The copolymer can self‐assemble with Cas9‐mRNA/sgRNA, indicating the excellent potential of nanoparticles to form a delivery carrier. This vehicle can efficiently release RNA in response to the hepatocytes carboxylesterase for genome editing. It was demonstrated that the mPEG‐b‐P(Met/n‐PMA)/Cas9 mRNA/sgRNA nanoparticles effectively accumulated in hepatocytes, lead to the inhibition of PCSK9, and lowered the levels of Low‐density lipoprotein cholesterol and total cholesterol in mouse serum down 20% of nontreatment. Interestingly, the nanoparticles even enable multiple functions in the regulation of blood glucose and weight. This study establishes a novel method to achieve complex CRISPR components stable loading, safe delivery, and fixed‐point release, which expand the application of CRISPR delivery systems.The broad application of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing tools is hindered by challenges in the efficient delivery of its two components into specific cells and intracytoplasmic release. Herein, a novel copolymer for delivery of Cas9‐mRNA/ single‐guide RNA (Cas9‐mRNA/sgRNA) in vitro and vivo, using carboxylesterase‐responsive cationic triadic copolymeric nanoparticles targeted proprotein convertase subtilisin/kexin type 9 (PCSK9) for hyperlipidemia amelioration is reported. A dimethyl biguanide derivative is designed and synthesized to form cationic block, and copolymerization onto prepolymer with propyl methacrylate, to fabricate a triadic copolymer mPEG This work reports a novel triadic copolymer‐based delivery vehicle to deliver Cas9‐mRNA and sgPCSK9 for gene editing both in vitro and vivo. Carboxylesterase‐responsive motifs are introduced in the vehicle, endowing the delivery vehicle enzymatic responsiveness in hepatocyte and disrupt the target PCSK9 gene and achieve a potent therapeutic efficacy for hyperlipidemia. Several CRISPR/Cas9‐based clinical trials are also underway or about to begin, such as chimeric antigen receptor T‐cell immunotherapy for cancer treatment and gene‐editing therapies for targeting the liver for lipid‐lowering therapy. However, the safe and effective delivery and release of CRISPR/Cas9 components into target organs, cells, and the nucleus remains a major challenge in genome editing‐mediated disease treatment, which will directly affect the success and therapeutic applications of this system.Genome‐editing system is becoming fundamental for biomedical research, gene‐drug development, and even gene therapy.1 Amon Compared with liposomes and inorganic nanoparticles, polymers show superior modularization and ease of functionalization and can be easily prepared from synthetic monomers or prefabricated polymers, which can precisely control the various properties of nanoparticles during preparation. Polymers also have variable drug delivery abilities, allowing polymer nanoparticles to deliver different drugs, including compounds with different molecular weights, such as small molecules, biomacromolecules, and proteins, which can meet the demands of different CRISPR/Cas9 codelivery forms . Thus, they are considered excellent delivery platforms for CRISPR/Cas9. In addition, studies on CRISPR/Cas9 polymers that affect gene delivery and editing processes have also attracted wide interest, including the type of surface charge, degree of PEGylation, type of CRISPR/Cas9, and location of targeted tissues.Liposomes, polymers, and inorganic nanoparticles have shown advantages in CRISPR/Cas9 delivery.6 Comp Several cationic copolymeric carriers have been developed for siRNA delivery. However, codelivering larger Cas9‐mRNA molecules or Cas9‐protein with short sgRNAs remains challenging.[2b] More importantly, cationic moieties tend to cause cytotoxicity owing to their interference with cell membrane integrity. To date, nearly no cationic polymer nanomaterials have been approved for clinical use by the Food and Drug Administration. Designing and synthesizing innocuous cationic copolymeric delivery systems to resolve these issues is necessary.Cationic copolymers dominate CRISPR/Cas9 delivery because of the high loading potential of CRISPR/Cas9 components and cellular uptake efficiency through electrostatic interactions, with the advent of reversible addition‐fragmentation chain transfer (RAFT) polymerization.12 Sev Recent reports have shown that polymers functionalized with guanidinium groups can readily adhere to nucleic acids via Gu+/PO34− electrostatic and hydrogen bond interactions. Therefore, metformin may be an ideal polymeric monomer for designing safe cationic gene carriers with intrinsic hypoglycemic activity. However, these interactions augment the stability between nucleic acids and guanidinium groups and usually result in poor intracellular release, which consequently leads to inefficient performance. Resolving the issue of release while maintaining the stability was also a desirable goal in our study.Metformin is the first‐line pharmacological treatment for type 2 diabetes, and this cationic drug has shown efficacy and a good safety profile.15 Rec For example, CES1 is abundantly expressed in the liver, whereas CES2 is primarily expressed in the small intestine and colon. In addition, CES1 preferentially metabolizes ester bonds, which contain a small alcohol group and a large acyl group. In contrast to CES1, CES2 preferentially hydrolyzes esters containing a relatively large alcohol group and a small acyl group. Therefore, we hypothesized that incorporating a simple CES1‐active propyl ester monomer in our cationic copolymer would achieve multiple effects. First, we added a hydrophobic force to the cationic copolymer to enable it to effectively encapsulate nucleic acids. Second, reducing some of the positive charges could further reduce the potential cytotoxicity. Third, this structure assists cationic copolymers in selectively releasing RNA from liver cells. Fourth, after activation by CES1, the propyl ester hydrolyzes into the corresponding alcohol and carboxylic acid and emits a negative charge. This negative charge can competitively interfere with the electrostatic and hydrogen bond interactions between biguanides and nucleic acids, promoting further release of nucleic acids.Carboxylesterase (CES) is an esterase enzyme family member that hydrolyzes carboxylesterase into its corresponding alcohol and carboxylic acid. Carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2) are two dominant enzymes involved in xenobiotic metabolism. Although CES1 and CES2 share some sequence homology, they exhibit different distributions and substrate specificities.18 For‐b‐P(Met/n‐PMA), also displayed as PEG‐PMet in the following figures) for the first time. Guanidine and propyl ester monomers were copolymerized onto methoxy PEG‐modified 4‐cyano‐4‐(phenyl thioformyl sulfur) valeric acid (mPEG‐CPADN) using RAFT polymerization. mPEG is another ideal choice for adding hydrophilic force to reduce the toxicity of cationic copolymers because it is commonly used to modify cationic copolymers to reduce protein adsorption and improve nucleic acid transfection efficiency. PEG composed of a comb‐like polymer does not exhibit any significant toxic effects.As shown in FigureHerein, we designed and fabricated a liver CES‐activated dimethyl biguanide triadic copolymer  monomers afforded mPEG‐b‐P(Met/n‐PMA) using mPEG‐CPADN and azodiisobutyronitrile (AIBN) as macroinitiators. The synthesis processes are described in the Experimental Section and are shown in Figure 1H nuclear magnetic resonance (1H‐NMR), 13C‐NMR, and infrared spectroscopy  was ≈10 000 MW with an UV absorption peak at 305 nm  was first synthesized Figure2A. Subs2.2‐b‐P(Met/n‐PMA) was ≈30–50 nm  was designed to be esterase‐sensitive. Figure ‐b‐P(Met/n‐PMA) in the presence of liver CES or denatured liver CES. In addition, mPEG‐b‐P(Met/n‐PMA) displayed good stability within 24 h of incubation with mouse serum  showed that the size of mPEGm Figure . mPEG‐b‐2.3‐b‐P(Met/n‐PMA) and RNA (mPEG‐b‐P(Met/n‐PMA)/RNA), the molecular dynamics (MD) simulation method was used to simulate the formation processes of mPEG‐b‐P(Met/n‐PMA)/RNA. In a typical simulation process, energy‐optimized RNA and mPEG‐b‐P(Met/n‐PMA) 3D models were constructed  molecules and one RNA molecule were solvated into a 15 cm × 15 cm × 15 cm cubic water box, which showed that all molecules self‐assembled into spherical particles after 100 ns  form hydrogen bonds with RNA, while methylene constitutes σ–π stacking with RNA . The RMSD of RNA fluctuates slightly in the entire process of simulation, indicates that the RNA molecule is relatively stable in the entire process of simulation. The RMSD of mPEG‐b‐P(Met/n‐PMA) changed significantly at the beginning of the simulation and tended to stabilize, indicating that mPEG‐b‐P(Met/n‐PMA) and RNA could assemble to form stable nanoparticles. Next, the energy magnitude between mPEG‐b‐P(Met/n‐PMA) and RNA was explored using the GROMACS energy module  and RNA, while that between RNA and water decreased, indicating that hydrogen bonding is the major driving force for the aggregation between mPEG‐b‐P(Met/n‐PMA) and RNA. In the self‐assembly of PEG‐PMet and RNA, the area exposed to the solvent gradually decreases; therefore, the solvent‐accessible surface area (SASA) can be used to evaluate the self‐assembly compactness of the spherical particles. Figure To gain insights into the mechanism and degree of self‐assembly between mPEGns Figure3A. The e Figure . This re2.4‐b‐P(Met/n‐PMA)/RNA complex was 20–30 nm in size  /RNA was increased to −3 mV compared with that of single mPEG‐b‐P(Met/n‐PMA) and single RNA /RNA nanoparticles, which were prepared at different N/P ratios from 0.5 to 4 /RNA decreased gradually, possibly due to the enhanced cooperative interactions between the cationic mPEG‐b‐P(Met/n‐PMA) polymer and anionic RNA. Figure ‐b‐P(Met/n‐PMA)/RNA at different N/P ratios. At an N/P ratio of 0.5:1, the mPEG‐b‐P(Met/n‐PMA)/RNA exhibited the highest negative zeta potential. Upon the addition of increasing amounts of the cationic polymer, the zeta potential values of the formed nanoparticles increased proportionally. At an N/P ratio of 4:1, a positive