-scroll attractors can be generated by selecting different switches. For example, closing W1, W2, and W3 and setting e1 = 2 V and e2 = 4 V, a six-scroll chaotic attractor can be generated from the designed circuit, as shown in Rc = 20 kΩ (c = 0.5), the initial boosting coexisting attractors can be realized by adjusting different initial capacitor voltages φv0. As shown in Rc = 13.9 kΩ; closing W2, W3, and W4; and setting e1 = 1 V, e2 = 3 V, and e3 = 5 V, a seven-scroll chaotic attractor can be obtained, as shown in Rc = 20 kΩ, seven coexisting chaotic attractors can be obtained as shown in The designed star memristive neural network circuit is verified by using the MULTISIM platform. When Chaos can be used for information encryption due to its high randomness and sensitivity ,45. ChaoThe encryption and decryption process is described in the following steps.a = 1, b = 0.01, c = 0.7, d = 1.3, µ = 0.01, N = 3, and initial states , the proposed SMNN is iterated N0 + mn times, and the last mn iterations are regarded as valid data. Each iteration can generate five chaotic seven-scroll sequences x1(i), x2(i), x3(i), x4(i), and φ(i), where i = .Step 1: Setting x) is the modulo operation and floor(x) is the flooring operation.Step 2: To obtain a pseudo-random sequence, the generated sequences are preprocessed asStep 3: A chaotic sequence is generated, which can be described asK(i) is used to encrypt the original image using the XOR operation, as follows:Step 4: To demonstrate the efficiency of the designed image encryption cryptosystem, three color images “Lena”, “virus”, and “chameleon” with a size of 512 × 512 are chosen as the encryption object. The experimental results and the security performance analyses including the histogram, correlation coefficient, information entropy, key sensitivity, data loss, and noise attacks are given in the following.(1) Histogram analysis:Histograms are used to evaluate the distribution of pixel intensity values in an image. In theory, a good image encryption system should produce a uniform histogram. x and y are the intensity values of two adjacent pixels. Here, 10,000 pairs of pixels were randomly chosen in horizontal, vertical, and diagonal directions from the original image and corresponding encrypted image to evaluate the correlation coefficient. As shown in (2) Correlation analysis: The relationship between adjacent pixels in an image can be described using correlation. In general, a stronger correlation indicates a more regular image and a weaker correlation indicates a more chaotic image. The correlation coefficient is computed by (14)ρxy=P(ix) denotes the probability of ix and 2N represents the number of the information source. The maximum theoretical information entropy is 8. (3) Entropy analysis: Information entropy can be used to evaluate the randomness of image information. The information entropy is calculated as (15)H(P)(4) Sensitivity analysis: Key sensitivity is an important indicator for measuring the security of encryption algorithms. A good image encryption scheme should be key-sensitive. The initial values are used as secret keys in this encryption algorithm. The decrypted images are shown in (5) Data loss and noise attacksData loss and noise attacks can seriously affect the decryption effect of encrypted images. To evaluate the ability of data loss and noise attacks, we cut off some parts of the encrypted image and then decrypt it. As shown in In this article, the chaotic dynamics of a star memristive neural network are studied. First, based on a Hopfield neural network with four neurons and a flux-controlled memristor, a star memristive neural network model is constructed. Then, its chaotic behaviors are revealed by using various numerical methods. Analysis results show that the star memristive neural network can generate abundant chaotic dynamics including chaos, multi-scroll attractors, and initial boosting coexisting behavior. Especially, the number of scrolls for the multi-scroll attractors can be changed by adjusting the memristor’s control parameters. In addition, the position and number of coexisting attractors can be changed by switching the memristor’s initial value. To further verify these results, an analog neural network circuit is designed and implemented. All numerical results are experimentally verified by MULTISIM circuit simulation. Finally, a color image encryption scheme based on the proposed star memristive neural network is designed. Simulation results such as histograms, correlation, information entropy, key sensitivity, and data loss and noise attacks demonstrate that the designed image encryption scheme has good security.With the rapid development of memristors, memristor-based neural networks have been widely used in various fields including memristive neurodynamics ,52, memr


