G′ and loss modulus G″ was observed. The plasticizing effect of trisilanolisooctyl POSS (HO-POSS) due to the presence of long isoctyl groups was confirmed. As a result, the crystallization of PCL was facilitated and the degree of crystallinity of cχ increased up to 50.9%. The damping properties and the values of tan δ for PCL/HO-POSS composition increased from 0.052 to 0.069. The TGA results point out the worsening of the PCL thermal stability, with lower values of T0.5%, T1% and T3%. Both HO-POSS and Gly-POSS facilitated the relaxation of molten PCL. The presence of Gly-POSS influenced the changes that occurred in the viscoelastic properties of the molten PCL due to the thermo-mechanical degradation of the material; a positive impact was observed. Polyhedral oligomeric silsesquioxane POSS nanoparticles can be applied as reinforcing additives modifying various properties of biodegradable polymers. The effects of aminopropylisobutyl POSS (amine-POSS), trisilanolisooctyl-POSS (HO-POSS) and glycidyl-POSS (Gly-POSS) on the viscoelastic, thermal properties and crystallization of biodegradable poly(ε-caprolactone) PCL were studied. The analysis of the viscoelastic properties at ambient temperature indicated that aminopropylisobutyl POSS (amine-POSS) and glycidyl-POSS (Gly-POSS) enhanced the dynamic mechanical properties of PCL. The increase in the storage shear modulus  In modern society, the use of nondegradable low-cost products based on polyethylene (PE), polypropylene (PP) and polystyrene (PS) is growing every day, generating unrecycled plastic waste . Most ofBiodegradable polyester materials: poly(lactic acid) PLA, poly(glycolic acid) PGA and polycaprolactone PCL are the class of polymeric materials that show unique properties, biocompatibility and degradation ability, together with excellent mechanical behavior ,8. Amongm in the range 59–64 °C, a glass transition temperature Tg of about −60 °C and elasticity at ambient temperature [Polycaprolactone is a high-strength, crystalline polymer, with a low melting temperature Tperature . Its bioperature , bone tiperature ,13, stenperature  and elecperature . The application of PCL as hot-melt adhesive for packaging and bookbinding due to its flexibility and thermo-plasticity properties was reported ,17. Polycaprolactone can be used as a biodegradable food packaging material, but its poor thermal stability and low melting point restrict its wider application . Here, tThe degradation of polycaprolactone PCL is relatively long due to the structure of PCL and its hydrophobicity preventing water uptake . In enviFor many application areas the performance of PCL should be improved, especially its mechanical properties, processing stability and resistance to thermo-mechanical degradation. The incorporation of nanoparticles, montmorillonites , graphen1.5)n, where n can be 8, 10 or 12 [Among various nanoparticles, polyhedral oligomeric silsesquioxanes (POSS) are very promising reinforcing additives . Silsesq10 or 12 . R can b10 or 12 ,27. Various POSS particles have been incorporated into thermoplastic polyolefins ,29, polyOrganic-inorganic hybrid medical biomaterials based on POSS have been developed . PolyhedPolyhedral oligomeric silsesquioxanes such as trans-cyclohexanediol isobutyl POSS were incorporated into poly(caprolactone-urea) urethane to obtain a scaffold for dermal replacement. The scaffold consists of two layers, the outer, removable POSS-PCU layer and the inner, biodegradable POSS-PCL layer . Electrically conductive nanocomposite material based on POSS-PCL/graphene was studied as a potential material in the field of neural tissue engineering . Trans-cHan et al.  reportedLee K.S. et al.  found thFrom the economic point of view, it is important to accomplish the best processing method during nanocomposite preparation. A solution-mixing method followed by film casting ,44 and tIn this work, reactive blending during processing was used to prepare POSS-PCL nanocomposites. The impact of three polyhedral oligomeric silsesquioxane POSS with various functional groups: aminopropylisobutyl-POSS; trisilanolisooctyl-POSS and glycidyl-POSS on the viscoelastic properties of POSS-PCL nanocomposites was studied. To our knowledge, the effects of these POSS on the properties of PCL have not been reported so far in the literature. Despite the fact that PCL is more stable during processing compared with other biodegradable polyesters and polylactide PLAs, the challenges associated with the use of biodegradable materials remains similar. Beside the need for mechanical property enhancement, the impact of the nanoparticles on the degradation that occurs in PCLs during the manufacturing process is important. The oscillation and rheological tests of melted PCL at higher temperatures are useful methods to estimate the change in viscoelastic properties resulting from the degradation of the material. With regard to the viscoelastic properties of PCL, it was shown that the presence of POSS particles enhanced not only the dynamic mechanical properties of PCL at ambient temperature, but it also influenced the relaxation behavior of melted PCL and the viscoelastic properties of the thermo-mechanical treated PCL. −1 of rotor speed) at 100 °C for 5 min. Then, the compositions were removed from Brabender and cooled. The flat films for rheological and tensile studies were compression-molded using hydraulic press at 200 bar and 100 °C for 5 min. The 3 wt.% of polyhedral oligomeric silsesquioxane POSS with various functional groups was incorporated into ε-polycaprolactone PCL . Aminopropylisobutyl POSS  with one aminopropyl group and seven isobutyl groups, further denoted as amine-POSS; trisilanolisooctyl-POSS , an open-cage POSS with three hydroxyl groups and seven isooctyl groups, further denoted as HO-POSS; a glycidyl-POSS  with eight glycidyl groups and epoxy equivalent weight 167, further denoted as Gly-POSS, were used. The melt-mixing method was applied to prepare PCL/POSS composites. For this purpose, the weighed amount of PCL was placed in mixer chamber  and was melted at 100 °C for 10 min. Then, appropriate weighed amount of selected POSS particles  was added to the melted PCL and mixed . Plate-plate geometry (diameter 25 mm) was used during measurements. −1.Procedures of the tests were: (1) the frequency sweep test at 100 °C, at constant value of oscillation amplitude 1%; (2) the frequency sweep test at 25 °C, at constant value of oscillation amplitude 0.02%; (3) the amplitude sweep test at 25 °C, at constant value of angular frequency 10 rad⋅sThe analysis of relaxation of the melted PCL containing various POSS particles was performed based on the frequency sweep test using various relaxation models. iλ, and values of relaxation modulus iG were calculated according to below equations.The Maxwell model ,49 was aG′ and loss shear modulus G″ with the level of correlation R2 = 0.999.Experimentally studied at 100 °C frequency range, the six Maxwell elements were sufficient for recalculation of the values of storage The continuous relaxation spectrum ,51,52 waH(lnτ) was discretized typically in the order of 100 steps. The spectrum represents all the pairs of fitted {iH,iτ} parameters.For the numerical computation the spectrum G′ and loss shear modulus G″ to the relaxation models was performed using the TRIOS® Software (TRIOS v3 1.5.3696) provided by TA Instruments .The fitting of storage shear modulus η0 and characteristic mean relaxation times τm were calculated from viscosity Cole-Cole plots  as proposed in the literature [Additionally, the zero-shear viscosity terature ,54,55,56−1 shear rate.To estimate the changes in the viscoelastic properties caused by the thermo-oxidative and thermo-mechanical degradation, time sweep tests followed by the separate frequency sweep tests were done. The procedures were: (1) for thermo-oxidative degradation-time sweep test: temperature 100 °C, air flow, time 30 min, 60 min, 90 min, 120 min; (2) for thermo-oxidative degradation-time sweep test, temperature 100 °C, air flow, time 30 min, additional mechanical stress, equivalent of 50 s−1. The first heating step was performed to eliminate the thermal history of the sample. The degree of crystallinity cχ was calculated according to the Equation (6) [mH—experimental melting enthalpy, −1 [w—the weight fraction of material. The DSC analysis was performed using DSC1 apparatus . All tests were performed under nitrogen atmosphere. The samples were subjected to three heating/cooling/heating steps from −90 °C to 200 °C with the heating rate 10 °C⋅mintion (6) ,15:(5)χc.5 J g−1 ), w—the −1.The TGA analysis was performed using TGA/DSC1  analyzer. Samples were heated from 25 °C to 600 °C in an argon with the heating rate 10 °C⋅minThe most popular polyhedral oligomeric silsesquioxane POSS are highly symmetrical silica cage-like structures with a size of approximately 1.5 nm  ,38. In pG′ for the linear viscoelastic region; the values of LVRG′ were as follows: PCL LVRG′ = 545.4 ± 2.4 kPa; PCL amine-POSS LVRG′ = 862.0 ± 3.4 kPa; PCL HO-POSS LVRG′ = 560.9 ± 2.9 kPa; Gly-POSS LVRG′ = 684.5 ± 2.9 kPa. The increase in the loss modulus G″ was also observed . The cross points G′ = G″ were as follows: PCL 118.5 kPa at 0.34%; PCL amine-POSS 122.3 kPa at 0.61%; PCL HO-POSS 117.1 kPa at 0.41%; Gly-POSS 131.0 kPa at 0.41%. The oscillation sweep test  at 25 °Cobserved . It was G′ for the linear viscoelastic region; the values of LVRG′ were as follows: PCL LVRG′ = 545.4 ± 2.4 kPa; PCL amine-POSS LVRG′ = 862.0 ± 3.4 kPa; PCL HO-POSS LVRG′ = 560.9 ± 2.9 kPa; Gly-POSS LVRG′ = 684.5 ± 2.9 kPa. The increase in the loss modulus G″ was also observed . The cross points G′ = G″ were as follows: PCL 118.5 kPa at 0.34%; PCL amine-POSS 122.3 kPa at 0.61%; PCL HO-POSS 117.1 kPa at 0.41%; Gly-POSS 131.0 kPa at 0.41%. The oscillation sweep test  at 25 °Cobserved . It was G′ for the linear viscoelastic region; the values of LVRG′ were as follows: PCL LVRG′ = 545.4 ± 2.4 kPa; PCL amine-POSS LVRG′ = 862.0 ± 3.4 kPa; PCL HO-POSS LVRG′ = 560.9 ± 2.9 kPa; Gly-POSS LVRG′ = 684.5 ± 2.9 kPa. The increase in the loss modulus G″ was also observed . The cross points G′ = G″ were as follows: PCL 118.5 kPa at 0.34%; PCL amine-POSS 122.3 kPa at 0.61%; PCL HO-POSS 117.1 kPa at 0.41%; Gly-POSS 131.0 kPa at 0.41%. The oscillation sweep test  at 25 °Cobserved . It was The values of tan δ at 25 °C as a function of growing deformation (oscillation strain %) are depicted at G′ and loss shear modulus G″ after the incorporation of amine-POSS and Gly-POSS increased. No significant effect of HO-POSS on the storage shear modulus G′ and loss shear modulus G″ of PCL was observed. The frequency sweep test at 25 °C confirmed the reinforcing effect of the selected POSS particles  leading G′ for filler/polymer systems mainly is a result of three phenomena: the hydrodynamic effect resulting from the fact that the filler is the rigid phase; the interphase filler–polymer interactions causing the occlusion of polymeric chains on the filler surface; and the filler–filler interactions. The