zeta potential value was obtained. Gel electrophoresis and UV spectroscopy demonstrated that the polymer effectively loaded RNA at an N/P ratio of 3:1 /RNA was investigated by monitoring the increase in the number of active complexes in the supernatant. To exclude the possibility of the self‐release of mPEG‐b‐P(Met/n‐PMA)/RNA, we monitored the stability of mPEG‐b‐P(Met/n‐PMA)/RNA at different temperatures. As shown in Figure ‐b‐P(Met/n‐PMA) did not degrade within a week. Under these conditions, esterase was added to mPEG‐b‐P(Met/n‐PMA)/RNA, and the UV spectrum could detect the release of RNA  Figure , becauseA Figure . Then, w1 Figure . The relA Figure , suggest2.5‐b‐P(Met/n‐PMA), using HepG2, HeLa, HEK293, and A549 cells . mPEG‐b‐P(Met/n‐PMA)‐delivered GFP mRNA showed higher fluorescence than GFP, GFP‐mRNA, and mPEG‐b‐P(Met/n‐PMA)/GFP. In addition, mPEG‐b‐P(Met/n‐PMA)/GFP‐mRNA showed a much higher internalization efficiency than the other controls   to evaluate the nuclear import of mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9 /Cas9‐mRNA/Cy5‐sgPCSK9 and Lipo3000/Cas9‐mRNA/Cy5‐sgPCSK9 was observed, which escaped from lysosomes, and were distributed in and around the nuclei. The mechanism of mPEG‐b‐P(Met/n‐PMA)‐mediated lysosomal escape may cause by the ATP‐depleting dependent way of polymetformin‐based polycationic micelles. As a control, mPEG‐b‐P(Met/n‐PMA), containing only Cy5‐sgPCSK9, exhibited negligible cytosolic delivery and nuclear import. In a cytotoxicity test using the CCK8 method, mPEG‐b‐P(Met/n‐PMA) was also shown to facilitate cell viability, while Lipo3000 showed cytotoxicity  increased with increasing dose, and even the dose of 500 µg mL−1 showed no cytotoxicity  might result from its biguanide structure. Metformin can facilitate the proliferation, migration, and differentiation of cells in vitro to a certain degree by pathways, such as ERK/AMPK.We evaluated the intracellular delivery of green fluorescent protein (GFP) and its mRNA (GFP‐mRNA), which were encapsulated into mPEG Figure 23). m23. m‐b) Figure. Further9 Figure . sgPCSK9y Figure . The proy Figure . As a me2.6Figure‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9 induced effective editing of the PCSK9 gene, whereas the control groups induced insignificant editing. Western blotting indicated that mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9 significantly downregulated the expression of PCSK9 /Cas9‐mRNA/sgPCSK9 improved the brightness of APC, suggesting that the LDL‐R content on the cell membrane surface increased significantly. Two other experimental groups detected an exogenous LDL‐C uptake effect of mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9 in HepG2 cells. The detection of total cholesterol levels /Cas9‐mRNA/sgPCSK9 promoted the uptake of exogenous cholesterol by HepG2 cells. In addition, we used the CRISPR Design server to identify eight sgPCSK9 off‐target sites. Then, we amplified these eight potential off‐target sites from mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9, treated them with HepG2, and evaluated CRISPR/Cas9 editing using the T7 endonuclease I (T7EI) assay. No off‐target cutting was observed at any off‐target site   was fabricated using RAFT polymerization, in which only the guanidine monomer was polymerized onto mPEG‐CPADN  was confirmed using 1H‐NMR  did not form nanoparticles, whereas self‐assembly occurred after RNA was added (mPEG‐b‐P(Met)/RNA). This might be attributed to the weakening of hydrogen bonds between RNA and water on the formation of new hydrogen bonds between RNA and mPEG‐b‐P(Met), which cause the properties of RNA to change from hydrophilic to hydrophobic and introduce hydrophobic interactions. UV spectra revealed the RNA loading and release abilities of mPEG‐b‐P(Met)  could not be triggered by esterase. Western blot assays showed that mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9 showed better effects on PCSK9 and LDL‐R than the nanoparticles that were non‐CES1‐responsive /Cas9‐mRNA/sgPCSK9 showed higher uptake ability for exogenous cholesterol than mPEG‐b‐P(Met)/Cas9‐mRNA/sgPCSK9 /Cy5, or mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/Cy5‐sgPCSK9. As shown in Figure‐b‐P(Met/n‐PMA)/Cy5 and mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/Cy5‐sgPCSK9 showed significant liver accumulation at 0.5 h postinjection, as strong fluorescence was evident in the liver, and a strong fluorescence signal was observed in the liver. 12 h after injection, the fluorescence in the liver showed a gradually decreasing trend. In contrast, the Cy5 group exhibited rapid and complete metabolism as fluorescence was observed in the intestine at 0.5 h and in the kidneys at 2 h. Biodistribution analysis confirmed that mPEG‐b‐P(Met/n‐PMA)/Cy5 and mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/Cy5‐sgPCSK9 had better accumulation and retention properties than free Cy5. In addition, the ex vivo fluorescence image of the excised organs further confirmed that mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/Cy5‐sgPCSK9 showed a higher fluorescence signal in the liver than free fluorescent molecules /Cas9‐mRNA/sgPCSK9 administration in mice /Cas9‐mRNA/sgPCSK9‐administered mice showed a PCSK9 level of 1470 pg mL−1, which was substantially lower than that in the other groups /Cas9‐mRNA/sgPCSK9‐administered mice had significantly lower levels of LDL‐C and total cholesterol (TC), with ≈20% reduction, compared to the control group /Cas9‐mRNA/sgPCSK9‐administered mice had lower levels of PCSK9 and higher levels of LDLR protein than control mice  in vivo, we found that mPEG‐b‐P(Met/n‐PMA) had an excessive effect on weight control, as shown in Figure ‐b‐P(Met/n‐PMA) attenuated high‐fat diet (HFD)‐induced body weight gain compared to Lipo3000 and metformin. Therefore, we monitored the effect of mPEG‐b‐P(Met/n‐PMA) on the blood glucose levels. First, we tested the glucose tolerance test (GTT) and insulin tolerance test (ITT) before administration and compared them with the normal chow diet (NCD) diet. The GTT and ITT of the HFD, mPEG‐b‐P(Met/n‐PMA), and Lipo3000 improved, suggesting that the mice developed mild glucose and insulin resistance. After 14 d of administration, the GTT and ITT of mPEG‐b‐P(Met/n‐PMA) decreased compared with those of Lipo3000 and HFD, indicating that mPEG‐b‐P(Met/n‐PMA) ameliorated glucose and insulin resistance , we found that mPEG‐b‐P(Met/n‐PMA) can formulate nucleic acids into stable nanomicelles via electrostatic and van der Waals interactions and that mPEG‐b‐P(Met/n‐PMA) can be selectively activated and disintegrated by CES in the liver. These nanomicelles can deliver nucleic acids to hepatocytes both in vitro and in vivo. This delivery strategy achieved efficient genome editing of PCSK9 and downregulation of serum LDL‐C. Notably, in addition to lipid‐lowering effects, this strategy has a dual effect on the regulation of blood glucose and weight. However, the mechanisms, pharmacokinetics, and pharmacodynamics of mPEG‐b‐P(Met/n‐PMA) in regulating blood glucose and weight remain unclear. Taken together, these data justify that further exploration of nucleic acid carriers with metformin is warranted. In addition, mPEG‐b‐P(Met/n‐PMA)‐mediated PCSK9 gene editing has the potential to ameliorate hyperlipidemia.By designing, synthesizing, and characterizing novel mPEG4N‐succinimidyl methacrylate, AIBN, and dicyandiamide were obtained from Macklin (China). Ethylenediamine was purchased from Sigma‐Aldrich (USA), and bis(thiobenzoyl) disulfide (CPADN) was purchased from TCI (Japan). MeO‐PEG (mPEG) was obtained from RuixiBio . The CCK8 kit was purchased from Beyotime (China). GFP mRNA, T7E1 endonuclease, and RNA Synthesis, and Purification kits were purchased from New England Biolabs (USA). Cas9‐mRNA was purchased from TriLink (USA). Antibodies against PCSK9 and LDLR were purchased from Abcam . Lipofectamine 3000 was purchased from Thermo Fisher Scientific (USA). Penicillin–streptomycin solution (100×), trypsin, fetal bovine serum (FBS), and Dulbecco's modified Eagle medium (DMEM) were purchased from Gibco (USA). High‐purity (Milli‐Q) water with a resistivity of 18.2 MΩ cm was obtained from an inline Millipore RiOs/Origin water purification system.The following instruments were used: a Bruker Avance III 600 MHz nuclear magnetic resonance (NMR) spectrometer ; a dynamic light scattering particle size analyzer , an ultraviolet‒visible (UV–vis) absorption spectrophotometer ; a Nicolet iS5  for Fourier transform infrared (FTIR) spectroscopy; a Nanodrop2000 ultramicrospectrophotometer ; an LC20 gel permeation chromatography ; a Nikon Eclipse Ti‐S inverted fluorescence microscope ; a field emission TEM ; an NCF950 laser scanning confocal microscope ; an Illumina HiSeq2500 high throughput sequencer ; and a flow cytometer .