Both mTOR and α-klotho play a role in the pathophysiology of renal disease, influence mineral metabolism and participate in the aging process. The influence of mTOR inhibition by rapamycin on renal α-klotho expression is unknown. Rats with normal (controls) and reduced (Nx) renal function were treated with rapamycin, 1.3 mg/kg/day, for 22 days. The experiments were conducted with rats fed 0.6% P diet (NP) and 0.2% P diet (LP). Treatment with rapamycin promoted phosphaturia in control and Nx rats fed NP and LP. A decrease in FGF23 was identified in controls after treatment with rapamycin. In rats fed NP, rapamycin decreased mRNA α-klotho/GADPH ratio both in controls, 0.6±0.1 vs 1.1±0.1, p = 0.001, and Nx, 0.3±0.1 vs 0.7±0.1, p = 0.01. At the protein level, a significant reduction in α-klotho was evidenced after treatment with rapamycin both by Western Blot: 0.6±0.1 vs 1.0±0.1, p = 0.01, in controls, 0.7±0.1 vs 1.1±0.1, p = 0.02, in Nx; and by immunohistochemistry staining. Renal α-klotho was inversely correlated with urinary P excretion . The decrease in α-klotho after treatment with rapamycin was also observed in rats fed LP. In conclusion, rapamycin increases phosphaturia and down-regulates α-klotho expression in rats with normal and decreased renal function. These effects can be observed in animals ingesting normal and low P diet. Streptomyces hygroscopicus, is the classic inhibitor of the mTOR (molecular target of rapamycin) pathway that integrates nutrient signals to control energy metabolism [Rapamycin, a macrolide antibiotic produced by tabolism . mTOR istabolism , 3. Rapatabolism  and it itabolism .Activation of the mTOR pathway is thought to participate in the pathophysiology of renal disease , 7, partRapamycin also has effects on mineral metabolism -e.g. rapamycin is known to promote phosphaturia in healthy mice by inhibiting renal tubular transport of phosphate (P) . This isIn addition, downregulation of the mTOR pathway may be involved in slowing aging and increasing lifespan . In factα-Klotho, a protein that is produced predominantly in the kidneys, has actions closely linked to those of mTOR, namely: a) it is related to kidney disease—α-klotho levels have been reported to decrease in a variety of renal disorders and α-klotho deficiency is known to facilitate progression of kidney disease ; b) it pThere is very little information on the effect of mTOR inhibition by rapamycin on renal α-klotho expression and the data available, which is to some extent contradictory, has been obtained mainly in renal transplant recipients who are receiving a variety of drugs –24. ThusAll experimental protocols were reviewed and approved by the Ethics Committee for Animal Research of the University of Cordoba and by Junta de Andalucia (Spain) . All protocols were carried out in accordance with the approved guidelines. They followed the guiding principle laid down by the Higher Council of Scientific Research of Spain following the normal procedures directing animal welfare and adhered to the recommendations included in the Guide for Care and Use of Laboratory Animals  and European laws . All animal welfare considerations were taken, including effort to minimize suffering and distress, use of analgesics and anesthetics and special housing conditions. All research personnel was trained and certified for animal experiments.Female Wistar rats provided by the Animal Housing Facilities of the University of Cordoba  were used in the experiments. Rats, aged 2 months at the beginning of the studies, were housed with a 12h/12h light/dark cycle and were fed a standard diet  containing normal amounts of calcium (Ca), and vitamin D: 0.6% of Ca and 500 IU/g of vitamin D. The Na content of the diet was 2480 mg/kg and the Cl content 1984 mg/kg. The diet had either normal (0.6%) or low (0.2%) P concentration. Daily food intake was recorded for each rat by averaging the amount of food eaten every week. Health and behaviour were monitored daily. Humane endpoints were used in the study. The specific criteria to determine when animals should be euthanized before ending the study included: complete anorexia, inability or extreme reluctance to stand, signs of severe organ system dysfunction, and pain and/or distress not responsive to analgesics. Animals were scheduled to be sacrificed 24 h after exhibiting any of these signs.Renal function was reduced by nephrectomy with ablation of 5/6 of renal mass (5/6Nx). Before performing surgery, rats were anesthetized using inhaled isoflurane . 