nature of the filler, and factors such as: the primary particle size, specific surface area, surface activity and tendency to form aggregate–aggregate associations are important [G′ observed for the PCL amine-POSS and PCL Gly-POSS samples. The HO-POSS, oppositely to amine-POSS and Gly-POSS, is an open-cage polyhedral oligomeric silsesquioxane that contains three hydroxyl groups attached to the same silicon Si atom as the isooctyl groups. Probably, the formation of the interphase interaction between the hydroxyl groups and polymeric chains was restricted due to steric hindrance and the covering effect of the isooctyl group preventing the penetration of the polymeric chains to the HO-group. As a result, the values of the storage modulus G′ for the PCL HO-POSS were lower as compared with the other PCL POSS composites. The amine-POSS and Gly-POSS can be applied as reinforcing additive-enhancing dynamic mechanical properties of PCL. The increase in the storage shear modulus mportant . For thecχ. Oppositely, the incorporation of HO-POSS enhanced the degree of crystallinity. The longer isooctyl groups present in HO-POSS had a plasticizing effect on the melted PCL, as analyzed further, and this probably facilitated crystallization.Here, also, the effect of the POSS on the crystallization of PCL was observed, as studied by DSC . Both amOther authors ,45 also g of PCL was shifted to higher values of temperature after the incorporation of selected POSS confirming the restricted mobility of the polymeric chains in the presence of POSS. Other authors [The glass transition temperature T authors  also obsm and crystallization temperature Tc of PCL. The DCS plots for the studied samples are compiled in The POSS less meaningfully changed the melting temperature TThe TGA studies  showed t50% for all PCL POSS compositions from 421 to 424 °C was observed. The authors [The HO-POSS, oppositely to amine-POSS and Gly-POSS, is an open-cage polyhedral oligomeric silsesquioxane. Bautista Y. et al.  showed t authors . Compari authors . The preη* at 100 °C. The opposite effect was observed for amine-POSS  are collected in Based on the storage shear modulus t 100 °C , the conBoth HO-POSS and Gly-POSS facilitated the relaxation of melted PCL. Oppositely, longer relaxation times were observed for amine-POSS PCL indicating the lower mobility of the PCL chains, probably due to interactions between the aminopropyl group of POSS and PCL chains. The observed facilitation of the relaxation of the melted PCL in the presence of HO-POSS and Gly-POSS can be an advantage during processing when high deformation (shear rate) on the material is applied.G′ and loss shear modulus G″ were observed. However, the cross points G′ = G″ determined from the frequency sweep tests were shifted to different values of angular frequency ω (The poly(ε-caprolactone) PCL belongs to a polyester that shows biodegradability. The ability to degrade under some conditions can be an ecological advantage facilitating the protection of the environment and waste management. From another point of view, polymer material should be stable during processing and not change viscoelastic properties. Frequency sweep tests were done for the melted PCL samples before and after thermal treatment at 100 °C for 30, 60, 90, 120 min. The melted PCL material was exposed only to the temperature and the oxygen present in the air; no mechanical stress was applied. The thermo-oxidative degradation of the melted PCL, up to 120 min, slightly changed the viscoelastic properties of the material. No significant changes in the values of the storage shear modulus quency ω , as the i and the relaxation times iλ after 30, 60, 90, 120 min of thermo-oxidative treatment are compiled in The calculated relaxation spectra before and after thermal treatment at 100 °C can give useful information about the change in materials due to thermo-oxidative degradation. The calculations according to the Equations (1) and (2), Maxwell’s discrete relaxation spectra for the samples before and after thermo-oxidative treatment for 120 min at 100°, are shown in i and the relaxation times iλ, recalculated based on similar angular frequency range, were observed for the PCL HO-POSS and PCL Gly-POSS composites indicating that the presence of both POSS-stabilized materials during thermo-oxidative treatment as compared with neat PCL , calculated based on the measurement of complex viscosity at constant temperature, can be used to compare the influence of various additives on the degradation of the material [R.M.I. is calculated according to the below equation (Equation (6)), using the values of complex viscosity η* determined from the viscosity plateau of the η* versus angular frequency ω plots for the samples before and after thermo-oxidative ageing over 30, 60, 90, 120 min at 100 °C in the presence of the oxygen from air.The material . The R.MR.M.I. parameter higher than 1 for samples after thermo-oxidative ageing indicated the decrease in the viscosity of the sample due to chain cleavage occurring during degradation. No meaningful change in the values of R.M.I. after thermo-oxidative treatment for all PCL samples was observed .The viscoelastic properties of the samples before and after thermo-mechanical treatment were studied. η* was stronger. The reduction of complex viscosity Δη* at 0.1 rad⋅s−1 was calculated according to Equation (7):As expected, the degradation of the PCL samples after the application of mechanical stress occurred to a higher extent, and the decrease in complex viscosity η* were as follows: Δη* = 1256 Pas for PCL; Δη* = 1320 Pas for PCL amine-POSS; Δη* = 1147 Pas for PCL HO-POSS; Δη* = 984 Pas for PCL Gly-POSS.The calculated values of ΔG′ and loss shear modulus G″  versus η′ (dynamic viscosity), are a useful tool to study the influence of the additives on the rheological behavior and the relaxation of the melted polymer. Some rheological parameters that can be derived from the Cole-Cole plots are the zero-shear viscosity η0 and characteristic relaxation time [The viscosity Cole-Cole plots, plots of ion time ,54,55,56η’ and higher value of angular frequency ω. The relaxation of the PCL HO-POSS was facilitated. Oppositely, the maximum of the semicircular arc for PCL amine POSS was shifted to a higher value of dynamic viscosity . The presence of amine-POSS disturbed the relaxation as compared with the neat PCL. All PCL compositions showed a semicircular shape of Cole-Cole plots, typical for homopolymers with monomolecular weight distribution. The incorporation of various POSS particles shifted the maximum of the arc as compared with the neat PCL. The relaxation behavior of the melted PCL was affected by the presence of POSS. The maximum of the semicircular arc was meaningfully shifted after the addition of the HO-POSS. It was observed at lower values of dynamic viscosity m (s) were: PCL τm = 0.0124 s; PCL amine-POSS τm = 0.0126 s; PCL HO-POSS τm = 0.0111 s; PCL Gly-POSS τm = 0.0124 s. The calculated values of mean relaxation time τη0 as compared with neat PCL or PCL or Gly-POSS samples. The calculated values of zero-shear viscosity η0 (Pas) were: PCL η0 = 2442 Pas; PCL amine-POSS η0 = 2587 Pas; PCL HO-POSS η0 = 2241 Pas; PCL Gly-POSS η0 = 2490 Pas.Moreover, the plasticizing effect of HO-POSS was observed. As it was described previously, the hydroxyl groups are covered by and attached to the same silicon atom as isooctyl groups. These isooctyl groups enhance the plasticization of PCL. The plasticizing effect of various POSS particles was observed by some authors  for otheη′ and the out of phase component of complex viscosity η″ for all PCL compositions due to PCL degradation and chain cleavage. The calculated values of zero-shear viscosity for samples after thermo-mechanical treatment were: PCLdegraded η0 = 1217 Pas; PCLdegraded amine-POSS η0 = 1246 Pas; PCLdegraded HO-POSS η0 = 1015 Pas; PCLdegraded Gly-POSS η0 = 1485 Pas. The Cole-Cole plots were significantly shifted to the left corner confirming the facilitated relaxation of the degraded materials as compared with these before the thermo-mechanical treatment. The calculated mean relaxation times τm for the samples after the thermo-mechanical treatment were: PCLdegraded τm = 0.0071 s; PCLdegraded amine-POSS τm = 0.0089 s; PCLdegraded HO-POSS τm = 0.0079 s; PCLdegraded Gly-POSS τm = 0.0113 s. Thermo-mechanical degradation over 30 min decreased the values of dynamic viscosity i decreased. The degradation of the PCL was taking place as a result of the exposure to temperature, oxygen and mechanical stress. However, the changes in the viscoelastic properties of the PCL occurred to a lesser extent in the presence of Gly-POSS in the composition.For all the PCL samples, thermo-mechanical degradation led to changes in the Maxwell relaxation spectrum . It confG′ and the loss shear modulus G″ of poly(ε-caprolactone), as measured in the function of the oscillation strain at 25 °C. The reinforcing effect was attributed to the stronger hydrodynamic effect and interphase interactions of POSS functional groups with polymeric chains. Aminopropylisobutyl POSS (amine-POSS) and glycidyl POSS (Gly-POSS) increased the values of the storage shear modulus η′ at 100 °C due to plasticizing effect. The plasticizing effect of HO-POSS was responsible for the facilitation of the crystallization leading to higher values of degree of crystallinity cχ. Oppositely, the presence of amine-POSS and Gly-POSS slightly reduced the degree of crystallinity cχ of the PCL as compared with neat PCL. Trisilanolisooctyl POSS (HO-POSS) decreased the values of dynamic viscosity The calculated continuous relaxation spectra showed that both HO-POSS and Gly-POSS facilitated the relaxation of melted PCL. Calculations from the Cole-Cole plots’ mean relaxation time τ also confirmed the facilitated relaxation of the melted PCL HO-POSS composition. Longer relaxation times were observed for amine-POSS/PCL composition. R.M.I. parameter suggested the higher stability of the melted PCL in the presence of HO-POSS and Gly-POSS. Thermo-oxidative degradation at 100 °C for up to 120 min slightly changed the viscoelastic properties of PCL. However, the lower values of the calculated η*, storage modulus G′ and loss modulus G″. The Maxwell’s relaxation spectra shifted to lower values of relaxation times λ, indicating that the material structure changed. The calculated values of the R.M.I. parameter for Gly-POSS/PCL confirmed that the reduction in viscosity due to chain cleavage in the presence of Gly-POSS was smaller. The thermo-mechanical degradation of PCL resulted in a decrease in complex viscosity These studies confirmed that polyhedral oligomeric silsesquioxane POSS, due to the presence of functional groups, can be used to modify the viscoelastic properties of poly(ε-caprolactone) at ambient and processing temperatures, and the relaxation behavior of melted PCL. Finally, the positive effect of Gly-POSS on the stability of the material during thermo-mechanical degradation is observed and provides potential for future studies.