‐b‐P(Met/n‐PMA)/RNA complex was minimized using the saddle point method. Then, 1 ns NVT and NPT equilibrium simulations were performed. Finally, an MD simulation of 100 ns was performed on the mPEG‐b‐P(Met/n‐PMA)/RNA complex, and the conformation was saved every 10 ps. Visualization of the simulation results was performed using VMD.MD simulations were performed using GROMACS 2018.4. Under constant temperature, pressure, and periodic boundary conditions, Amber14SB was used as the full atomic force field, and TIP3P was used as the water model. All the hydrogen bonds were restrained using the LinCS algorithm with an integral step size of 2 fs. The electrostatic interactions were calculated using the particle mesh Ewald. The cutoff of nonbonded energies was 10 Å and was updated every 10 steps. The v‐rescale temperature‐coupling method was used to control the simulated temperature to 298.15 K, and the Parrinello–Rahman method was used to control the analog pressure to 1 bar. To eliminate the effect of close contact between atoms, the energy of the mPEGN‐Succinimidyl methacrylate  was dissolved in 10 mL of anhydrous tetrahydrofuran in an ice bath, and ethylenediamine  was added dropwise to the reaction. The resulting solution was then stirred at room temperature for 12 h. After the completion of the reaction, the white precipitate was removed by suction filtration, and the filtrate was concentrated by rotary evaporation. After drying under vacuum, a pale‐yellow sticky solid of N‐(2‐aminoethyl)methacrylamide (217 mg) was obtained with a yield of 84.7%.N‐(2‐aminoethyl)methacrylamide  and dicyandiamide  were dissolved in 10 mL of a 2% hydrochloric acid solution. The reaction temperature was increased to 90 °C, and the mixture was stirred for 3 h. Then, it was concentrated via rotation‐evaporation, and N‐(2‐(3‐carbamimidoylguanidino)ethyl)methacrylamide was obtained in 80.3% yield.2  and NHS‐modified CPADN  were dissolved in 10 mL of dry DCM and stirred overnight at room temperature. The solution was then recrystallized from cold diethyl ether thrice and centrifuged to obtain a pink precipitate. Finally, the precipitate was dried by lyophilization to form a pink powder.MeO‐PEG‐NHn‐propyl methacrylate , N‐(2‐(3‐carbamimidoylguanidino)ethyl)methacrylamide , and AIBN  were dissolved in dried dimethyl sulfoxide (DMSO) and sealed in a flask under nitrogen. The reaction proceeded at 70 °C for 24 h. Then, the solution was cooled to room temperature, extracted with methyl tertiary‐butyl ether, and dialyzed against water for 1 d. The final product, mPEG‐b‐P(Met/n‐PMA) , was obtained via freeze‐drying.mPEG‐CPADN , ‐b‐P(Met/n‐PMA) , was obtained via freeze‐drying.mPEG‐CPADN , N‐(2‐(3‐carbamimidoylguanidino)ethyl)methacrylamide , and AIBN  were dissolved in dried DMSO and sealed in a flask under nitrogen. The reaction proceeded at 70 °C for 24 h. Then, the solution was dialyzed against water for 1 d. The final product, mPEG‐b‐P(Met/n‐PMA) was mixed with Cas9‐mRNA and sgPCSK9 (weight ratio of 2:1) at a Gu+/PO34− molar ratio of 3:1 in HEPES buffer  and incubated at room temperature for 30 min. The resulting complex was denoted as mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9. The size, size distribution, and morphology of the mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9 cells were measured via DLS and TEM.A solution of mPEG‐b‐P(Met/n‐PMA) was evaluated using agarose gel electrophoresis. The solution of mPEG‐b‐P(Met/n‐PMA) was mixed with sgPCSK9 at different molar ratios of Gu+/PO34− in HEPES buffer  and incubated for 30 min. The incubated mixture was electrophoresed on a 15% polyacrylamide gel at 110 V in TBE for 2 h. Gel images were obtained using a Molecular Image.The RNA‐binding ability of mPEG‐b‐P(Met/n‐PMA) solution was added to a dialysis bag (cutoff Mn = 10 000 Da) containing esterase or denatured esterase (30 U mL−1). The mixture was then kept at 37 °C under constant shaking at different times.One milliliter of the mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9 solution was added to a dialysis bag (cut‐off Mn = 10 000 Da) with or without esterase (30 U mL−1). The mixtures were maintained at different temperatures under constant shaking for 4 h.The mPEG−1 penicillin, and 100 mg mL−1 streptomycin.HepG2, HEK293T, and HeLa cells were purchased from ATCC (USA). The cells were cultured in 10% FBS‐supplemented DMEM, 100 U mL‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9, and Lipo3000/Cas9‐mRNA/sgPCSK9 was added to the cells and incubated for 48 h at 37 °C with 5% CO2.HepG2 cells were seeded on six‐well cell culture clusters at a density of 60%. Next, Opti‐MEM‐reduced serum medium containing Cas9‐mRNA/sgPCSK9, mPEGGFP and its mRNA were used to express green fluorescence. Cyanine 5 (Cy5) was used to label sgPCSK9 (cy5‐sgPCSK9), and 4′,6‐diamidino‐2‐phenylindole (DAPI) was used to label nuclei. HepG2, HeLa, HEK293, and A549 cells were seeded in confocal culture dishes at a density of 60% and transfected as described above. The medium was then removed, and the cells were washed with PBS thrice and observed under a confocal microscope.The transfected cells were rinsed thrice with PBS, trypsinized, and collected. The cells were then resuspended in PBS and analyzed using a flow cytometer.‐b‐P(Met/n‐PMA), and Lipo3000 for 48 or 72 h. Subsequently, the cells were washed thoroughly with PBS, and the CCK‐8 solution was added to each well to a concentration of 10%. The plate was incubated at 37 °C, and the absorbance was measured at 450 nm.HepG2 cells were seeded in a 96‐well plate at densities of 4000 and 5000 cells per well and cultured for 24 h. The cells were then exposed to a series of concentrations of HEPES buffer, metformin, mPEG−1 were seeded into six‐well plates. The cells were then transfected with Cas9‐mRNA/sgPCSK9 and mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9 and cultured at 37 °C for 48 h. Genomic DNA was isolated and harvested using a genomic DNA extraction kit , according to the manufacturer's instructions. Targeted DNA fragments at the PCSK9 locus were amplified from the genomic DNA of wild‐type or transfected cells. A pair of optimized PCR primers was designed, and the annealed PCR products were 396 bp and ranged from upstream to downstream at the targeted site.T7EI mutated HepG2 cells at a density of 60% cells mLPrimer:PCSK9 sense primer: 5′ ‐ACCCACCTCCTCACCTTTCC‐3′PCSK9 antisense primer: 5′‐CCCTGACCTCGTGTTTCCTC‐3′After amplification, 5 µL amplicon was diluted with 1.1 µL 10× T7E1 buffer and 4.4 µL nuclease‐free water. The amplicon was denatured at 100 °C in boiling water and allowed to cool naturally to room temperature for rehybridization. Next, 0.5 µL of T7E1 was added to the tube and incubated at 37 °C for 30 min. Then, 1 µL of proteinase K was added and mixed well. Samples were electrophoresed using 5% polyacrylamide gel.β‐actin. The primary antibody was used at a 1:8000 dilution. The antibodies were validated by molecular mass analysis using a positive control sample.Proteins from cell and tissue lysates were extracted using RIPA buffer. A total of 20 µg of protein was used for western blotting. The following primary antibodies were used: anti‐PCSK9, anti‐LDL‐R, and anti‐−1 LDL. Cells were then washed twice in PBS, and intracellular lipids were extracted in isopropanol, dried under vacuum, and TC was measured using an enzymatic assay and normalized to total cell protein as determined by the modified Lowry assay. For morphological examination, HepG2 cells were fixed for 30 min with 5% formalin solution in PBS, stained with Oil Red O for 30 min, and counterstained with hematoxylin for 5 min. Finally, the cells were examined under a light microscope.Transfected HepG2 cells in six‐well plates were cultured for 12 h in serum‐free medium with or without 50 µg mLn = 6). The mice were maintained under a constant 12‐h light/dark cycle with unrestricted access to food and water.All animal procedures were performed in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. C57BL/6J mice and an ethical approval statement (2021015) were provided by the Experimental Animal Center of Chongqing Medical University. Age‐matched (6–8 weeks old) male wild‐type (WT) C57BL/6 mice received either a NCD or a HFD containing 60 kcal% fat for 12 weeks (‐b‐P(Met/n‐PMA) and mPEG‐b‐P(Met/n‐PMA)/Cy5‐sgPCSK9 nanoparticles were prepared as described above. The mPEG‐b‐P(Met/n‐PMA) nanoparticles were stained with Cy5. The nanoparticles were administered via intravenous injection and tracked for 24 h using an in vivo Imaging system.The mPEG‐b‐P(Met/n‐PMA), mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9, Cas9‐mRNA/sgPCSK9, or Lipo3000/Cas9‐mRNA/sgPCSK9. Cas9‐mRNA and sgPCSK9 were used in a weight ratio of 2:1. These formulations were administered intravenously every 7 d. Immediately after euthanasia of the animals 28 d after receiving saline, saline, mPEG‐b‐P(Met/n‐PMA), mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9, Cas9‐mRNA/sgPCSK9, and Lipo3000/Cas9‐mRNA/sgPCSK9 were administered and ELISA was performed to evaluate PCSK9 protein, LDL‐C, HDL‐C, TC, TG, ALT, AST, BUN, S‐Cr, and triglyceride levels in plasma samples.Male C57 mice (6–8 weeks) were randomized for treatment with saline, mPEG‐b‐P(Met/n‐PMA), mPEG‐b‐P(Met/n‐PMA)/Cas9‐mRNA/sgPCSK9, Cas9‐mRNA/sgPCSK9, and Lipo3000/Cas9‐mRNA/sgPCSK9, they were sacrificed, and the major organs, including the liver, heart, spleen, lung, and kidney, were extracted. Then, the tissues were fixed with 10% formalin solution, embedded in paraffin, and sectioned to ≈5 µm. The sections were stained with H&E.After being injected with saline, mPEG−1 body weight d‐glucose. For ITTs, the mice were fasted for 4 h and were intraperitoneally injected with 0.14 units kg−1 body weight insulin. Blood glucose levels were measured at 0, 15, 30, 60, and 120 min in tail vein blood using a GA‐7 Advantage glucometer (Sannuo).For GTTs, the mice were fasted for 12 h and then intraperitoneally injected with 1 g kgP < 0.05. Experimental data were compared using one‐way analysis of variance (ANOVA).Statistical analyses were performed using SPSS software . The results are depicted as the mean ± SD, and statistical tests are indicated in the figures. Differences were considered statistically significant at The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