5/6 Nx was performed in two steps. In the first step a 5- to 8-mm incision was made on the left mediolateral surface of the abdomen. The left kidney was exposed, and the two poles  were ablated. The kidney was inspected and returned to an anatomically neutral position within the peritoneal cavity. The abdominal wall and skin incisions were closed with sutures, and the rat was placed back into its home cage. After 1 week of recovery, in the second step, the animal was reanesthetized, a 5- to 8-mm incision was made on the right mediolateral surface of the abdomen. The right kidney was exposed and unencapsulated, the renal pedicle was clamped and ligated, and the kidney was removed. The ligated pedicle was returned to a neutral anatomical position and the abdomen and skin incisions were closed with suture materials. Fentanyl, 0.2 mg/kg, ip  was used as analgesic agent.Two sets of experiments were conducted. In the first one rats were fed a normal P diet (NP) while in the second experiment rats were fed a diet with low P content (LP). The design was identical in both experiments. Thus, in each experiment, rats with intact renal function and with reduced renal function (Nx) were allotted to 2 groups (n = 16–20). Half the rats received placebo treatment  and the other half were treated with rapamycin (Rap) at a dose of 1.3 mg/kg/day   was administered daily in unflavored gelatin tablets (80 mg of pure gelatin dissolved in 1 ml of distilled water). The placebo groups were fed gelatin tablets without rapamycin. Visual confirmation of complete consumption of the gelatin tablet was always obtained. Rats received the treatments for 22 days. None of the animals reached the humane endpoints and no animal died spontaneously before the end of the experiments. Accordingly, all animals were euthanized at the end of the study. Euthanasia was performed, after overnight fasting, by exsanguination under general anesthesia . Blood samples, drawn from the abdominal aorta, and kidney samples were collected. Urine samples were obtained in the last 3 days prior to sacrifice by placing the rats in metabolic cages.Immediately after obtaining the blood sample, plasma was separated by centrifugation and stored at –20°C until assayed. Plasma concentrations of urea, creatinine, Ca, and P as well as urine Ca and P were measured by spectrophotometry . ELISA tests were used to quantify plasma concentrations of intact FGF23 , parathyroid hormone (PTH) , and cystatin C .-ΔΔCt method.Total RNA was isolated from renal tissue using TRIzol reagent protocol  and a treatment with DNase I amplification Grade  was done according to the manufacturer’s instruction. Quantification was performed by spectrophotometry . The sequence of primers used for RT-PCR is shown in Supplementary data, Proteins were isolated from renal tissue by using a lysis buffer HEPES (10 mmol/l), KCl (10 mmol/l), EDTA (0.1 mmol/l), EGTA (0.1 mmol/l), DTT (1 mmol/), PMSF (0.5 mmol/l), protease inhibitor cocktail (70 μg/ml), and I-Gepal CA-630 (0.6%), pH 7.9 . Protein concentration was determined by the Bradford method. For Western blot analysis, 50 μg of protein was electrophoresed on a 4–20% SDS-polyacrylamide gel  and electrophoretically transferred  from the gels onto nitrocellulose membranes . The following steps were performed with gentle shaking. Membranes were incubated in Tris-buffered saline with Tween 20 (TBST) solution (20 mM Tris-HCl (pH 7.6), 0.2% Tween 20, 150 mM NaCl) , and 5% nonfat dry milk  at room temperature for 1h to avoid nonspecific binding. Membranes were then washed with TBST buffer and incubated overnight at 4°C with an anti-⍺-klotho antibody . The membranes were then washed with TBST buffer and immunolabeled using a peroxidase-conjugated secondary antibody . Finally, they were revealed on autoradiographic film using ECL Western Blotting Detection Substrate . GAPDH  was used as a housekeeping protein to ensure equal loading of the gels. Protein levels were quantified using ImageJ software .