Objective: For meropenem 40%T > MIC is associated with optimal killing of P. aeruginosa and E. coli. However, it is unknown how the distribution of %T > MIC through a treatment day impacts the antimicrobial effect in vitro. Therefore, we investigated the in vitro antibiotic activity of meropenem, precisely if 40%T > MIC is achieved in one single long period (single dose), 2 × 20% periods (dosing-bid), or 3 × 13.3% (dosing t.i.d.) thereby keeping the overall period of T > MIC constant.Material/Methods: Time kill curves (TKC) with P. aeruginosa-ATCC-27853 and E. coli-ATCC-25922 and five clinical isolates each were implemented over 24 h in CAMHB with concentrations from 0.25×MIC-32×MIC. Periods over and under MIC were simulated by centrifugation steps (discarding supernatant and refilling with fresh CAMHB). Double and triple dosing involved further addition and removal of antibiotic. Complementary growth controls (GC) with and without centrifugation steps were done and the emergence of phenotypical resistance was evaluated (repeated MIC-testing after antibiotic administration).Results: No impact of centrifugation on bacterial growth was seen. TKC with P. aeruginosa showed the best killing in the triple dosage, followed by the double and single dose. In multiple regimens at least a concentration of 4×MIC was needed to achieve a recommended 2-3 log10 killing. Likewise, a reduction of E. coli was best within the three short periods. Contrary to the TKCs with P. aeruginosa we could observe that after the inoculum reached a certain CFU/mL (≥10^8), no further addition of antibiotic could achieve bacterial killing (identified as the inoculum effect). For P. aeruginosa isolates resistance appeared within all regimens, the most pronounced was found in the 40%T > MIC experiments indicating that a single long period might accelerate the emergence of resistance. Contrary, for E. coli no emergence of resistance was found.Conclusion/Outlook: We could show that not solely the %T > MIC is decisive for an efficient bacterial eradication in vitro, but also the distribution of the selected %T > MIC. Thus, dividing the 40%T > MIC in three short periods requested lowers antibiotic concentrations to achieve efficient bacterial killing and reduces the emergence of resistance in P. aeruginosa isolates. The distribution of the %T > MIC did impact the bacterial eradication of susceptible pathogens in vitro and might play an even bigger role in infections with intermediate or resistant pathogens. In antimicrobial therapy the efficacy of antibiotics is not only dependent on the drug itself, but also on the patient’s physiology including disease state, comorbidities or age, and the variety of the bacterial species .Enterobacteriaceae, especially Klebsiella pneumoniae (∼1%); Pseudomonas aeruginosa (15–17%); Acinetobacter baumannii (∼29.5%), and methicillin resistant Staphylococcus aureus (MRSA) (11.8–13.5%)  .Two important aspects for a successful antibiotic treatment are the unbound antibiotic concentration at the target site and the effect the drug has on the bacterial pathogen, which is best explained by pharmacokinetic (PK) and pharmacodynamic (PD) indices. The three most important PK-PD indices are the time over the MIC (T > MIC), the peak drug concentration over MIC (Cmax/MIC), and the 24 h area under the concentration curve over MIC (AUC/MIC) ratios . DiffereFor intensive care unit (ICU) patients it might be beneficial to target a free drug concentration of a beta-lactam of 100%T > MIC or even 100%T > 4×MIC, whereas in patients with normal renal and hepatic function the serum drug concentrations should exceed the MIC of the causative pathogen for at least 40–70% of the dosing interval .However, four main concerns arise when using threshold values for PK/PD parameters.First, the PK/PD threshold target seems to vary between different beta-lactam antibiotics as described by Ambrose. et al. and Craig, which has been summarized in Second, the PK/PD threshold might vary depending on the magnitude of effect that is targeted, e.g., for more serious infections a 2-3 log reduction in CFU/mL might be targeted , while for others bacteriostasis might be sufficient  . Third, Fourth, the PK/PD indices only insufficiently consider the shape of the concentration time curve of the antibiotic. Thereby, a high Cmax/MIC ratio might or might not be accompanied by a high AUC/MIC value. Likewise for T > MIC all concentrations below the MIC are equally ineffective and similar to all concentrations above the MIC are treated as equally effective. However, concentrations that are just below the MIC might show some anti-infective activity compared to others close to zero and concentrations slightly above the MIC might not display the maximum effect that is only achieved with higher concentrations .in vitro antimicrobial activity over 24 h.Studies such as from Dandekar et al. have already proven that treatment success with meropenem might be optimized by prolonged infusion and 3 times daily doses  . NeverthP. aeruginosa and E. coli ; For susceptibility testing and pharmacodynamic experiments meropenem , dissolved in dimethyl sulfoxide (DMSO), was used.Cation adjusted Mueller Hinton Broth (CAMHB)  was used as liquid growth media containing 17.5 g/L casein acid hydrolysate, 3 g/L beef extract, 1.5 g/L starch, 20–25 mg/L calcium, and 10–12.5 mg/L of magnesium with a pH of 7.3 ± 0.2.P. aeruginosa and E. coli strains.Columbia agar plates  containing 5% sheep blood were used as solid growth media for P. aeruginosa 0.008–2 mg/L and E. coli 0.008–0.06 mg/L.Broth microdilution for evaluating the minimal inhibitory concentration (MIC) of the test strains was done according to the performance standards for antimicrobial susceptibility testing of the Clinical and Laboratory Standards Institute (CLSI) . The approximate concentration range was set based on EUCAST ranges for All TKC analyses and growth controls (GC) were performed in triplicate over 24 h in a shaking water bath  at 37°C under aerobic conditions. The bacterial suspension was adjusted to 1.5 × 10^8 CFU/ml in NaCl, corresponding to a McFarland standard of 0.5, 100 µL was added to the test tubes filled with CAMHB to a final volume of 10 ml. After 1 h of pre-incubation, 100 µl aliquots were taken of each falcon tube to determine the CFU/mL at time point 0 h before the addition of the antibiotic. The samples were pipetted in the first row of a 96-well microtiter plate. Subsequently, seven serial dilution steps with a volume of 20 µL were carried out in the 96-well microtiter plates filled with 180 µL of 0.9% NaCl in rows two to seven. Aliquots of 20 µL of each concentration were dropped onto Columbia blood agar plates and incubated at 37°C under aerobic conditions for 24 h. This procedure was also done for subsequent time points.From the meropenem stock dilutions were made (freshly prepared before every dosing) to achieve final concentrations several fold above and below the respective MIC of the pathogens (0.25×MIC to 32×MIC) by always adding 100 µL of the stock to the 14-ml tubes. To simulate 40%T > MIC, 2 × 20%, and 3 × 13.3%T > MIC centrifugation steps at 37°C for 5 min at 1300 g were done at certain time points to create antibiotic free time. The supernatant was discarded, falcon tubes were refilled with fresh CAMHB and vortexed to resuspend the bacterial pellet. Double and triple dosing involved further addition of antibiotic. Complementary GCs with and without centrifugation steps were done.0, 2, 9.6, and 24 h. Underlined time points indicate meropenem administration and bold time points represent the centrifugation time points.In the 40%T > MIC setting after 9.6 h the samples were centrifuged to create a meropenem free time. The CFU/mL was determined at 0, 2, 4.8, 12, 14, 16.8, and 24 h.To simulate 2 × 20%T > MIC centrifugation of the tubes was done after 4.8 h after each antibiotic administration. Samples were taken after 0, 2, 3.2, 8, 10, 11.2, 16, 18, 19.2, and 24 h.Finally, to achieve 3 × 13.3%T > MIC the removal of meropenem was done after 3.2 h after the antibiotic administration. Aliquots were drawn after For penems, a bactericidal concentration is commonly defined as a 3 log10 reduction in cell counts within 12–24 h. Thus, we set a threshold of 2-3 log10 reduction of CFU/mL in our experiments to better compare the impact of distributing the 40%T > MIC on bacterial killing through a treatment day.The emergence of phenotypical resistance was evaluated by repeated MIC testing after antibiotic administration. Thus, up to 3 CFU from the Columbia agar plates of 0 and 24 h of each tested concentration and of all three dosing regimens were collected with a sterile Q-tip and broth microdilution was performed as mentioned above. Ratios of the evaluated MIC of 24 and 0 h were calculated to evaluate a potential emergence of resistance.No impact of centrifugation on bacterial growth of all bacterial strains was seen.P. aeruginosa ATCC-27853 are depicted in P. aeruginosa isolates is also shown in TKC with In all settings the concentration of 1×MIC could slow down bacterial growth or even reduce the initial bacterial count until the first antibiotic removal. Overall, best killing over 24 h was achieved in the three short periods with 3 × 13.3%, followed by the 2 × 20%T > MIC and the 40%T > MIC for ATCC-27853 and the clinical isolates. In multiple regimens at least a concentration of 4×MIC was needed to achieve a 2-3 log10 killing which was defined as the threshold according to the prescribing information , at least a concentration of 4×MIC was needed. Nevertheless, similar to the previous data with P. aeruginosa strains a reduction of E. coli ATCC-25923 within the first interval of dosing was best with the triple dosage compared to the other dosing regimens. Thus, as mentioned before a concentration of 4×MIC or higher achieved a 2-3 log10 killing , no exposure to antibiotic could achieve bacterial killing. This was identified as the inoculum effect, which was confirmed in additional experiments (data not shown).Contrary to P. aeruginosa ATCC-27853 in the 40%T > MIC experiment a value of 3.2, in the 2 × 20%T > MIC experiment a value of 2.7, and in the 3 × 13.3%T > MIC experiment a value of 0.7 was calculated, confirming the best growth inhibition in the three short periods with a concentration of 4×MIC. Moreover, the highest reduction of CFU/mL was achieved with a concentration of 16×MIC with values of 0.8 in the 40%T > MIC, -0.6 in the 2 × 20%T > MIC, and -1.5 in the 13.3%T > MIC experiments, confirming again best killing in the three short periods. Contrary to E. coli ATCC-25923, the concentration of 4×MIC could not reduce the CFU/mL in any setting, as values of 2.2, 2.9, and 2.5 were obtained for 40%, 2 × 20%, and 3 × 13.3%, respectively.Another possibility to evaluate the bacterial killing of the three different experimental settings is the comparison of CFU/mL at the start and at the end of the experiment. Applying a log10 on the ratio of CFU/mL of 24 h and CFU/mL of 0 h for the tested concentrations results in either positive or negative values in the different settings. Positive values indicate bacterial growth and negative bacterial killing. For P. aeruginosa and E. coli isolates in P. aeruginosa isolates the emergence of phenotypical resistance was present within all regimens. Concentrations at 1×MIC or higher in the 40%T > MIC setting showed up to 18-fold higher MIC values indicating that single administration favors emergence of resistance compared to the other dosing regimens for P. aeruginosa isolates. Contrary, for E. coli no emergence of resistance was found.The median ratios of the MIC values (24/0 h) are depicted for in vitro, but also the distribution of the selected %T > MIC. Our data indicate that three short periods of 13.3%T > MIC, which equals 3 × 3.2 h, above a concentration of at least 4×MIC of meropenem achieved best bacterial killing within 24 h compared to the continuous 40%T > MIC, which equals a period of 1 × 9.6 h.According to the current paradigm, the most important PK-PD parameter for beta-lactam antibiotics, such as meropenem, is the percentage of time it exceeds the MIC . Our TKCPrevious studies have already shown that, for example, in critically ill patients the %T > MIC for beta-lactams might need to be adjusted from 40–70%T > MIC to 100%T > MIC to meet the target attainment . MoreoveP. aeruginosa and E. coli isolates is not present in one single long period but distributed over the treatment day, thereby keeping the overall period of T > MIC constant?Despite these relevant considerations we raised another question; what happens if the recommended 40%T > MIC of meropenem against P. aeruginosa ATCC-27853, the five clinical isolates of P. aeruginosa, and for E. coli ATCC-25922. In detail, within the 3 × 13.3% experiments the bacterial count could be kept low  for a longer time (up to 20 h) compared to the continuous 40%T > MIC (up to 9.6 h). One explanation could be that the bacteria are more often and more evenly exposed to meropenem during the 24 h. Namely every 8 h for 3.2 h in the 3 × 13.3% setting compared to once for 9.6 h in the continuous 40%T > MIC. Therefore, the