However, reinfection rates were higher among patients who received nitrofurantoin . Among patients younger than 40 years, patients treated with nitrofurantoin had more reinfections . Treatment failure rates were mildly higher in patients treated with fosfomycin, despite having less reinfections. We suggest that this effect is related to a shorter duration of treatment (one vs. five days) and encourage clinicians to be more patient before declaring fosfomycin failure and prescribing another antibiotic.In this study, we compared the failure rates of fosfomycin and nitrofurantoin for uncomplicated urinary tract infections. We used Meuhedet Health Services’ large database to collect data on all female patients, older than 18 years, who were prescribed either antibiotic during 2013–2018. Treatment failure was a composite endpoint of hospitalization, emergency-room visit, IV antibiotic treatment, or prescription of a different antibiotic, within seven days of the initial prescription. Reinfection was considered when one of these endpoints appeared 8–30 days following the initial prescription. We found 33,759 eligible patients. Treatment failure was more common in the fosfomycin group than the nitrofurantoin group (8.16% vs. 6.87%,  Urinary tract infections (UTI) are one of the most common infections in primary care settings , where eSeveral comparisons between the clinical outcomes of urinary infections with these antibiotics were conducted, without a clear advantage for a specific one ,5,6,7. TThe aim of this trial was to evaluate the factors affecting failure among patients with UTI attending primary care in a large group of patients in real-life settings. We used data extracted from electronic medical records (EMRs) of Meuhedet Health Services (MHS), a large Israeli Health Maintenance Organization (HMO), to assess the frequency of different outcomes that constitute treatment failure, according to the antibiotic regimen that was given by primary care physicians. The “off-label” practice of prescribing several consecutive doses of fosfomycin in urinary infection is not allowed in the electronic prescribing forms used by Meuhedet Health Services and therefore was not studied.Meuhedet is the third largest healthcare provider in Israel, serving over 1,200,000 patients that are representative of the general population of Israel. Meuhedet’s computerized database includes real-time input from all physician visits, diagnoses, medical prescriptions and dispensing data, laboratory results and hospitalizations.In this retrospective study, we collected data of all female patients aged 18 or above who were diagnosed with urinary tract infection (UTI), simple cystitis  and were prescribed either nitrofurantoin or fosfomycin between January 2013 and December 2018. Data included the patient’s age, serum creatinine level—if measured 90 days (or less) prior to UTI diagnosis—and the following information regarding the 30 days after diagnosis: emergency room (ER) visits, admissions to hospital, visits in a community emergency clinic, intravenous (IV) treatment (gentamicin or ceftriaxone), diagnosis of pyelonephritis, and changes in antibiotic treatment.Patients were excluded if they had one or more of the following: a positive HCG test in the 270 days prior to UTI diagnosis; a diagnosis of UTI less than 90 days prior to the current diagnosis; diagnosis of pneumonia, upper respiratory tract infection, cellulitis, or fever of unknown origin within the 30 days following the UTI diagnosis.Treatment was considered a failure if one or more of the following events (ordered from the most severe to the least severe) occurred within seven days from the index UTI diagnosis: hospitalization, emergency room or emergency clinic visit, and IV antibiotic treatment or prescription of a different antibiotic than the initial one. As a change in antibiotic treatment might reflect reasons other than treatment failure, such as an allergic reaction, a change in working diagnosis, and issues with pharmacy supply, we performed an additional analysis of treatment failure while disregarding this endpoint.Similarly, a patient was considered as having a reinfection if one or more of the following events (ordered from the most to the least severe) happened within eight to thirty days from the initial diagnosis: hospitalization, emergency room or emergency clinic visit, another visit at the primary care physician with a diagnosis of UTI, or a prescription of any antibiotic treatment.In addition, a patient who had undergone more than one of the events that was defined as a treatment failure was counted as one failure, classified according to the most severe event. The same classification was performed for re-infected patients. Patients defined as treatment failure could not be defined again as re-infected.p-value of less than 0.05 was considered statistically significant. In addition to p-values, the absolute effect size  was calculated; the effect size of two proportions was defined according to Cohen’s h [p-value of less than 0.2 in a univariate analysis were considered as potential confounders . These were time from last UTI event, age, and creatinine levels.Statistical analysis was performed using R software . The packages ‘ggplot2′, ‘dplyr’, ‘zoo’, ‘data.table’, and ‘tidy’ were used in addition to the default R packages. Categorical variables were summarized as counts and percentages. Continuous variables were summarized as means and standard deviations (SD). Univariate analysis was performed using chi-squared test to compare categorical variables, and Welch’s t-test to compare means of continuous variables. The study was approved by Meuhedet internal ethics board on 17 February 2020, approval code 01-29-01-20.Between 1 January 2013 and 31 December 2018, there were 569,897 visits of women aged 18 and above with a diagnosis of UTI at Meuhedet. Cases were excluded if one or more of the following occurred: (1) no antibiotic treatment was prescribed, (2) the patient had a sequential diagnosis of upper respiratory tract disease, fever of unknown origin, cellulitis, or pneumonia within 30 days from original diagnosis, (3) the patient had a positive HCG test in the past 270 days, or (4) when there was a previous case of UTI 90 days prior to the index event. In addition, patients that were treated with an antibiotic different than fosfomycin or nitrofurantoin were excluded. After these exclusions, 33,759 patients remained who were treated with nitrofurantoin or fosfomycin .p-value < 0.001) and their previous documented diagnosis of UTI (by the same UTI definitions) was closer in time   . BMI andp value < 0.0001). However, reinfection rates were higher among patients who received nitrofurantoin   . The magp-value = 0.008, beta = 0.7377, McFadden R2 = 0.85)  .p-value = 0.044, beta = 1.4147, McFadden R2 = 0.857).When excluding a change of antibiotic treatment from the criteria of failure events, a similar multivariate logistic regression controlling the same variables showed that nitrofurantoin was associated with more treatment failures . In this age group, patients treated with nitrofurantoin had more cases of reinfection , derived mainly from a higher likelihood of turning to an ER, and were admitted within seven days of diagnosis. They were, however, less likely to have a change in antibiotic treatment .Among patients younger than 40, nitrofurantoin and fosfomycin had similar rates of treatment failure , but reinfection was more common with nitrofurantoin  . Regarding reinfections, however, in a multivariate logistic regression controlling for age, creatinine, and time from last UTI diagnosis, there was no significant difference between the two treatments .Among patients 40 years and older, treatment failure was more common with fosfomycin (8.31% vs. 6.25%  0.0004) . In a muPrescribing antibiotics to women presenting uncomplicated UTI to primary care clinics is not as simple a decision as it seems. Despite the known antimicrobial activity of the many options, additional considerations must be made—the predicted adherence of the specific patient to the suggested drug being one of the most important ones.In a large multinational, open-labeled, analyst-blinded, randomized clinical trial study published in 2018, Huttner et al.  had compOur real-life retrospective study included 33,759 female patients with uncomplicated UTI in the community settings and compared the clinically relevant outcomes of two common antibiotics, fosfomycin and nitrofurantoin, from 1 January 2013 to 31 December 2018. Despite the 4 years that passed since the conclusion of the data collection, these two antibiotics are still the mainstay of treatment for simple cystitis in the community.p-value = 1); however, patients treated with nitrofurantoin had more cases of reinfection . In contrast, in patients older than 40 years, treatment failure was more common in patients treated with fosfomycin , and reinfection was again more common with nitrofurantoin . When calculating the absolute effect size, it was minor in all subgroups. Practically speaking, the benefit of one antibiotic over the other was small, resulting in a high NNT.We showed that in women 40 years old and younger, fosfomycin and nitrofurantoin had similar rates of treatment failure  in fosfomycin group and 364 (3.79%) in the nitrofurantoin group. These patients might not be truly failing the first course of treatment, but present longer symptoms which might have caused the physician to consider the option of failure . IntuitiReal world studies have advantages and disadvantages versus randomized and controlled studies. Presumably, patients’ adherence to recommended treatments are much higher in prospective trials, where the investigators can actively encourage the patients to follow the study protocol. Insufficient compliance is supposedly higher with nitrofurantoin due to the need to take the medication several times every day, for five days, compared to the single dose prescription of fosfomycin. However, the number of patients that can be recruited and followed prospectively is limited by financial and logistic barriers, where retrospective studies using a large database retrieved from electronic medical records can include vast numbers of patients. The population included can be much more heterogenic in these studies and be more representative of the relevant population. Another important caveat of retrospective studies is our inability to control many confounders, e.g., sexual activity, menopause status and medical conditions like diabetes and immune deficiency. In a recent study by Martischang et al. , risk faA recent meta-analysis of four studies  found noThe limitations of this study are the retrospective nature which did not allow us to establish causality for our results. For example, the endpoint of hospitalization could represent a true failure of the treatment but could also be a coincidental allergic reaction. In addition, we did not have microbiological data as most of the patients in the community setting are treated empirically, as per guidelines . AnotherIn summary, real-world data shows better outcomes of fosfomycin for uncomplicated UTI in patients 40 years of age and younger, with minor absolute effect, whereas in older women, fosfomycin was associated with less reinfections but more treatment failures.