-klotho antibody  at 1:100 dilution. After rinsing, the sections were incubated for 30 min at room temperature with the ImmPRESS Universal Antibody polymer reagent  and treated with diaminobenzidine substrate for 1 min . Every step was followed by three washes with phosphate-buffered saline for 5 min. Finally, sections were counterstained with haematoxylin . Assessment of α-klotho was performed in 8 randomly selected fields per kidney by measuring optic density (units) in renal tubules expressing α-klotho, using ImageJ software .Kidney samples were fixed in 10% formalin and embedded in paraffin. Three μm sections were deparaffinized, rehydrated and microwave-treated in 0.01 mmol/l citrate buffer (pH 6) for 20 min. Then, samples were incubated with endogenous enzyme blocking solution  for 10 min. Sections were blocked with normal horse serum 2.5%  for 20 min. After that, sections were incubated overnight at 4°C in a humidified chamber with primary mouse anti-⍺Values are expressed as the mean ± standard error (SE). The difference between means for two different groups was determined by t-tests; the difference between means for three or more groups was assessed by ANOVA. Fisher LSD test was used as a post-hoc procedure. A correlation study was carried out using the Pearson test. A p<0.05 was considered significant.Although there were no differences in food intake between groups , in the An increase in plasma creatinine, 0.97 ± 0.04 mg/dl, urea, 70.4 ± 5.3 mg/dl, and cystatin C, 2362 ± 84 ng/ml, was observed in NxNP rats when compared with NP rats, 0.60 ± 0.02 mg/dl, 31.9 ± 0.9 mg/dl and 1279 ± 63 ng/ml. Rapamycin treatment did not influence renal function parameters in rats with normal renal function and only produced a small increase in cystatin C in Nx rats .Plasma P concentration was not affected by Nx but tended to be decreased in rats treated with rapamycin and the differences reached significance in Nx rats, 5.6 ± 0.3 vs 6.6 ± 0.3 mg/dl, p = 0.02 . UrinaryPlasma Ca concentration did not show intergroup differences . ChangesPlasma concentrations of FGF23 were much higher in Nx rats, 1358 ± 324 pg/ml, than in rats with intact renal function, 311 ± 17 pg/ml, p = 0.0001. Treatment with rapamycin significantly (p<0.05) reduced FGF23 in rats with intact renal function, 259 ± 16 pg/ml, but not in Nx rats, 1246 ± 176 pg/ml . An incrRenal α-klotho expression is presented in To obtain a wider understanding of the role of rapamycin-induced phosphaturia on the observed changes in renal α-klotho expression, additional experiments were performed in rats that had a very low urine P concentration secondary to feeding a diet with low (0.2%) P content.In these experiments, rapamycin treatment also reduced body weight gain in rats with normal renal function 15.3 ± 23 vs 29.2 ± 3.2 g, p = 0.009, but not in Nx rats 6.2 ± 4.0 vs 10.0 ± 3.3 g.Changes in biochemical parameters elicited by Nx and by treatment with rapamycin in rats fed LP were in agreement with what had been observed in the experiments with NP. No intergroup differences were observed in either plasma Ca or P. Both FGF23 and PTH were much higher in Nx groups than in rats with normal renal function and rapamycin treatment decreased FGF23 only in rats with intact renal function, 205 ± 19 vs 310 ± 30 pg/ml, p<0.05 .Urinary excretion of P was unaffected by Nx but was significantly increased by rapamycin both in rats with normal renal function, 2.04 ± 0.46 vs 0.47 ± 0.08 mg/day, p = 0.04, and in Nx rats, 2.46 ± 0.57 vs 0.44 ± 0.08 mg/day, p = 0.009. Urinary excretion of Ca was increased only in Nx rats receiving rapamycin, 5.26 ± 0.42 vs 3.11 ± 0.44, p = 0.02 .Rapamycin treatment reduced renal α-klotho expression in rats with intact and reduced renal function fed 0.2% P at both mRNA and protein levels. Thus, mRNA α-klotho/GADPH ratio was significantly lower, p = 0.0001, in LP-Rap rats, 0.4 ± 0.01 than in LP rats, 1.0 ± 0.1; and it was also lower, p<0.0001, in NxLP-Rap rats 0.1 ± 0.03 than in NxLP rats, 0.9 ± 0.1 . At the This study demonstrates downregulation of renal α-klotho expression in rats after treatment with rapamycin. The results were consistent both at the mRNA and protein levels and were observed in rats with