bacteria might not be able to adapt that fast on the presence of meropenem compared to the continuous setting. In the single long period 14.4 h of antibiotic free time is generated between antibiotic removal and the end of the experiment. Contrary, in the three short periods there are only 4.8 h of antibiotic free time between antibiotic removal and the next antibiotic administration. Thus, recurrent short antibiotic exposure of 3 × 13.3% seems to eradicate the bacteria over the 24 h better than the continuous 40%T > MIC as less selection pressure on the bacteria might be present and adapted resistances of the pathogens to survive meropenem exposure might not evolve that rapidly. This has been discussed as well by Baker at al., as they state that the presence of antibiotics creates a selection pressure for antibiotic resistant microbes, and large populations of bacteria are more likely to harbor drug resistance than small populations .Nevertheless, the study has several limitations. First, we did not test how 100%T > MIC, sometimes recommended for critical settings, would have impacted killing and the emergence of resistances.Moreover, additional sampling time points within all experiments would have been an advantage to further compare killing and growth between the different settings. However, since the experiments had to run day and night for 24 h it has been hampered by feasibility.Another critical point to mention is the washing procedure as it has been performed only by one centrifugation step, discarding old CAMHB and resuspending the bacterial pellet with new CAMHB. Thus, additional washing with CAMHB, PBS, or NaCl would have been assured to decrease the risk of residual antibiotic in the samples.Furthermore, we only performed phenotypical evaluation of resistance and did not investigate potential genotypical mechanisms.Last, we wanted to test our hypothesis of distributing %T > MIC in an extreme setting, by adding and removing antibiotic through centrifugation steps creating an all or nothing situation instead of a simulation dedicated to clinically used dosing regimens.in vitro, but also the distribution of the selected %T > MIC. Thus, dividing the 40%T > MIC in three dosing intervals required lower antibiotic concentrations to achieve efficient bacterial killing and reduced the emergence of resistance in P. aeruginosa isolates. The distribution of the %T > MIC did impact the bacterial eradication of susceptible pathogens and might play an even bigger role in infections with intermediate or resistant pathogens.In conclusion we could show that not solely the %T > MIC is decisive for an efficient bacterial eradication Hence, this study shows that defined PK-PD targets such as the T > MIC should be examined more closely and the shape of the concentration vs. time curve deserves more attention in future investigations. Subsequent studies should be performed to challenge Cmax/MIC and AUC/MIC values in comparable settings.


The confinement of concrete enhances its strength and ductility by restraining lateral dilation. The accuracy of a confinement model depends on how well it captures the dilation of concrete. In the current paper, a mesoscale model is established to study the dilation properties of concrete in active confinement, where the heterogeneity of concrete is considered. The stress–strain and lateral–axial strain curves of concrete in active confinement were used to demonstrate the validity of the mesoscale model. Subsequently, the distribution of lateral strain and the influences of the strength grade and confinement ratio on the dilation of concrete were investigated in a simulation. The results show that the distribution of the lateral strain along the radial or longitudinal directions is not uniform on the specimen when compressive failure occurs. The confinement ratio has a more significant influence on the concrete’s transverse dilation than the strength grade. Finally, an expression of the lateral–axial strain relationship of concrete in active confinement is proposed. The proposed formula can reflect the simulation results of the mesoscale model and is in good agreement with the prediction of existing formulas. The mechanical properties of confined concrete have been extensively studied in recent years, illustrating that confinement can enhance concrete’s strength and ductility by inhibiting transverse dilation ,2,3,4. MThe properties of concrete in active confinement have been experimentally studied recently ,9,10,11.This paper aims to develop a new method for studying the dilation properties of concrete in active confinement. To accomplish this objective, the concrete is regarded as a combination of the mortar phase, aggregate phase, and interfacial transition zone (ITZ), and then, a numerical concrete model is developed according to the theory of random distribution. The validity of the mesoscale model is first verified, and then a parameter analysis is conducted to study the influences of strength grade and confinement ratio on the dilation properties. Based on the simulation results, the lateral–axial strain curve of the concrete column in active confinement is proposed, and then it is compared to the existing models presented in the literature.Considering the heterogeneity of concrete at the mesoscale, existing studies ,18,19,20The “take and place” method was used to generate aggregate particles, which was similar to the previous studies ,22. The p and vertical displacement u were applied to the specimen and upper loading plate, respectively.The damage plasticity model developed by Lee and Fenves  is extenGrote  highlighExperimental results show that the elastic modulus, peak strain, and decreasing index of the mortar phase differ from those of concrete with the same strength . A paramfm/100.7 .fm, and the confining pressure p. In a word, the elastic and plastic damage models were used for the aggregate and mortar phases in the random aggregate structure. Meanwhile, the ITZ phase can be regarded as a mortar phase with reduced strength. The main parameters of the constitutive model include the compressive strength of mortar To illustrate the accuracy of the mesoscale model, the stress–strain curves of concrete columns were compared between the simulation results and specification results, as presented in In this section, the numerical model of concrete with different strengths was established. The proposed model was efficient in describing the compressive stress–strain curve of unconfined concrete. Based on this model, it is valuable to study the dilation property of concrete in active confinement by applying the confining pressure.In order to verify the prediction accuracy of the mesoscale model on the performance of actively confined concrete, concrete specimens with four strengths, such as 35.8, 40, 50, and 60 MPa, were collected from the literature ,11 to crThe lateral–axial strain relationship is an important curve reflecting the dilation properties of concrete. Nevertheless, the strain measurement presents considerable discreteness due to the influence of the tester, equipment, and strain gauge displacement. The numerical model previously established above can overcome the shortcomings of tests and provide an effective means for studying concrete’s lateral–axial strain curve. The analysis described above shows that the proposed mesoscale model can make a good estimation on the stress–strain and lateral–axial strain curves of concrete in active confinement, illustrating that it is an efficient tool for investigating the mechanical and deformational behavior of concrete.fc = 30 MPa, 40 MPa, 50 MPa, and 60 MPa. Moreover, the confinement ratios were p/fc = 0, 0.1 0.2, 0.3, and 0.4.To study the dilation properties of concrete in active confinement, the simulations of a concrete cylinder with different strengths and confinement ratios were conducted based on the mesoscale model. The strengths of the concrete cylinder were The failure modes of the concrete cylinder having different confinement ratios are presented in The failure of the concrete cylinder with no or low confinement ratios was mainly concentrated in the middle of the specimen, where the dilation was the most significant; thus, the measurement of the lateral strain in the test was focused here. To investigate the strain distribution along the longitudinal direction, nine sections were selected along the specimen’s height in the simulation. The total height of the concrete cylinder was 200 mm, and the spacing between sections was 25 mm. ous work . Figure Generally, the distribution of the lateral strain along the circumference and the height of the specimen is not uniform when compressive failure occurs in concrete. Therefore, great discreteness exists in the strain measurement in the tests. In order to reduce the influence of discreteness, the average strain was used in the analysis of the simulation results. Specifically, the lateral strain at each section is the average of 100 points along the circumferential direction, and the average strain at several sections can be regarded as the specimen’s lateral strain. Considering that the failure of the concrete cylinder with no or low confinement ratio was prone to local damage, while the failure of the specimen with a high confinement ratio was relatively uniform, the difference was considered in the current study when obtaining the average value. By trial and error and comparing with the test data, the authors observed that if 3, 5, and 7 sections were selected for the specimens with low (0 or 0.1), middle (0.2), and high (0.3 or 0.4) confinement ratios, respectively, presented in Overall, the strength grade influenced the development of concrete’s secant strain ratio, especially for concrete specimens with no or low confinement ratios.εco. When the axial strain is three times the peak strain of plain concrete, the secant strain ratio is 2.25, 0.81, 0.54, 0.40, and 0.25 as the confinement ratio is set as 0, 0.1, 0.2, 0.3, and 0.4, respectively. Moreover, the initial secant strain ratio is 0.177, 0.156, 0.130, 0.113, and 0.070 when the confinement ratio increases from 0 to 0.4, illustrating that the confining pressure can inhibit the development of the secant strain ratio. The analysis indicates that the confinement ratio is an important parameter affecting the transverse dilation of concrete, which should be considered in the formula.The influence of the confinement ratio on the dilation of the specimen with different strengths is similar. The simulation results show that the confinement ratio has a more significant influence on transverse dilation than that of concrete’s strength, as presented in α equals 1.0. The confinement ratio p/fc can be set as zero for unconfined concrete. Previous studies [β reflects the influence of the confinement ratio, and a satisfactory prediction can be achieved when it is set as 6.0. As the formula can apply to unconfined concrete, the value of parameter  studies ,7,39 havTo illustrate the performance of the proposed lateral strain equation, it was compared with the simulation results presented in In general, the proposed lateral strain formula can reflect the simulation results of the mesoscale model, and it is consistent with the prediction of the existing formulas. Therefore, it is an effective formula for predicting the dilation properties of actively confined concrete.The simulation results of the mesoscale model were in good agreement with the stress–strain curves of the plain concrete from codes, based on which the strength relationship between the concrete and mortar was established.The mesoscale model made a good estimation of the stress–strain and lateral–axial strain curves of the concrete in active confinement, illustrating that it was an efficient tool for investigating the concrete’s mechanical and deformational behavior.The distribution of the lateral strain along the circumference and the height of the specimen was not uniform when compressive failure occurred in the concrete.The confinement ratio had a more significant influence on the transverse dilation than that of the concrete’s strength. With the increase in the confinement ratio, the transverse dilation was inhibited, and then the concrete’s secant strain ratio slowly developed.An expression of the lateral–axial strain curve of the concrete in active confinement was proposed, which can reflect the simulation results of the mesoscale model, and it was consistent with the prediction of the existing formulas.A mesoscale concrete model was established to investigate the dilation properties of actively confined concrete in the current study. The following conclusions were formulated through the analysis:Mesoscopic analysis can reflect the heterogeneity of concrete and the interaction among different phases, which is an efficient method to study the mechanical and deformation behavior of concrete. At present, the content of our research is limited, and we anticipate conducting further research using this approach in the future, such as the influence of nano silica or fiber on the performance of concrete, and the interaction mechanisms of the steel tube and concrete core.