Textile-reinforced concrete (TRC) is highly anticipated as an alternative to reinforced concrete due to its ability to enable lightweight design, free formability, and improved ductility. In this study, TRC panel test specimens were fabricated and four-point loading flexural tests were performed to examine the flexural characteristics of TRC panels reinforced with carbon fabric, and to investigate the effect of the fabric reinforcement ratio, anchorage length, and surface treatment of fabric. Furthermore, the flexural behavior of the test specimens was numerically analyzed using the general section analysis concept of reinforced concrete and compared with the experimental results. Due to bond failure between the carbon fabric and the concrete matrix, the TRC panel showed a large decrease in flexural performance in terms of flexural stiffness, flexural strength, cracking behavior, and deflection. This low performance was improved by increasing the fabric reinforcement ratio, anchoring length, and sand–epoxy surface treatment of the anchorage. Comparing the numerical calculation results with the experimental results, the deflection of the experimental results was approximately 50% larger than the numerical calculation results. This is because the perfect bond between the carbon fabric and the concrete matrix failed, and slip occurred. Reinforced concrete is a globally used construction material that consists of homogeneous reinforcing bars and heterogeneous concrete. It is a composite material that offers various advantages in terms of material and structure by complementing different mechanical properties through the interaction of compression and tension. Based on these advantages, reinforced concrete plays a significant role in building modern society. With the recent developments of technology, there is a trend towards constructing larger and ultra-high reinforced concrete structures. However, this trend also leads to an increase in load due to the weight of the structure and external force, which can cause cracks on the surface of the reinforced concrete, leading to corrosion of the reinforcement and concrete. Corrosion of reinforcing bars and concrete can result in a deterioration of durability and stability, leading to significant repair and reinforcement costs. Therefore, there is a need for a replacement material for reinforced concrete to prevent a reduction in the durability and stability of structures, and to promote sustainable development in the construction industry; further, the interest in alternative materials is also increasing ,2.Textile-reinforced concrete is being explored as an alternative material for reinforced concrete. It is a composite material composed of a high-performance cement-based matrix, usually with a small aggregate maximum size, and a bundle of high-toughness continuous multifilaments composed of glass, carbon, polymer, or other materials . TextileTextile-reinforced concrete has numerous advantages and efficient application targets, but clear design criteria and guidelines have not yet been established, resulting in experiments being conducted based on the experience of researchers. As it is a composite material composed of two different materials, there are difficulties in understanding the material properties compared to conventional construction materials. Furthermore, because the adhesion behavior between the two materials, textile and concrete, has not been clearly identified, there are many limitations in analyzing the composite behavior and applying it to actual structures ,9.According to the literature, the main failure of TRC is caused by the slippage of the textile and the separation of the matrix and the textile, and this failure mode is related to the incomplete bonding of the textile and the matrix ,11,12. AIn this study, to examine the difference in flexural behavior according to the textile reinforcement ratio, anchorage length, and surface treatment of concrete composite panels reinforced with carbon fabric, textile-reinforced concrete was fabricated and a flexural test performed. Differences in ultimate load, center deflection, and crack distribution ac-cording to each variable were compared and evaluated. In addition, the bending behavior was numerically calculated according to the nominal flexural strength calculation method of general reinforced concrete members and the deflection calculation method using the effective second moment of inertia, and the results were compared with the experimental results.The experiments were divided into two parts: Group I and Group II. A total of 15 variables were set, with 5 variables in the Group I experiment and 10 variables in the Group II experiment. Three specimens were produced for each variable, resulting in a total of 45 TRC panel specimens that were manufactured and tested. Details of the TRC panel specimens are presented in There were three parameters: the number of carbon-fiber textile layers, the anchorage length of the textile, and the presence or absence of sand–epoxy surface treatment on the textile. The number of carbon-fiber textile layer parameters was set to 0 (NF), 1 (C1)∼4 (C4) to examine the effect of the textile reinforcement ratio on the flexural behavior of the TRC panel. Section details according to the number of textile layers are shown in h) was based on 40 mm, but its height slightly varied depending on the number of textile layers. The actual measured height of the specimen and the location of each textile layer are presented in detail in The width of the TRC panel specimens was fixed at 100 mm, and the height Sections perpendicular to the axis of bending that are plane before bending remain plane after bending.(2)The concrete matrix and textile are fully bonded, so the strain in the textile is equal to the strain in the concrete matrix at the same level.(3)The stress–strain relationship of carbon-fiber textile is perfectly linear.(4)The tensile strength of concrete and compressive strength of carbon-fiber textile are neglected in flexural strength calculations.(5)Stress induced by flexural load acts only on the warp of the textile reinforcement.For the numerical calculation, the Hognestad  model waAccording to the assumption that the plane section remains plane, the strain of each textile layer and the compressive strain of concrete are proportional to the distance away from the neutral axis, as shown in Equation (4) can be derived using the force equilibrium condition, and When the compressive strain of the matrix reaches the ultimate compressive strain, the TRC panel fails and the resistance moment at that time corresponds to the nominal moment, The deflection of the carbon-fiber textile-reinforced concrete panel was calculated using the elastic deflection equation. As for the moment of inertia applied to the elastic deflection equation, the effective moment of inertia, ttee 440  as a metIn order to predict the deflection of reinforced concrete flexural members, ACI Committee 318  uses thethe span ,31. In tThe load–deflection curves were illustrated for the three TRC panel specimens for each variable resulting from the flexural test. The load–deflection relationships for the three specimens were averaged and are presented as one graph for each variable. For instance, in the case of the A4SE series test specimens, The NF-I and NF-II test specimens, which did not have fabric reinforcement, showed a linear increase in vertical deflection as the load increased and failed as soon as a flexural crack occurred between the two-point loading parts, with an average ultimate load of 3.68 kN and 3.01 kN, respectively. The difference in flexural strength between the NF-I and NF-II test specimens appears to be due to the difference in the compressive strength of the concrete matrix .The carbon fabric-reinforced TRC panel specimens exhibited the same initial behavior as the NF specimens, then developed flexural cracks and continued to behave without failure. The first flexural cracking load did not show any variation across the variables. All of the TRC specimens showed a sudden drop in load immediately after the first flexural crack occurred, a phenomenon resulting from the brittle stress redistribution from the concrete matrix to the carbon fabric . This phThe average load–deflection relationship between the A0 series, which had the shortest anchorage length, and the A4SE series treated with sand–epoxy, is shown in The maximum load was the highest for the C4L1A0 test specimen in the A0 series, with an average of 13.51 kN, and for the C4L1A4SE specimen in the A4SE series, with 27.45 kN. Both the A0 series and the A4SE series demonstrated higher maximum loads as the number of carbon fabric reinforcing layers increased . The C4LThe crack patterns observed after completing the flexural test on the TRC panel specimens are presented in The load–deflection curve according to the difference in anchorage length is shown in The number of cracks in the C2L1A0, C2L1A1, C2L1A2, C2L1A3, and C2L1A4 specimens were 2.3, 2.3, 2.7, 3.7, and 3.7, respectively, indicating an increase in the number of cracks with longer anchorage lengths. For the C2L1A0 specimen, large crack widths were observed, and carbon fabric slip between the cracks was visible. However, as anchorage length increased, crack widths decreased.The C1L1A4SE and C2L1A4SE specimens experienced flexural failure, while the C3L1A4SE and C4L1A4SE specimens developed flexural shear cracks that subsequently led to flexural shear failure. Flexural cracks occur due to tensile stress that arises in the vertical direction at the bottom of the specimen. The reduction in cross-sectional area resulting from the occurrence of flexural cracks causes an increase in inclined stress and shear stress at the tip of the flexural crack, which expands and leads to a flexural shear crack. The C1L1A4SE and C2L1A4SE specimens had a low carbon fabric reinforcement ratio, resulting in long flexural cracks towards the compression fiber, which made it difficult for them to expand into inclined cracks. In contrast, the C3L1A4SE and C4L1A4SE specimens could easily expand into inclined cracks due to the controlled expansion of flexural cracks, as the carbon fabric received the tensile stress adequately due to the increased carbon fabric reinforcement ratio. Upon examining the crack patterns of the SE series, the number of cracks increased, while the crack width decreased due to the sand–epoxy surface treatment.The load–deflection relationship curves of the TRC panel specimens were plotted using the two equations proposed by Branson and Bischoff. The flexural moment corresponding to the assumed The initial behavior of the numerical calculation and the experimental results was very similar in all specimens. The behavior before the first flexural crack occurred showed identical results, but the time of occurrence of the first flexural crack was slightly different. This was because the load at which the first flexural crack occurred in the TRC panel test specimen was mostly higher than the flexural strength of the concrete matrix itself .The behavior of all test specimens after the first flexural crack was closer to the experimental results when the Bischoff formula was applied than when the Branson formula was used. As the carbon fabric reinforcement ratio increased, the difference between the application results of the Branson formula and the Bischoff formula decreased . The behIn the case of maximum load, the experimental results were 8% to 22% higher when compared to the nominal strength by numerical calculation, except for the C2L1A4SE specimen. This difference can be attributed to the variation in ultimate compressive strain assumed in the numerical calculation and the difference in the first cracking load.To investigate the effect of the fabric reinforcement ratio, anchorage length, and surface treatment on concrete composite panels reinforced with carbon fabric, textile-reinforced concrete specimens were fabricated and flexural tests conducted. In addition, the flexural behavior of the TRC panel specimens was numerically calculated and compared with the experimental results, and the following conclusions were drawn.(1)All TRC specimens exhibited a sudden drop in load immediately after the first flexural crack occurred. This is due to the brittle stress redistribution from the concrete matrix to the carbon fabric, and it can be reduced by improving the bond between the matrix and the carbon fabric and increasing the fabric reinforcement ratio.(2)With an increase in the carbon fabric reinforcement ratio, from one layer to four layers, the degree of load reduction and reduction in flexural stiffness at the time of flexural cracks decreased, and the maximum load and number of cracks increased. This is because the carbon fabric rapidly contributes to the tensile stress acting on the bottom of the concrete matrix.(3)As the anchorage length increased from 50 mm to 275 mm, the flexural stiffness after the first cracking of the TRC panel increased, and the number of cracks increased.(4)Sand–epoxy surface treatment and an increase in anchorage length simultaneously contributed to the improvement of the load-carrying capacity of the TRC panel up to 110%. However, the effect of increasing the anchorage length was limited, and additional load-bearing capacity could be improved through sand–epoxy surface treatment.(5)Comparing the numerical calculation results with the experimental results, it was found that the deflection of the experimental results was approximately 50% larger than the numerical calculation results. This is because the bond between the carbon fabric and the concrete matrix failed, and slip occurred.To prevent slip of the fabric, it is necessary to secure a reliable bond between the carbon fabric and concrete using methods such as surface improvement through sand–epoxy surface treatment, sufficient anchorage length, and sufficient concrete thickness. In addition, it is necessary to accurately identify the bond behavior and set limitations for TRC material performance to design structures applying TRC.