intact and decreased renal function and with different dietary loads of P. The effect of rapamycin on klotho seems to be partly mediated by changes in phosphaturia.Rapamycin is best known for its immunosuppressive effects  and for Additionally, rapamycin also has an impact on mineral metabolism. In an elegant study, Kempe et al. demonstrated that rapamycin influences phosphate balance by promoting phosphaturia in healthy mice . Althougα-Klotho is a protein, mainly expressed in the kidneys, that is essential for the phosphaturic action of FGF23 and may have an important role in the pathogenesis of several kidney diseases , 19. WheThe effect of rapamycin on renal α-klotho has been previously explored but the few reported data, which are somewhat contradictory, are difficult to interpret because in many instances rapamycin was administered together with other drugs. Han et al. investigated the effect of cyclosporine, tacrolimus, and rapamycin on renal α-klotho expression in mice. They found that both cyclosporine and tacrolimus decreased α-klotho but rapamycin did not. However, when rapamycin was added to either cyclosporine or tacrolimus a further decrease in α-klotho was identified . InfanteWe observed a consistent decrease in renal α-klotho expression (mRNA and protein) after treatment with rapamycin, both in rats with intact and decreased renal function. A possible explanation for this unexpected finding may be related to the phosphaturic effect of rapamycin. An increased tubular load of P may lead to tubular injury through the formation of Ca-P microcrystals that bind to Toll-like receptor 4 (TLR4) . In factTo further investigate the role of urine P on the decrease in renal α-klotho the studies were repeated in rats fed low P diet. The results obtained in rats fed low P diet were practically identical to those previously recorded with normal P diet. Urinary excretion of P was very low in rats fed low P but was also increased by rapamycin. Again, a consistent decrease in renal α-klotho expression was detected in rats treated with rapamycin. Overall, these results would lend support to the hypothesis that the increase in urinary P down-regulates α-klotho. On the other hand, it seems clear that such regulation would not require a high level of P in urine, because even though urine P was increased when compared with placebo treated rats, it was still very low in the rats fed 0.2% P and treated with rapamycin.It is interesting to note that renal α-klotho has been shown to increase in rats subjected to caloric restriction . CaloricIn conclusion rapamycin increases phosphaturia and down-regulates α-klotho expression in rats with normal and decreased renal function. These effects can be observed in animals ingesting normal and low P diet.S1 FigCa, calcium; LP, rats with intact renal function fed a low phosphorus diet; LP-Rap, rats with intact renal function fed a low phosphorus diet and treated with rapamycin; NP, rats with intact renal function fed a normal phosphorus diet; NP-Rap, rats with intact renal function fed a normal phosphorus diet and treated with rapamycin; NxLP, nephrectomized rats fed a low phosphorus diet; NxLP-Rap, nephrectomized rats fed a low phosphorus diet and treated with rapamycin, NxNP, nephrectomized rats fed a normal phosphorus diet; NxNP-Rap, nephrectomized rats fed a normal phosphorus diet and treated with rapamycin; P, phosphorus.(TIF)Click here for additional data file.S2 FigScatterplot showing correlation between renal mRNA α-klotho expression and daily urinary excretion of phosphorus (P) in rats fed normal P diet.(TIF)Click here for additional data file.S1 Raw imagesImages of the unprocessed nitrocellulose membranes used for Western Blot analysis. LP, rats with intact renal function fed a low phosphorus diet; LP-Rap, rats with intact renal function fed a low phosphorus diet and treated with rapamycin; NP, rats with intact renal function fed a normal phosphorus diet; NP-Rap, rats with intact renal function fed a normal phosphorus diet and treated with rapamycin; NxLP, nephrectomized rats fed a low phosphorus diet; NxLP-Rap, nephrectomized rats fed a low phosphorus diet and treated with rapamycin, NxNP, nephrectomized rats fed a normal phosphorus diet; NxNP-Rap, nephrectomized rats fed a normal phosphorus diet and treated with rapamycin; P, phosphorus.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.