Stable complexes were formed between SL and Cu2+, Fe3+, and V5+, and the ligand-to-metal binding stoichiometry was found 2 : 1 in the SL-Cu2+ and SL-Fe3+ complexes, and 1 : 1 in the SL-V5+ complex. The results of LOD and bending constant were , , and  for Cu2+, Fe3+, and V5+, respectively.The ligand 3-(-(2-hydroxyphenylimino) methyl)-4H-chromen-4-one (SL) has been synthesized and examined as a chemosensor for some metal ions in aqueous solutions based on colorimetric analysis. Color changes were monitored using UV–visible spectroscopy. Binding stoichiometry and limit of detection (LOD) were estimated using titration experimentation based on UV–visible absorbance and Job's plot. The synthesized ligand was tested for selectivity in the presence of several cations and was examined for possible utility as a chemosensor in real water samples. The results indicated sensing ability and selectivity for Cu It is well established that some metal ions have important beneficial roles and functions in biological and chemical processes, and on the other hand, overexposure to some metal ions causes toxicity. Transition metals are abundant in nature, particularly in soils and aquatic systems and releases of these elements, to the environment are associated with industrial processes. However, their extreme presence in water resources causes serious contamination and can be considered hazardous pollutants at some levels, and creates a threat to human beings and other biota because of their bioaccumulation in food chains and toxicity effects , 2. Ther2+, Fe3+, and V5+. Information of the high levels of exposure to copper from diet and drinking water results in gastrointestinal and hepatic systems illness as it is absorbed rapidly by the stomach and intestine .2H2O, [Fe(SL)2].3H2O, K3[VO2SL], (NH4)3[VO2SL] complexes, respectively.In order to prove and support the formation of metal complexes and the binding of SL with Cu3+ and V5+ complexes are listed in −1 in the FT-IR spectrum of the free organic ligand (SL) is due to ν(C=N) vibration , K3[VO2SL], and [Fe(SL)2].3H2O, respectively, due to the involvement of the nitrogen atom of the azomethine group in coordination with metal ions [In order to confirm the complexation of the SL ligand with Fe8.15 ppm . The peatal ions . Moreovetal ions .2].2H2O, [Fe(SL)2].3H2O  molar ratio in the case of copper and iron) and K3[VO2SL] or (NH4)3[VO2SL]  molar ratio in the case of vanadium), as shown in According to the observations of Job's plot, UV–visible titration, mass spectra, FTIR measurements, and NMR spectral analysis, the binding possibility of the tested Schiff base ligand (SL) to the targeted metal ions can be suggested, as shown in the proposed feasible structures of the complexes with general formulae [Cu(SL)2+, Fe3+, and V5+ ion detection in the presence of different metal ions such as Cr3+, Fe2+, Ni2+, Ca2+, Co2+, Mg2+, Zn2+, Mn2+, Hg2+, Pb2+, Ba, and Al3+ was investigated. For this purpose, a competitive titration experiment was performed, and each of the interfering metal ions was added separately to the solution of SL- Cu2+, SL-Fe3+, or SL-V5+, and the resulting solutions were scanned for UV–vis absorption spectra, and the results are expressed as absorbance intensity versus the composition of the sensing solution , and the UV–visible spectra were recorded every 8 hours. The observations showed that the intensity of UV absorbance remained constant over the entire period, which is a sign of the stability of the examined SL chemosensor.The interference of the coexisting ions during the practical utility of the examined chemosensor SL for Cu Figures –10. It i2+, Fe3+,or V5+ metal ions, experimental tests were performed with distilled and real water samples taken from tap water of domestic water supply and from the Al-Aqiq water reservoir dam located at Al-Baha region, Saudi Arabia. Each water sample was spiked with a known amount of Cu2+, Fe3+, or V5+ (70 µM), and then their concentrations were estimated from the calibration curve. The observations  hydrazone as a selective and sensitive probe for Alesponses . The rep2+ ions in semiaqueous media, which showed sensing ability for copper ions with different concentrations ranging from 10−3 to 10−7 in aqueous solutions and showed excellent applicability in real water samples [3+, Cu2+, Fe3+, and Al3+ ions simultaneously in DMF/H2O (v/v = 1/1) solution with LOD of 3.37 × 10−7 M, 4.65 × 10−7 M, 3.58 × 10−7 M, and 4.89 × 10−7 M, respectively [2+, Zn2+, and CN− ions in aqueous media with excellent accuracy and precision with detection limits of 5.50 × 10−7, 8.70 × 10−7 and 1.56 × 10−6 M, respectively, which are lower than the permissible levels recommended by WHO for safe drinking water [2+ ions only by fluorescence quenching with a LOD of 3.95 × 10−6 M [2+, Fe3+, and V5+ ions, in aqueous media with excellent accuracy and precision with detection limits of 3.322 × 10−5 M, 2.065 × 10−5 M, and 1.782 × 10−5 M, respectively, which are lower than the permissible level recommended by WHO for safe drinking water.Another chromone-based colorimetric sensor was developed and synthesized by the condensation reaction between 3-formyl-6-methylchromone and 2-aminobenzamide for highly selective detection of Cu samples . Colorimectively . A chromng water . A chrom× 10−6 M . Compare2+, Fe3+, and V5+. Experimental results showed 2 : 1 (SL:M) binding stoichiometry in the case of SL-Cu2+ and SL- Fe3+ complexes, while 1 : 1 stoichiometry was observed in the SL-V5+ complex. The LOD and bending constant were found to be , , and  for Cu2+, Fe3+, and V5+, respectively.We have synthesized the Schiff base ligand 3-(-(2-hydroxyphenylimino) methyl)-4H-chromen-4-one (SL) and examined it as a chemosensor for the detection of metal ions in aqueous solutions. The tested compound (SL) exhibited a remarkable selectivity and sensitivity response toward three metal cations: Cu


Concrete is one of the most widely used structural construction materials and has significantly influenced industrial development. Recently, various efforts have been made to reduce greenhouse gases worldwide, with the concrete industry also making efforts to move toward sustainable development. In particular, owing to the immense growth of the global concrete industry, the shortage of natural aggregates has become a serious problem. Therefore, a considerable amount of social and research interest has focused on finding alternative aggregate materials to replace natural aggregates. Considering that aggregates comprise a large proportion of the concrete volume, they significantly influence the mechanical properties and durability of the concrete. Therefore, when replacing the existing natural aggregate with a substitute aggregate material, there should be no deterioration in the performance of the concrete; in this way, sustainable concrete development can be achieved.From this perspective, this Special Issue is a collection of ten papers focused on various alternative aggregate materials for sustainable concrete development, including substitute aggregate materials, artificial aggregates, recycled aggregates, and functional concrete materials. In the Editors’ opinion, each article presents a novel, clear, scientific idea from various standpoints , thus representing a major contribution to our understanding the mechanical behavior of various aggregates and concrete materials. The Editors hope this article collection can contribute to the continuous research for a more thorough and reliable understanding of the mechanical behavior or durability characteristics of sustainable aggregates and concrete materials.Among the experimental contributions, Wenhao et al.  evaluateSe-Jin et al.  synthesiSung-Ho et al.  investigMaria et al.  examinedJi-Hwan et al.  applied In two papers, 3D-printed capsules were researched. Taeuk et al.  producedTianyu et al.  examinedFinally, Se-Jin et al.  evaluate


In this review, we describe previous basic and clinical studies on autonomous aldosterone production. Over the past decades, mineralocorticoid receptor antagonists (MRAs) have been found to concentration-dependently inhibit steroidogenesis in different degrees. However, many studies have proven the suppressive effects of MRAs on the activities of hormone synthase. The probable factors of cytochrome P-450 reduction, both in microsomes and mitochondria, have also been considered: (1) one of the spironolactone metabolite forms had destructive function, except canrenone, (2) 7α-thio-spironolactone was an obligatory intermediate in the spironolactone-induced CYP450 decrease, and (3) the contributing steroids should have 7α-methylthio or 7α-methylsulfone groups. In previous clinical research, spironolactone-body-containing cells showed a type II pattern of enzyme activity , and the subcapsular micronodules composed of spironolactone-body-containing cells also exhibited a type II pattern and excess aldosterone secretion, indicating that the subcapsular micronodules might be the root of aldosterone-producing adenoma. Moreover, combined with the potential impeditive function to aldosterone secretion, a few cases of spontaneous remission of primary aldosteronism, with normal ranges of blood pressure, plasma potassium, plasma renin activity, and aldosterone renin ratio, have been reported after long-term treatment with MRAs. Primary aldosteronism (PA) is a leading form of secondary hypertension, a disease that presents with hypertension due to autonomous aldosterone hypersecretion from the adrenal glands. Compared to patients with essential hypertension (EHT), PA is associated with a three- to four-fold higher frequency of cardiovascular complications  and has SL has been widely used as an MRA for different types of hypertension since its discovery in the mid-1950s. SL is used for the oral treatment of PA as an MRA. SL has been reported to inhibit aldosterone synthesis in the adrenal cortex and aldosterone antagonism in the periphery. Inhibition of hormone synthesis has been reported for 11β-hydroxylase ,7,8,9, 2By comparing the effects of SL, canrenone, and potassium canrenoate on cytochrome P450 in human and bovine adrenal glands, Cheng et al.  found thIn addition, another report focusing on aldosterone synthesis influenced by different MRAs, SL, prorenone, SC 19886, SC 26304, and SC 27169, by Netchitailo et al.  revealedAccording to these studies, canrenone, one of the major active forms of SL, is often used to compare its effects with SL. SL is a prodrug with a short half-life (1.4 h), and the long half-life of canrenone 16.5 h) has also led to the conclusion that canrenone might be the main therapeutic form of SL treatment [.5 h has In addition to aldosterone secretion from the zona glomerulosa, the production of cortisol from the zona fasciculata has begun to attract attention. A study designed by Rourke and Colby  used adrThrough further studies, we can clearly see that SL has the ability to reduce the production of aldosterone and cortisol in several ways. On the one hand, the enzyme activities of 21-hydroxylase, 11β-hydroxylase, and 18-hydroxylase will be affected by SL, leading to the reduction in aldosterone; on the other hand, the selective inhibition of 17α-hydroxylase will certainly decrease the production of cortisol. SL, canrenone, and 7α-thio-SL were potent inhibitors of aldosterone synthesis and cortisol production. The in vitro experiment by Menard et al.  showed tKossor and Colby  used a hInterestingly, they found that a high dose of SL could decrease the concentrations of testosterone in testicular and peripheral venous plasma, and the concentration of cortisol in adrenal venous plasma, by 60–75% and 50–65%, respectively. In contrast, canrenone and potassium canrenoate did not cause any significant changes. Although some of the positive results were meaningful and helpful for the study of SL, the difficulty between practical usage and experimental results is worth noting. Ye et al.  reportedMRAs, such as canrenone or potassium canrenoate, SL, and EP, play an essential role in hypertension because of their function in reducing blood pressure (BP) and improving hypokalemia. However, with further studies on MRAs , especiaAiba et al.  focused n = 54). Similarly, a clinical study by Fourkiotis et al. [n = 29), corroborated this conclusion by revealing a PAC increase from 58.3 ± 9.0 to 159.3 ± 35.9 ng/L. However, there are also some interesting cases reporting the decrease in PAC during treatment. A 51-year-old female patient with PA and a 3-year history of hypertension showed improvements in the PAC and aldosterone secretion after two periods of SL treatment [Generally, during MRA treatment, plasma aldosterone concentration (PAC) is expected to concomitantly increase in patients with PA. We  found ths et al. , which creatment . The firreatment , high-dereatment , and salreatment . The impp = 0.045). Moreover, they claimed that 12 of 15 patients had normal aldosterone/PRA ratios, which might be due to the repaired sensitivity of the adrenal gland to Ang II caused by long-term therapy with aldosterone receptor blockers. This hypothesis was further supported by a subsequent study performed by the authors [Armanini et al.  recruite authors , which fIn addition to potassium carenoate, some research focusing on SL has also been conducted. Fischer et al.  studied However, there were some different opinions in the study by Lucatello et al. , who thoIn this review, we outlined the findings of basic and clinical studies regarding the remission phenomenon with MRAs in PA. These studies have elucidated the inhibitory effects of MRAs on steroidogenesis and aldosterone secretion, which might lead to spontaneous remission of PA after long-term treatment with MRAs. Further studies of PA remission are required.