The experimental load-deflection diagrams are reproduced by modifying the trilinear diagram used in the cohesive model, which helps to understand how the trilinear diagram parameters are affected by high temperature exposure. Finally, some expressions are proposed to adapt the initial trilinear diagram (obtained with specimens not exposed to high temperature) in order to numerically reproduce the fracture behaviour of PFRC affected by high temperature exposure.Fibre-reinforced concrete (FRC) has been used for decades in certain applications in the construction industry, such as tunnel linings and precast elements, but has experienced important progress in recent times, boosted by the inclusion of guidelines for its use in some national and international standards. Traditional steel fibres have been studied in depth and their performance is well-known, although in recent years new materials have been proposed as possible alternatives. Polyolefin macro-fibres, for instance, have been proven to enhance the mechanical properties of concrete and the parameters that define their behaviour  have been identified. These fibres overcome certain traditional problems related to steel fibres, such as corrosion or their interaction with magnetic fields, which can limit the use of steel in some applications. The behaviour of polyolefin fibre-reinforced concrete (PFRC) has been numerically reproduced with success through an embedded cohesive crack formulation that uses a trilinear softening diagram to describe the fracture behaviour of the material. Furthermore, concrete behaves well under high temperatures or fire events, especially when it is compared with other construction materials, but the behaviour of PFRC must be analysed if the use of these fibres is to be extended. To this end, the degradation of PFRC fracture properties has been recently experimentally analysed under a temperature range between 20 °C and 200 °C. As temperature increases, polyolefin fibres modify their mechanical properties and their shape, which reduce their performance as reinforcements of concrete. In this work, those experimental results, which include results of low (3 kg/m Concrete is a building material usually reinforced with steel rebars, which confer a good tensile strength to a structural element. This makes reinforced concrete one of the most versatile materials in building and civil engineering works. Nevertheless, in the last decades, fibres have become an interesting complement to steel rebars as a reinforcement and there are a wide variety of fibres that can be added, depending on the properties that need to be enhanced.When strength needs to be improved, steel fibres have been traditionally employed and there is considerable research on their usage ,2,3,4,5.On another note, the influence of high temperatures on the mechanical properties of concrete is also of paramount importance in some applications. The use of reinforced concrete in nuclear reactors boosted the interest in how high temperatures and fire affect concrete mechanical performance . This inRegarding other types of fibres, polyolefin macro-fibres have proven to provide remarkable structural properties to concrete. Polyolefin-fibre-reinforced concrete (PFRC) has been extensively studied in recent times and its performance in vibrated or self-compacting concrete as well as the influence of many parameters on the PFRC mechanical properties or their performance under shear stresses are already well-known ,22,23,24From the numerical point of view, concrete fracture has long been successfully simulated using cohesive formulations, which relies on a softening diagram that drives the fracture process by progressively reducing cohesive forces between both cracking surfaces as they move apart from each other. PFRC fracture has been successfully reproduced using this approach by means of an embedded fracture model that had been used in the past with concrete and masonry . To adapThe present study seeks to adapt the trilinear softening cohesive model to account for high-temperature exposure of the PFRC elements. To do this, the results obtained in  are usedThe experimental results used here as a reference correspond to an experimental campaign that can be consulted in . Two setAll specimens were manufactured with 60 mm long fibres, the appearance of these fibres as well as their properties can be seen in After manufacturing, all the specimens were introduced in a convection oven at a heating rate of 2.80 °C/min, remaining at the desired temperature for 3 h before progressively cooling for 7 h inside the oven. The bending tests were performed once the cooling process was completed.In the case of HF3 specimens, three temperatures were used for characterisation: room temperature (20 °C), 150 °C and 200 °C, while in the case of HF10 specimens, six temperatures were chosen: room temperature (20 °C), 150 °C, 165 °C, 175 °C, 185 °C and 200 °C. Temperatures over 150 °C correspond to values at which fibres showed progressive degradation due to heating (see ).The mechanical behavior of the specimens was analysed with a three-point bending test as described by the standard RILEM TC-187-SOC  with notThis section presents the numerical work carried out to reproduce the experimental diagrams obtained for the HF3 and HF10 specimens. These simulations use an embedded crack model that is briefly described in A fracture is reproduced with the finite element method (FEM) using an element formulation that is based on the cohesive zone concept developed by Hillerborg . This foThis formulation can only be used in constant strain triangular elements, which implies using a single integration point per element, and only allows three crack orientations, which are parallel to the sides of the element see .A stands for the element surface area, h for the height of the triangle with respect to the opposite side of the solitary node, L is the crack length and At the onset of fracture, the element is divided into parts ned with , where Ale, thus  becomes found in .(2)t=AhS denotes the symmetric part of the resulting tensor.The crack displacement vector pression . To do tned with . SuperscBy using the expression of t  and the dsymbolσ , the folt, k, r and f). This trilinear diagram can be expressed with the following equations :(6)σ=fcThe three-point bending test on notched specimens shown in t, k, r and f observed in In this section, trilinear diagrams for each mix and high-temperature exposure are obtained. To do this, the coordinates of all four points t, since the elastic modulus of concrete is much higher than the elastic modulus of the fibres, and given that the volume of fibres is almost negligible if compared with concrete, this point is defined as the tensile strength of concrete. Following the experimental observations, this value will be gradually reduced as temperature increases, thus reproducing a gradual material degradation. Point k is related to the crack opening value at which fibres start to transmit meaningful stresses across the fracture plane. The reinforcing action of fibres stops the load drop and can even lead to a load recovery. Both coordinates of point k have been estimated considering that the contribution of fibres is negligible up to this instant, therefore, they have been obtained with the softening diagram of a plain concrete (without fibres). The reinforcing capacity of fibres decays when a certain crack opening is reached, which is related to point r in the trilinear diagram. On the one hand, the abscissa f identifies the crack opening value at which fibre reinforcement is negligible, this point depends on the length of the fibres and, in this case, using previous experiences, has been fixed with a value of 7.5 mm for all temperatures.Regarding point eriences , on the Following these criteria, the initial softening diagram, adjusted for room temperature (20 °C), was modified so that the load-deflection diagrams fitted the experimental results. In the case of HF3, the initial peak load is correctly reproduced for all three temperatures. The same can be noted in regards to the minimum load after peak and the subsequent remnant peak load that takes place around a deflection of 4 mm for all temperatures.T = 150 °C, the peak load in the numerical model takes place at a slightly earlier deflection than in the experimental curve. Nevertheless, in the authors’ opinion this does not make the numerical simulation inaccurate, since only one experimental value for each temperature is available and, as past experience shows, if a higher number of specimens had been tested for each case, the experimental envelopes would fit all numerical results.In the case of HF10, the minimum load after peak and the subsequent remnant peak load are well-reproduced for all temperatures. In some cases, especially in the case of t, k and r reduce their values as the temperature increases, which can be a consequence of moderate concrete and fibre degradation. In the case of T = 150 °C is smaller than at T = 20 °C, which would suggest a decrease as the temperature increases, but peak loads at 165 °C and 175 °C and 185 °C are higher than at 150 °C .(7).(7)σt, While expressions  and 9)  9) provipression  seems tot, k and r of the trilinear diagram (A trilinear diagram calibrated with specimens not exposed to high temperature can be adapted for high temperature exposure by modifying the ordinates of points The load drop after the initial peak becomes higher as temperature increases. This drop is relevant beginning from a temperature exposure of 150 °C. A function for defining The load drop of the remnant peak load that takes place after the initial peak load can be only observed for temperatures over 175 °C. A function for This work has studied how high temperature exposure affects the parameters of a trilinear softening diagram that allows for numerically reproducing the behaviour of PFRC specimens through a cohesive model. To do this, the experimental results of  have bee


Background: Ventilator-associated lower respiratory tract infectious complications in critically ill patients cover a wide spectrum of one disease process (respiratory infection), initiating from tracheal tube and/or tracheobronchial colonization, to ventilator associated tracheobronchitis (VAT) and ventilator-associated pneumonia (VAP). VAP occurence has been associated with increased intensive care unit (ICU) morbidity  and ICU mortality. Therefore, treatments that aim at VAP/VAT incidence reduction are a high priority. Aim: The aim of the present review is to discuss the current literature concerning two major aspects: (a) can aerosolized antibiotics (AA) administered in a pre-emptive way prevent the occurrence of ventilator-associated infections? and (b) can VAT treatment with aerosolized avert the potential evolution to VAP? Results: There were identified eight studies that provided data on the use of aerosolized antibiotics for the prevention of VAT/VAP. Most of them report favorable data on reducing the colonisation rate and the progression to VAP/VAT. Another four studies dealt with the treatment of VAT/VAP. The results support the decrease in the incidence to VAP transition and/or the improvement in signs and symptoms of VAP. Moreover, there are concise reports on higher cure rates and microbiological eradication in patients treated with aerosolized antibiotics. Yet, differences in the delivery modality adopted and resistance emergence issues preclude the generalisability of the results. Conclusion: Aerosolized antibiotic therapy can be used to manage ventilator-associated infections, especially those with difficult to treat resistance. The limited clinical data raise the need for large randomized controlled trials to confirm the benefits of AA and to evaluate the impact on antibiotic selection pressure. Lower respiratory tract infections are well known entities, including ventilator-associated pneumonia (VAP) and ventilator-associated tracheobronchitis (VAT). VAP has been described since 1965, while VAT is a relatively newer clinical entity ,3. US epBoth VAP and VAT significantly affect patient outcomes because they increase the duration of mechanical ventilation, length of intensive care unit (ICU) stay, similarly to pneumonia, and healthcare costs, provoking antimicrobial resistance due to increased antibiotic consumption ,5; VAP h“Ventilator bundles”, targeting the reduction of respiratory infection incidence in the ICU, may significantly decrease the reported rates of infections ,12. TherAerosolized/nebulized or tracheobronchially instilled antibiotics have a long history, and they have been evaluated in studies on hospital-acquired respiratory tract infections in the intensive care units (ICUs) since the 1990s ,17. NotaThe purpose of the present narrative review is to describe the role of nebulized antibiotics in the management of ventilator-associated infections in critically ill patients. More specifically, we aimed to review the literature regarding two related questions: (a) whether prophylactic antibiotics administered through the respiratory tract can prevent ventilator associated infections and (b) whether nebulized antibiotic treatment of VAT could affect the potential evolution of VAT into VAP.We used the PubMed, Cochrane Database of Systematic Reviews, and Google Scholar research databases to search for published articles using the following terms: antibiotics AND aerosolized OR nebulized AND ventilator infections. The search was narrowed down to titles and abstracts. Abstracts were then examined, and studies that included the key terms in the title or abstract were considered. The abstracts and/or full texts of selected, potentially relevant papers were further evaluated. The selection was limited to surveys published in English up to 2022. Exclusion criteria for articles included: no accessible full text, publications with only abstracts, and editorials and studies that did not report specific quantitative outcomes.Ventilator-associated pneumonia (VAP) may occur in patients who are managed with mechanical ventilation for more than 48 h, and the diagnosis requires radiologic evidence of pneumonia (new infliltrates) and may include the presence of purulent tracheal secretions, fever, signs of inflammatory reaction, respiratory distress, and the presence of microorganisms ,10. VAT Early and appropriate therapy of VAP has been consistently demonstrated to decrease mortality, but the efficacy of treatment has been challenged by the presence of multidrug-resistant pathogens. More emphasis has been given to the prevention of VAP, instead of VAT, since any preventive measure has been considered cost-beneficial . HoweverVarious pathophysiologic mechanisms have been described as potential mechanisms for ventilator-associated infections . MechaniVentilator bundles cannot lead to “zero VAP” due to the fact that microaspiration and biofilm formation cannot be fully prevented, and this is also because systemic antibiotics cannot reach the endotracheal tube and biofilms to kill bacteria. Local application of antibiotics administered through the endotracheal tube might have the advantage of directly presenting the antibiotic in contact with the tubing system, enabling possible penetration into the biofilm. Moreover, the antibiotic is directly administered to the target organ quickly, while intratracheal antibiotic concentrations can greatly exceed the minimum inhibitory concentration of the pathogens. This might enable the eradication of multiple pathogens, including those with difficult-to-treat resistance ,26,27.The earliest modality of antibiotic delivery to the tracheal tube, the trachea, and the lung parenchyma was intratracheal installation to the tube . Direct Jet nebulizers use the already available compressed gas (air or oxygen) from a wall system or the ventilator to produce aerosol. Apart from their low cost, another advantage of jet nebulizers is the capability of synchronization with each breath. Disadvantages include long treatment time, high residual drug volume (0.8–2 mL), possible risk for denaturing medication due to shear stress generated from a highly turbulent flow, and the effect on the tidal volume that the patient receives by the additional delivery of 6–8 L of gas into the ventilator circuit. Generally, it is considered the lowest-efficiency nebulizer ,31,32. UVibrating mesh nebulizers generate aerosol by vibration of a plate, producing particles 1–5 μm in size. This nebulizer has the highest efficiency for drug delivery among all others, has a short treatment time, has the lowest residual volume (0.2 mL), and minimally affects the solution temperature and, therefore, the drug inactivation. The only disadvantage is its high cost ,31,32.Lung pathophysiology, breathing pattern, and the severity of disease can affect the particle deposition in the lungs. Larger antibiotic particles deposit centrally in the upper respiratory tract, while smaller particles  are more likely to be exhaled . SpontanThe antibiotic selection depends on inherent properties to ensure maximal efficacy and safety. The ideal aerosolized antibiotic should be sterile, nonpyrogenic, preservative-free, with a pH level of 4.0–8.0, and adjusted tonicity and osmolarity of 150–1200 mOsm/L . TherefoBased on data from recent studies, nebulized antibiotics cannot treat VAP or decrease mortality, as shown by the recent Phase 2 and 3 trials  ,37. HoweK. pneumoniae prevented the onset of bronchopneumonia, whereas intramuscular administration was not protective. Further experimental studies demonstrated that the endotracheal administration of polymyxin B or gentamicin prevented ventilator-associated pneumonia (VAP), providing the rational for the conduction of clinical studies [In a non-human model, the aerosolized kanamycin administered before intratracheal instillation of  studies ,40.P. aeruginosa and A. baumannii, the most common pathogens causing VAP in Europe and Asia [P. aeruginosa was 1.6% during the polymyxin treatment  and 9.7% during the placebo cycles (370 patients) (p < 0.01). Similarly, three patients in the polymyxin and 17 in the placebo groups acquired P. aeroginosa pneumonia (p < 0.01). Feeley and co-workers [P. aeruginosa pneumonia during a seven-month period. The study included 292 intubated and spontaneously breathing patients in a respiratory–surgical ICU. They concluded that daily use of polymyxin B aerosol appears to be a dangerous form of therapy. The mortality rate for acquired pneumonia was 64%, higher than that in previous studies in which no polymyxin was used, although there was only one episode of acquired pneumonia due to P. aeruginosa. Moreover, 10 patients acquired pneumonia caused by a polymyxin-resistant organism, while seven cases were caused by organisms not frequently pathogenic to man . Although these results were not repeated, they prompted the medical community towards skepticism concerning the intratracheal instillation of drugs to prevent VAP.Polymixin and amikacin often remain the only antimicrobial agents available against and Asia ,42. The and Asia . Initialand Asia , in a prand Asia  evaluatep < 0.01). Moreover, significantly fewer patients suffered bacteriologically confirmed respiratory tract infections (p < 0.01). Bacteria isolated from patients treated with gentamicin were slightly more resistant to gentamicin than the microorganisms recovered from control patients, concluding that endotracheal administration of gentamicin may be a helpful adjunct treatment, but special attention should be paid to the possibility for resistance emergence.Klastersky et al.  studied p = 0.07), at the 30-day follow up. Regarding the secondary outcomes, the intervention resulted in a lower VAP incidence density rate  (p < 0.01) and less VAP episodes either from Gram-negative bacteria (p = 0.03) or MDR bacteria (p = 0.04). There was also no development regarding ICU or hospital mortality. However, among all patients who developed VAP (n = 39), those having received prophylactic nebulized colistin presented a significantly lower ICU mortality (p = 0.016). The authors hypothesize that the control of airway inflammation during the VAP episode may explain the survival benefit. This is also supported by the findings of lower SOFA score on the infection day and the white blood cell count following VAP diagnosis in the Col group vs. the NS group. In this study, neurosurgical admission was a risk factor for GNB-VAP, whereas GNB-VAP was lower in the neurosurgical Col group. Thus, the prophylactic administration of inhaled colistin may become attractive, especially in neurocritical patients. Interestingly, during the study period (19 months), there was no difference in the development of colistin-resistant bacteria or multidrug resistance between the two groups.In a two-arm, randomized, open-label, controlled trial , prophylp = 0.02), and the pooled odds ratio (meta-odds ratio) for developing pneumonia was 0.53 compared to control. The antibiotic prophylaxis did not increase the development of VAP due to MDR pathogens. The ICU mortality was not different between the compared groups with or without prophylaxis. It is evident that, if this treatment modality is selected, the antibiotics should be delivered by aerosolization to achieve more uniform distribution and deeper penetration into the lung parenchyma [Povoa and colleagues  conducterenchyma . Whetherp = 0.021), and it was 54% lower for the entire ICU stay . No clinically significant changes in bacterial culture and sensitivity patterns were observed. In addition, the proinflammatory response in the lung was attenuated; the frequency of VAP was directly related to changes in TNF-alpha and IL-beta. In addition, the subjects receiving empiric antibiotics received less systemic antibiotics compared with those receiving placebo. In another single-institution double-blind, randomized trial [Others studied aerosolized cephalosporines administered through the respiratory tract–endotracheal tube. Wood et al.  evaluateed trial , the pated trial  comparedP. aeruginosa in experimentally model, which may be an alternative safe protection method for VAP in the future [P. aeruginosa or nebulized P. aeruginosa + specific polyclonal anti-P. aeruginosa IgY antibodies. Tracheal growth of P. aeruginosa increased in both groups during the experiment, but with lower growth in the Pa-IgY-treated group. After 12 h, the treatment effect was diminished and bacterial growth increased in both groups. However, in this porcine model, Pa-IgY antibodies decreasd the airway bacterial colonization, although transiently, leaving a promising potential for the future [More recently, inhaled specific polyclonal antibodies have been used to protect against tracheal colonization by specific microbes, and specifically e future . The pose future .Similarle future .Aerosolized antibiotics (AA) as a monotherapy for VAT have been investigated in few original studies, as shown in P. aeruginosa and A. baumannii [The paucity of available data has been underlined in the literature. Systematic reviews of published data do not clarify further this issue. Russell et al.  suggesteaumannii . Larger aumannii . Moreoveaumannii .Actually, the guidelines and position paper from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) on the use of nebulized antimicrobials for the treatment of respiratory infections in adult patients under mechanical ventilation recommended to avoid the additional use of aerosolized antibiotics for the treatment of VAT because the quality of available data is rather low .The emergence of MDR bacteria, following tracheobronchial instillation/aerosolization of antibiotics, is a major concern when implementing strategies using antibiotics to prevent infections. Resistance emergence has not been commonly identified in the studies using aerosolized antibiotics. Karvouniaris et al. reported a reduced risk for VAP due to MDR pathogens when nebulized colistin was used (OR 0.33 CI 95% 0.12–0.88) . In accoHowever, concerns regarding the emergence/worsening of antibiotic-resistance when antibiotics are widely used in ICU critically ill patients still remain. The concerns are further implicated when considering the strict administration criteria of inhaled antibiotic strategies, such as optimal formulations, as well as length and dosage of treatment regimens. Moreover, whether ultrasonic, vibrating-mesh, or jet nebulizers in mechanically ventilated patients are preferred has not yet been determined. Furthermore, the pharmacokinetics/pharmacodynamics of inhaled therapies might be challenging. International scientific societies remain skeptical for the use of inhaled antibiotic strategies. The American Thoracic Society guidelines for the management of adults with hospital-acquired and ventilator-associated pneumonia do not recommend the use of aerosolized antibiotics to prevent VAP , and theThe presence of adverse effects associated with the use of aerosolized antibiotics is another concern. Irritation and airway inflammatory reaction may lead to cough, bronchospasm, bronchoconstriction-wheezing, desaturation, and even hypoxemia. Among inhaled antibiotics, colistin is more responsible for these effects ,61. The Regarding our view, the role of antibiotic aerosolized therapy to manage ventilator associated infections, especially those due to MDR Gram-negative bacteria is promising, but clinical data are limited, and there are still questions that require further research in order to be answered. This is most important when considering the marked heterogeneity in clinical practice, with significant differences in the use of aerosolized antibiotics between patients. First, large randomized controlled trials should be conducted to confirm the benefits of AA and to explore the impact on antibiotic selection pressure with the use of aerosolized antibiotics. Research may also need to focus on early (pre-emptive) treatment of VAT to prevent evolution to VAP, using appropriate nebulized antibiotics, especially in those at high risk for VAP development. Moreover, future research should focus how to refine aerosolized treatment in terms of delivery device technology, adequate antibiotic dosing, and specific protocol design.