This study investigated recycled aggregates of Jeju Island—where porous basalt exists as a natural aggregate—and is a study aimed at verifying the applicability of the basalt-based recycled aggregate in the field. To this end, the application properties of concrete were analyzed using the quality improvement of the recycled aggregate (PRA), the type of fine aggregate received in the region, and the cement content as variables. In an experiment using concrete in which 50% of the PRA was replaced with basalt (BA), the air content of the fresh concrete decreased due to the increasing solid content of the aggregate when PRA and fine aggregates (with an even particle size distribution) were used. Regarding the properties of the hardened concrete, when the PRA and fine aggregate (with a high fineness modulus) were used, the compressive strength was 33.6 MPa and the modulus of elasticity was 23.1 GPa, which are higher than those of the BA specimen. The resistance to carbonation increased due to the improved quality of the PRA specimen. Pores that are 0.3 mm in size or larger decreased when the PRA and fine aggregates of even particle sizes were used. This form of pore reduction was found to have a direct correlation with the improvement of mechanical properties. Large-scale capital development projects on Jeju Island, including the construction of Jeju’s second airport and its new port, are scheduled, all of which require enormous volumes of construction materials, such as cement and aggregate . Consequ2 to CaCO3 by injecting CO2 into recycled aggregate containing old cement paste. Various methods for enhancing the quality of recycled aggregates by applying or combining these properties are being studied [The abrasion rate, absorption rate, and density of recycled aggregate can affect concrete quality, as discussed in the literature. Various studies on their physical, chemical, and curing properties have been proposed to improve the quality of recycled aggregates. Typically, such researched physical methods aimed at impacting old cement paste using crushing and grinding equipment, with chemical methods, such as removing old cement paste using strong acid solutions, suggested. Unlike the above two methods, the curing method reinforces the matrix by transforming Ca(OH) studied ,14,15,16 studied ,18. In a studied . The res studied ,21. The  studied ,23. The  studied ,25,26. H studied ,28.In a recent study on recycled Jeju aggregates, a recycled aggregate with an absorption rate of 3–5% was produced using a physical method that considered time and costs, in which its concrete properties of mix strength (50 MPa) and performance  were verified . However3, respectively. For these three variables and three factors, nine mix proportions were selected using the Taguchi method of experimental planning, the selected mix proportions of which are listed in This study examined mixes of fine aggregates and binders for the practical use of recycled aggregates for concrete on Jeju Island. The cement used in the experiment was ordinary Type 1 Portland cement from ‘S’ Company . The physical and chemical properties of the cement are shown in 2 concentration of 5 ± 0.2% [We conducted experiments in accordance with ASTM standards to analyze the fresh properties and hardened properties of the concrete. Accordingly, a slump of the fresh concrete was measured using a slump cone with a base diameter of 200 mm, top diameter of 100 mm, and height of 300 mm based on ASTM C143:2020  specific5 ± 0.2% . The con5 ± 0.2% . The expx represents the compressive strength, and y describes the modulus of elasticity of concrete.The relation equation of compressive strength and modulus of elasticity of basalt-based recycled aggregate concrete in Jeju Island is as follows:However, there is no decrease in compressive strength and modulus of elasticity with an increase in the replacement rate of recycled aggregate mentioned in many studies. It is known that mechanical performances deteriorate when recycled aggregates containing a relatively large number of pores are used. However, the results of this study suggest that the increase in the concrete’s fillability of the concrete owing to the improvement of the aggregate shape has a greater effect. Moreover, it can be considered that the weak part of the old paste does not act at the general strength of 30 MPa. In addition, the increase in pores due to the type of fine aggregate appears to have a greater effect.x and y represent the air content of fresh concrete and the modulus of elasticity of hardened concrete, respectively.The relation equation of the modulus of elasticity and air content of basalt-based recycled aggregate concrete in Jeju Island is as follows:The compressive strength does not exhibit any correlation with the air content in non-hardened concrete. However, the modulus of elasticity decreases as the air content increases, indicating an inversely proportional relationship. This suggests that the modulus of elasticity could be more directly affected by the concrete pores than its compressive strength.2 supply, with the porosity of the old paste acting as a weak part for carbonation.2 value of the relationship between the depth of carbonation and air content after 13 weeks is as high as 0.80, and the correlation of the air content with the depth of carbonation decreases marginally after 26 weeks. This can be considered to be because the carbonation resistance decreases as the old paste of RRA is neutralized. It can be surmised that if the recycled aggregate contains a large volume of old paste, carbonation resistance will decrease as the age increases.2 value of 0.75. It can be considered that the ratio of 0–0.2 mm pores due to the aggregate would be high, and the image shown in x and y represent the air content of fresh concrete and the porosity of image analysis of hardened concrete, respectively.The relation of porosity and air content of basalt-based recycled aggregate concrete in Jeju Island is described as follows:2 value of 0.889. Consequently, the pore-related characteristics—such as the unconsolidated air content, depth of carbonation, and modulus of elasticity—exhibit similar tendencies.The air content of fresh concrete causes the collapse of trapped pores before hardening, thus decreasing the pore content of the hardened concrete. However, the main aggregate used in this experiment is basalt, and the air content partially increases because a large number of pores  are included in the aggregate. The correlation between the air content of the unhardened concrete and the porosity of the hardened concrete is high, with an RConcrete, Mindess et al. suggested that in order to reliably prevent freeze–thaw, the specific surface area of pores should be at least 25 mm2/mm3 and the spacing factor should be less than 200 μm. Furthermore, the Kansas DOT claimed that the spacing factor for concrete before hardening should be 250 μm or less [2/mm3. In the case of the formulation containing RRA and PRA, the specific surface area of the pores can be found to approach 25 mm2/mm3 owing to the influence of the old paste. Consequently, it can be expected to have a relatively high resistance to freeze–thaw. or less . In thisp < 0.05), and the explanatory power of the regression model was approximately 98.7% (modified R squared was 97.3%) . Because the Durbin–Watson statistic was close to 2 (2.364), there was no problem with the assumption of the independence of residuals. In addition, the variance inflation factor (VIF) was less than 10 for all instances; therefore, there was no multicollinearity problem. As a result of the significance test of the regression coefficient, cement , PRA , and C sand  all had a significant negative effect on carbonation depth, and RRA  had a significant positive effect. In other words, it was found that the higher the cement, PRA, and C Sand, the lower the depth of accelerated carbonation value; moreover, the higher the RRA, the higher the depth of accelerated carbonation value. Comparing the sizes of the standardization coefficients, it was verified that the depth of accelerated carbonation was greatly affected, in the order of greatest to least effect, by C sand (=−0.532), PRA (=−0.502), RRA (=0.411), and Cement (=−0.242).A multiple regression analysis was conducted, as shown in The non-standardized regression equation of cement, RRA, PRA, and C sand for the depth of accelerated carbonation is as follows:p > 0.05). In contrast, C sand had a significant negative correlation with compression strength, −0.710; flexural strength, −0.706; and modulus of elastic, −0.806 (p < 0.05). In addition, the correlation coefficients of general sand with compression strength, flexural strength, and modulus of elasticity were 0.727, 0.723, and 0.814, respectively, indicating a significant positive correlation (p < 0.05). That is, it was found that the concrete mixing ratio of water, cement, and basalt did not significantly affect the compressive strength, flexural strength, and elastic modulus of concrete only for the mixing ratio presented in this paper. For the depth of accelerated carbonation, water, cement, BA, S sand, and C sand were not statistically significant (p > 0.05), whereas RRA and PRA were found to be statistically significant (p < 0.05). The correlation coefficient of RRA and PRA was 0.675 and −0.724, respectively. It was found that when the proportion of RRA increased in the mixing concrete, the depth of accelerated carbonation increased, and when the proportion of PRA increased in the mixing concrete, the depth of accelerated carbonation decreased. For PRA, porosity was the only mechanical property that was statistically significant (p < 0.05). Considering that the correlation coefficient between porosity and PRA was −0.759, the porosity of concrete decreased as the proportion of PRA increased.Pearson’s correlation analysis was conducted to analyze the correlation between the mechanical properties of concrete such as compressive strength, flexural strength, modulus of elasticity, the depth of accelerated carbonation, and raw materials, and the results are shown in This study analyzed changes in the mechanical properties of concrete using the type of recycled aggregate, the type of fine aggregate, and the unit cement content as variables for the application of porous basalt-based recycled Jeju aggregate in the field. The findings are as follows.The change in slump based on the replacement of the improved basalt-based recycled aggregate in the properties of non-hardened concrete was not significant. The slump increased as the fineness modulus of the fine aggregates decreased, and the unit cement content increased. Furthermore, the air content increased considerably when sea sand was used.Regarding the strength properties of concrete, there was no major deterioration in quality due to the use of basalt-based recycled aggregate in concrete with a strength of 30 MPa at 28 days of age. The compressive strength of the concrete increased owing to the quality improvement of the basalt-based recycled aggregate and the even particle size distribution of the fine aggregates. The maximum compressive strength of concrete using recycled aggregate was 33.6 MPa, whereas the compressive strength of concrete using natural aggregate was 29.5 MPa for the concrete of 28 days of age.The modulus of elasticity of concrete increased slightly due to the increased concrete’s fillability when the concrete was mixed with improved basalt-based recycled aggregates. The highest modulus of elasticity of 23.1 GPa was observed when uniform fine aggregates were used at 28 days age. The change in the modulus of elasticity using fine aggregates had a greater effect than the use of recycled aggregates.The depth of accelerated carbonation of concrete decreased as the concrete matrix density increased when the improved basalt-based recycled aggregate was used. However, when RRA was used, the depth of carbonation increased rapidly with age. The depth of carbonation was large when fine aggregates of uneven grain size were used because dense concrete could not be formed.2/mm3.The concrete pores increased in proportion to the pore content of the basalt and recycled aggregates. Pore content of 0.3 mm or larger decreased when improved basalt-based recycled aggregates and fine aggregates of even particle size were used. The decrease in pore content was directly related for the improvement of carbonation resistance and modulus of elasticity. Using recycled aggregates can be expected to increase the freeze–thaw resistance because the pore spacing factor was 200 μm or less and the specific surface area of the pore was close to 25 mmIn multiple regression and correlation analyses, it was found that the quality improvement of the fine and recycled aggregates improved the depth of carbonation resistance and the mechanical properties and durability index of concrete, respectively.3 at a concrete mixing strength of 30 MPa due to the improvement in quality. Compared to basalt—a local natural aggregate—the improved recycled aggregate did not exhibit many differences in concrete quality. Consequently, it would be necessary to select a fine aggregate of good quality in the consideration of concrete quality and economic feasibility. Although additional verification of the mechanical properties and durability, such as dry shrinkage of recycled aggregate concrete, is required, its use in structural concrete products of 40 MPa or less was confirmed. It is believed that, with continued research, recycled aggregates mixed with valid industrial byproducts can be used as an effective substitute for basalt natural aggregates.The recycled aggregate used in this experiment did not meet the domestic recycled aggregate standard of 3% or less. However, there was no deterioration in quality even with a unit cement content of 300 kg/m


Pseudomonas antibiotics were compared. The DOT value was 9.94, and the relative DOT were 34.8% for carbapenems, 32.1% for piperacillin/tazobactam, 24.3% for fourth generation cephalosporins/ceftazidime/aztreonam, and 8.9% for fluoroquinolones. Although no correlation was found between the total DOT and the resistance rate for any bacterium, a significant negative correlation was found between the heterogeneity indices and resistance rates for Pseudomonas aeruginosa and Klebsiella pneumoniae. The significant cutoffs that discriminate the risk of resistance were 0.756 for the AHI and 0.889 for the modified AHI for K. pneumoniae. Antibiotic diversity is more important in preventing AMR than overall antibiotic use. The ideal ratio of broad-spectrum antibiotics should be studied for diversified use to prevent AMR.Increased antibiotic use and antibiotic homogeneity cause selective pressure. This study investigated the correlation between antibiotic diversity and antimicrobial resistance (AMR) in Gram-negative organisms. The days of therapy/100 patient-days (DOT) for four broad-spectrum antibiotic classes were evaluated for 2015–2022. The antibiotic heterogeneity index (AHI) for the equal use of four classes (25%) and the modified AHI for the equal use of three classes (30%), excluding fluoroquinolones (10%), were measured (target: 1.0). Quarterly antibiotic use markers and the resistance rates against ≥2 anti- Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa were responsible for 3.57 million deaths associated with AMR in 2019 ) and were compared using Because there was more than one episode at which AS could be initiated in certain patients, the number of events was evaluated instead of the number of patients. The mean annual number of events for AS was 1113.7 ± 89.2, and the mean annual cumulative number of interventions or supervisions for consultation, including timeouts, was 9688.4 ± 695.9. Thus, the number of interventions or supervisions required for one event was 8.71 ± 0.15. A summary of the patient background information for each year between April 2015 and March 2022 is presented in The annual number of events according to the reasons for the intervention/supervision is presented in The DOT value was 9.94 for the consecutive 7-year study period. As a measure of risk-adjusted antibiotic use, the SAAR of adult broad-spectrum antibacterial agents predominantly used for hospital-onset infections in the Department of Respiratory Medicine, the Department of Colorectal Surgery, and the ICU is demonstrated in In the evaluation of diverse antibiotic use, an AHI of >0.8 and a modified AHI of >0.9 were almost achieved between 3Q/2017 and 1Q/2019. However, a decrease in both antibiotic heterogeneity indices was observed from 4Q/2020 onwards, when more than 2000 new daily cases of COVID-19 were being reported for the first time in Japan. Although the total DOT for the evaluated broad-spectrum antibiotics gradually increased from 2Q/2015 (8.2) to 2Q/2017 (11.2), with the start of the prospective audit and feedback, the DOT decreased to 9.3 in 4Q/2017 and was sustained within the range of 9 to 10 until 2Q/2021. However, the DOT increased to approximately 11 after 2Q/2021. P. aeruginosa, and A. baumannii are presented in P. aeruginosa was 7.5%, and a slight increase was observed from April 2021–March 2022 (12.0%). The isolation of carbapenem-resistant A. baumannii was rare . The resistance rates against each antibiotic with anti-Pseudomonas activity among Gram-negative species are presented in The resistance rate against ≥2 antibiotic classes was 14.5% and that against ≥3 antibiotic classes was 7.2% in glucose non-fermenting Gram-negative rods and 14.1% and 3.8% in Enterobacteriaceae spp., respectively . The rat2 0.176, p = 0.026; modified AHI: R2 0.320, p = 0.002) or ≥3 antibiotic classes  in glucose non-fermenting Gram-negative rods. A significant negative correlation was observed in the resistance rate against ≥2 antibiotic classes in Enterobacteriaceae spp. . By contrast, no correlation was found between the total DOT for broad-spectrum antibiotics and the antibiotic resistance rate in either of the Gram-negative rod groups (Using the quarterly data for 7 years (28 terms), the correlation between each antibiotic marker and the resistant rate against ≥2 or ≥3 antibiotic classes was evaluated . The AHId groups .P. aeruginosa and K. pneumoniae  or the modified AHI  in K. pneumoniae. However, no significant correlation was found between the total DOT and the resistance rates against ≥2 and ≥3 antibiotic classes for any organism.Among the Gram-negative rod species, a significant negative correlation between the AHI or modified AHI and the antibiotic resistance rates was observed for eumoniae . NotablyP. aeruginosa against ≥2 classes of antibiotics was less than 15% between 4Q/2015 and 1Q/2018, the resistant rate reached 20% several times after 2Q/2018. The resistance rate for K. pneumoniae against ≥2 classes of antibiotics was approximately 5% until 3Q/2019, and, in general, the rate increased steadily thereafter. There was a tendency toward an increase in the antibiotic resistant rates in these organisms during the COVID-19 pandemic.The time series of the resistant rate of these organisms are demonstrated in P. aeruginosa. By contrast, the significant cutoff values were 0.756 for the AHI  and 0.889 for the modified AHI  for resistant strains against ≥2 antibiotic classes in K. pneumoniae. However, the high accuracy of these antibiotic heterogeneity indices cannot be expected to discriminate the risk for resistant strains because of the low-level AUC.In the analysis for the ROC curve, significant cutoffs of antibiotic heterogeneity indices that discriminate the isolation of resistant strains were not determined for K. pneumoniae against ≥2 classes of antibiotics in the AHI, modified AHI, and overall DOT for broad-spectrum antibiotic classes is shown in p = 0.206). The ROC curve that demonstrated the detection ability of resistant Klebsiella sp. Troughton et al. [E. coli and K. pneumoniae against carbapenems increased during the implementation of the AS program. Determining the changes in antibiotic administration and the likely effect on resistance levels at hospitals is important to identify the priorities in terms of intervention. Veličković-Radovanović et al.  reportedn et al.  demonstrn et al.  found thn et al.  reportedP. aeruginosa resistance to carbapenems but also the rate of three anti-pseudomonal agents, including carbapenems. They observed a negative correlation between resistance and the diversity of antibiotic use, as measured by the Peterson index. Sandiumenge et al. [A. baumannii and ESBL-producing Enterobacteriaceae. Interestingly, compared with other strategies including antibiotic cycling, greater antimicrobial diversity was achieved following treatment by well-trained physicians with patient-specific empirical antimicrobial selection considering the length of hospitalization and recent antibiotic exposure. Plüss-Suard et al.  reportede et al.  reportedP. aeruginosa and A. baumannii decreased to less than one third that of the baseline rate. In P. aeruginosa, the resistance rates to cefepime and piperacillin/tazobactam decreased soon after the start of PAMS, and the resistance rate to imipenem decreased 6 months after the start of PAMS.We previously developed a strategy called periodic monitoring and supervision (PAMS).  RecommenPseudomonas antibiotics. The total broad-spectrum antibiotic use was not a significant factor in increasing the resistance rates, but a decrease in the antibiotic heterogeneity indices caused an increase in the resistance rates for P. aeruginosa and K. pneumoniae. Because high-level antibiotic heterogeneity was achieved and the restriction of carbapenems or piperacillin/tazobactam was not required between September 2007 and March 2008, PAMS was discontinued in June 2008 in our institution. Although the restriction of piperacillin/tazobactam was announced in the hospital between November 2017 and March 2018 because of a sustained high % DOT for this antibiotic, no strict strategic control of antibiotic use was implemented, and prospective audit and supervision and consultation services with frequent timeouts for the appropriate antibiotics in each patient have been conducted. In the present study, the 7-year study period was divided into 28 terms, and the quarterly overall DOT for broad-spectrum antibiotic classes and two antibiotic heterogeneity indices were compared with the resistance rate against ≥2 and ≥3 anti-K. pneumoniae against ≥2 tested antibiotics were 0.756 in the AHI and 0.889 in the modified AHI, respectively. Although the modified AHI appears to be a feasible index in Japan, there was no significant difference in the AUC between the original AHI (0.636) and the modified AHI (0.616). If the discrimination ability for the increased risk of antibiotic resistance is 100%, the AUC is 1, whereas if the discrimination ability is 0%, the AUC is 0.5. Thus, we were unable to conclude which was the better index for balanced antibiotic use to decrease the risk of the isolation of resistant Gram-negative rods.Based on the DOT % for each broad-spectrum antibiotic derived from our 7-year time series, a rate of 25% use of fluoroquinolones, which is the target for the AHI , might be too high to achieve in clinical practice in Japan. Hence, we proposed a modified AHI specifying 10% use of fluoroquinolones and 30% use of each of the remaining three antibiotic classes. A higher value was achieved using the modified AHI compared with the original AHI. The cutoff values that discriminated the isolation of resistant P. aeruginosa and K. pneumoniae were compared with the AMR rates derived from Japanese nationwide surveillance [P. aeruginosa isolated from patients with respiratory tract infections [K. pneumoniae isolated from postoperative intra-abdominal infections [P. aeruginosa . However, variable differences between the non-susceptibility rates in this study and that of nationwide surveillance were found in K. pneumoniae . In previous studies, considerable differences in AMR rates have been reported for different countries ,38,39,40eillance ,42. The fections  were 17.P. aeruginosa and K. pneumoniae were observed from 2Q in 2020, both of which had a substantial effect on the results of this study. The annual number of interventions or supervisions has been sustained during the pandemic. However, a reduced adherence rate for intervention might decrease the antibiotic diversity, but this rate was not available. The increased % DOT with carbapenems should have been recognized and corrected earlier. Third, although the resistance rate for K. pneumoniae had a substantial impact on the results, only seven of the isolated strains of this organism produced carbapenemase. Fattouh et al. [K. pneumoniae from clinical isolates were found to produce IMP-type carbapenemase in Japan. Furthermore, 83 of 104 CPE isolates were IMP-6 producers, the majority of which simultaneously produced CTX-M type ESBL. Meropenem MIC <2 μg/mL rates were 75.0% in IMP-6-positive K. pneumoniae, suggesting that several IMP-6-producers might not have been identified by routine screening [This study has several limitations. First, the results obtained from the day-to-day activity of the AS program were analyzed retrospectively. Second, the COVID-19 pandemic may have impaired the activities of the AS program ,44 and mh et al.  reportedcreening . TherefoWe propose the following measures for the balanced use of broad-spectrum antibiotics to decrease the AMR based on the results. First, the establishment of a comprehensive AS program, including education and a prospective audit and feedback, aimed at achieving appropriate antibiotic use in each patient is required. There is a tendency for the prolonged use of broad-spectrum antibiotics, such as carbapenems and piperacillin/tazobactam, which causes selection of AMR organisms with resulting poor susceptibility to these antibiotics. Hence, planned changes to other antibiotics should be considered within 7 to 10 days based on the culture results and the patient’s clinical course. Second, even with appropriate antibiotic selection or duration of administration in each individual patient, hospital-wide antibiotic use may also become unbalanced over time. Therefore, the early detection of the excess use of particular antibiotics by periodic antibiotic monitoring is required to optimize hospital-wide antibiotic use. Third, from our experience, substantial full-time equivalents for AS activity of the core members should be sustained during the ongoing COVID-19 pandemic because AS activity might be impaired as a result of the additional roles gained by staff during this time .The balanced use of broad-spectrum antibiotics and antibiotic diversity are more important in preventing AMR among Gram-negative rods than the total use of broad-spectrum antibiotics. This study underscores the importance of the routine monitoring of antibiotic heterogeneity indices, which are easy to calculate as a convenient tool for the AS metric. Further studies are required to determine the ideal ratio of broad-spectrum antibiotic classes for diversified use to prevent AMR.


The tyrosine kinase receptor encoded by the MET oncogene has been extensively studied. Surprisingly, one extracellular domain, PSI, evolutionary conserved between plexins, semaphorins, and integrins, has no established function. The MET PSI sequence contains two CXXC motifs, usually found in protein disulfide isomerases (PDI). Using a scrambled oxidized RNAse enzymatic activity assay in vitro, we show, for the first time, that the MET extracellular domain displays disulfide isomerase activity, abolished by PSI domain antibodies. PSI domain deletion or mutations of CXXC sites to AXXA or SXXS result in a significant impairment of the cleavage of the MET 175 kDa precursor protein, abolishing the maturation of α and β chains, of, respectively, 50 kDa and 145 kDa, disulfide-linked. The uncleaved precursor is stuck in the Golgi apparatus and, interestingly, is constitutively phosphorylated. However, no signal transduction is observed as measured by AKT and MAPK phosphorylation. Consequently, biological responses to the MET ligand—hepatocyte growth factor (HGF)—such as growth and epithelial to mesenchymal transition, are hampered. These data show that the MET PSI domain is functional and is required for the maturation, surface expression, and biological functions of the MET oncogenic protein. The MET proto-oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor(HGF). MET is an essential gene involved in organ development, angiogenesis, and wound healing ,2,3. In MET is translated into a precursor protein of 175 kDa that matures through proteolytic cleavage by furin in the Golgi apparatus . The extThe MET PSI domain is a cysteine-rich conserved sequence with two non-canonical CXXC motifs observed in disulfide isomerases ,16. We dSequence alignment analyses show that the PSI domain is highly conserved between MET, RON, integrins, PLEXINA1, and SEMA4D, which are all molecules involved in cell growth and invasion. Notably, seven cysteines and two CXXC motifs display complete conservation between the proteins mentioned above, highlighting their potential functional importance B. IndeedWe explored whether the MET PSI domain has a similar enzymatic property. We engineered and produced the extracellular domain of MET (MET ECD) and purified it by affinity chromatography. As anticipated, Coomassie stainings display a single 90 kDa β and 50 kDa α chains under reducing conditions, and a single 140 kDa α-β heterodimer under non-reducing conditions A. KD ≈ 0.3 ± 0.1 µM, α2/β3 (ITGA2B3) and the isolated PSI domain of integrin β3  and transfected it in MET-negative TOV112D cells. wt_MET constructs were used as control. The proteins translated from wt_MET is resolved in two bands, a major corresponding to the mature MET β chain (145 kDa) and a minor corresponding to the precursor (175 kDa). By contrast, in MET_∆PSI-transfected cells, the relative ratio between the two bands is inverted, suggesting that the cleavage is hampered C. SimilaWe next investigated the subcellular localization of the MET precursor in CXXC mutants. As expected , confocaSince the precursor accumulates, we investigated if tyrosine kinase is constitutively phosphorylated by clustering in the Golgi. To avoid constitutive phosphorylation due to uncontrolled overexpression and clustering of the kinase  in transMET activates multiple signal transducers, including PI3K/AKT  and MAPK (proliferation). The subsequent activation of those pathways results in epithelial–mesenchymal transition (EMT) ,31. We, S. cerevisiae) were purchased from Sigma-Aldrich . Sarcosine was purchased from Thermo Scientific™ . HGF was purchased from R&D , and 16F16 was purchased from Selleck Chemicals LLC . All antibodies used in this study are listed in Ribonuclease A (RNAse) from bovine pancreas , dithiothreitol (DTT), ethylendiamminotetreaacetic acid (EDTA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), L-glutathione (GSH), oxidized glutathione (GSSG), and ribonucleic acid (RNA) from baker’s yeast (cDNA sequences of human MET ectodomain (MET ECD: Δ25–932) and mouse β3 integrin PSI (1–56) were C-terminally tagged with Flag-His. The engineered soluble receptor was produced by transfection of HEK293T cells and purified from cell supernatants by affinity chromatography using HisTrap HP columns  according to the manufacturer’s instructions. Large-scale protein production and purification were performed by U-Protein Express BV . Human α2β3 integrin was purchased from R&D .w/v) in 50 mM sodium acetate buffer pH 5.0. Restored activities were monitored with a spectrophotometer at 25 °C with λ = 300 nm. Since different batches of sRNAse could give slightly different results [The scrambled RNAse was prepared as previously described . Titrati results , analyseKD value for the interaction between MET ECD and sRNase [KD was slits of 5–8 nm, excitation wavelength of 295 nm, and emission spectra of 300–400 nm. The spectra of MET ECD (0.51 µM) in 50 mM sodium phosphate buffer, pH 7.5, were recorded in the presence of different sRNase concentrations (from 0.1 to 2 µM) at 25 °C. The maximum intensities of each spectrum (351 nm) were subtracted from the maximum intensities (361 nm) acquired for NATA in the same conditions.The fluorescence analysis of MET ECD was performed on a FluoroMax-4 Horiba spectrofluorometer  with a quartz cuvette of 1 × 0.4 cm path length. Quenching of the intrinsic fluorescence was used to calculate the d sRNase . The setThe human ovarian carcinoma TOV112D , the human lung squamous cell EBC1 , and the human embryonic kidney HEK293T  cell lines were purchased and cultured according to manufacturer’s instructions in a humidified atmosphere at 37 °C. Cells were passed and subcultured to ~80/90% of confluence and were regularly verified for the absence of mycoplasma contamination.http://n2t.net/addgene:37560; RRID:Addgene_37560; accessed on 12 December 2021). The MET PSI domain complete deletion and amino acid substitutions were performed using the QuickChange II Site-Directed Mutagenesis Kit  using primers listed in The lentiviral plasmid containing the complete cDNA of the human wild-type MET was a gift from David Rimm , and expressed mutagenesis for SXXS mutants were commercially performed by GenScript  in the pcDNA3.1(+)-myc-His A plasmid. Plasmids were used for transient transfection of HEK293T cells using Lipofectamine 2000 reagent .Cells were lysed in Laemmli buffer as previously described . ProteinFormaldehyde-fixed cells were permeabilized with Triton X-100 (intracellular FACS) or not (membrane proteins), washed with PBS-1%BSA and incubated with antibodies listed in TOV112D cells were plated in 8 wells Nunc™ Lab-Tek II™ Chamber Slides™. Immunofluorescence experiments were carried out as previously described . Images ® Non-Radioactive Cell Proliferation Assay  for 48 h. Results are mean +/− SEM of N = 6 replicates.Cells were plated in 96-well plates at a density of 5000 cells/well. After 24 h starvation, cells were treated with HGF. Cell viability was assessed using CellTiter 96p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. Data are reported as mean ± SEM of N ≥ 3 independent experiments. ANOVA with Tukey’s post hoc test was performed to compare different groups with ns: not-significant; *: Assays were performed as previously described, according to a procedure we refer to as redox immunohistochemistry ,27. BrieMET is an oncogene encoding the tyrosine kinase receptor of the hepatocyte growth factor. It is translated as a single chain 175 kDa precursor protein, cleaved in the Golgi apparatus into α (50 kDa) and β (145 kDa) chains by the protease furin . Three iMET contains a highly conserved extracellular domain , whose function is still unexplored. Notably, this domain contains two conserved CXXC motifs found in the catalytic site of PDIs ,16. Thiscis”, we cannot exclude the possible function(s) in “trans”. Indeed, we reveal that the PSI domain profoundly affects the cellular thiol–redox equilibrium. Therefore, the MET PSI domain may serve as a chaperone for the folding of other proteins. In addition, MET PSI domain is exposed at the cell surface. It may act as a disulfide isomerase towards other cell surface proteins or components of the extracellular matrix. Mass-spectrometry-based approaches could identify such potential substrates and open new insights into MET signalling and, subsequently, tumor biology. Deletion of the PSI domain, or site-directed mutations in CXXC motifs, hampers the cleavage of the single-chain MET precursor. The uncleaved precursor is trapped in the Golgi apparatus. As demonstrated in furin-negative cells, the lack of cleavage per se is not essential for MET translocation to the plasma membrane ,37. ThusRecently, it was established that the inhibition of integrin PDI activity reduced platelet aggregation in vitro and thrombus formation in vivo ,47,48,49