Immunoglobulin G4-related disease (IgG4-RD) is a systemic fibro-inflammatory disease with the potential to involve virtually all organs, including the pancreas, kidneys, lungs, and pleura, amongst others. IgG4-RD pleural involvement may cause diverse complications such as pleural effusion, pleural thickening, pleural nodules, and additional lesions, which can be presented in many clinical diseases. However, isolated cases of pleurisy are still rare in IgG4-RD. We report a 72-year-old patient who was admitted to our hospital with cough, expectoration, and fatigue. He had a right-sided pleural effusion, and the tissue evaluation of the pleural biopsy by medical thoracoscopy met the diagnostic criteria of IgG4-RD. His serum IgG4 levels were elevated and he was finally diagnosed with IgG4-RD pleural involvement. He was subsequently started on prednisone 40 mg daily and his pleural effusion was almost disappeared 2 weeks later. This paper reported a case of IgG4-RD who had exclusive involvement of the pleura and highlighted the significance of considering IgG4-RD as a potential diagnosis in patients with unexplained pleural effusion. InitialHerein, we present a case report of IgG4-RD accompanied by the presence of right pleural effusion, highlighting the necessity of including IgG4-RD in the etiological diagnosis of idiopathic pleural effusion.29/L (neutrophils accounted for 74.1%), hemoglobin 110 g/L (130–175 g/L), platelet 358 × 109/L (125–350 × 109/L), C-reactive protein (CRP) 60 mg/L (0–3.48 mg/L), albumin 32.30 g/L (35–55 g/L), and globulin 46.50 g/L (20–35 g/L). Serum tumor marker tests, rheumatic immune series, anti-neutrophil cytoplasmic antibody (ANCA) and interferon-γ release test (TSPOT.TB) were all normal. Further imaging studies, including echocardiography, abdominal ultrasound (US), US of thyroid glands, the parotid and bilateral submandibular glands, and abdomen computed tomography (CT) scan showed no abnormalities. A chest CT revealed patchy lesions in the upper lobe of the right lung, as well as a right pleural effusion with pleural thickening . He was currently smoking 10 cigarettes per day. Upon admission, his body temperature, pulse rate, respiratory rate and blood pressure were 36.5°C, 80/min, 20/min, and 120/85 mmHg, respectively. Bronchoscopy and bronchoalveolar lavage outside the hospital showed no microorganisms, and cytological analysis ruled out malignancy. Laboratory tests on admission showed: white blood cell count 7.97 × 10ickening . Right terformed  and histerformed . Serum lBased on these findings, a diagnosis of pleural involvement of IgG4-RD was made. The patient was commenced on prednisone 40 mg daily and a follow-up CT 2 weeks later showed significant improvement with almost complete resolution of the lung lesions and pleural effusion . Serum I3IgG4-RD is indeed a fascinating clinical entity. It was first reported in Japan and has been found to be involved in a proportion of patients with undiagnosed pleural effusion . KasashiWe conducted a search of MEDLINE database for English literature from the beginning year of inclusion to September 2023, using the keywords: “IgG4 related diseases” AND “pleural effusion” OR “pleural involvement.” We analyzed a total of 37 cases of IgG4-RD with pleural involvement after excluding studies with mismatched clinical data and non-case reports . The aveIt seems to be interesting that our patient had a history of asthma, which aligns with previous case reports on this matter , 23. KasRegarding serology, it is worth noting that 34 91.89%) of the patients in our collected cases exhibited elevated serum IgG4 levels . Increas9% of theIn IgG4-PL patients, it is typically observed that CRP levels are within normal range. However, in the cases we analyzed, 10 individuals demonstrated a mild increase in CRP levels , 36, 37.It is noteworthy that elevated levels of adenylate deaminase (ADA) in pleural fluids have been reported in some IgG4-PL cases in the studies we retrieved , 11–13, Our case needs to be differentiated from tuberculous pleurisy, malignant pleural effusion, and MCD. Acute symptoms such as fever, chest pain, and dry cough are often associated with tuberculous pleurisy . A combiAlthough nonspecific, imaging serves as a crucial component of the diagnostic approach for various organs. In cases with pleural involvement, imaging manifestations may include pleural effusion, pleural thickening, pleural calcification, and pleural nodules . RadioloPleural lesions in IgG4-RD can sometimes be directly visualized via thoracoscopy. However, not all cases of IgG4-RD involving the pleura appear abnormal on thoracoscopic examination . HistopaGC are the first-line therapeutic drugs for IgG4-RD . The dos4In summary, IgG4-RD involving the pleura is a clinically uncommon and heterogeneous entity. The main patterns of pleural involvement consist of pleural nodules, pleural thickening, and pleural effusion. Medical thoracoscopy serves an important role in diagnosing pleural involvement in IgG4-RD, especially in cases of pleural effusion. A definitive diagnosis of IgG4-related pleural lesions relies on the histological identification of a lymphoplasmacytic infiltrate with abundant IgG4+ plasma cells, storiform fibrosis and obliterative phlebitis in the pleurae and on the exclusion of all other possible causes. The possibility of IgG4-RD should be considered in patients with unexplained pleural effusion charactered by the presence of IgG4-positive plasma cell in pleural biopsies. Generally, IgG4-PL respond well to systemic corticosteroid therapy.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients was not required to participate in this study in accordance with the national legislation and the institutional requirements. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.AZ, XL, ZG, YJ, and DL provided the ideas for this case report. AZ and DL prepared and revised the manuscript. DL reviewed the article. XL, ZG, and YJ confirmed the authenticity of all original data. All authors have read and approved the final version of the manuscript.


To exploretheeffects of physical, mechanical, anti-deterioration properties of graphene oxide (GO) on cement-based cementitious materials, GO sheet dispersions areprepared by the improved Hummers method and ultrasonic dispersion method. The influence of theGO content on the compressive and flexural strengths of cement paste is investigated, and the penetration process of chloride ions in graphene oxide concrete is discussed by the electric accelerated erosion method. Combined with the rapid freeze-thaw test, the deterioration of graphene oxide concrete ismethodically analyzed. Theobtained results reveal that an appropriate amount of GO improves both the compressive and flexural strengths of cement pastev. In the chloride environment, the chloride diffusion coefficient of 0.03% GO concrete is 18.75% less than that of ordinary concrete.Under the action of freeze-thaw cycles, with the increase of salt freezing times, the deterioration mode of GO concrete is a combination of mortar shedding, micro-crack expansion, denudation, and block shedding; The stress-strain curve of the specimen tends to be flat with the growth of salt freezing times. The peak stress gradually lessens, the peak strain gradually grows, and the elastic modulus remarkably reduces. Compared with ordinary cement paste, theGO is capable of promoting the growth of cement paste hydration crystals, changing the size and shape of crystals, and realizingthe regulation of cement paste microstructure. Incorporating an appropriate amount of theGO could promote the cement hydration process and enhance the chemical water-binding amount in the cement paste. The optimal GO content is reported to be 0.03% of the cement mass. Concrete is extensively utilized worldwide due to its abundant source of materials, low price, and simple construction. With the long-term development of domestic infrastructure construction, the durability of concrete structures has become a hot issue in civil engineering. The structure’s durability is directly related to the project’s safety and even the national economy’s social benefits, so it paid more and more attention , 2. MehtIn the Northeast China, buildings are often subjected to freezing disaster because of the low temperature in winter temperature, and when the temperature rises, the freezing and concreting buildings begin to melt, and the buildings are subjected to repeated freezing and thawing cycles, make making buildings more vulnerable to damage –3. Becau2, nano-CaCO3, andnano-TiO2) to cement concrete and examined their influences on the concrete strength, microstructure, cement hydration process, and whether it is beneficial to improve the durability of concrete. With an in-depth realizing the basic theory of cement-based materials at the nanoscale , nanoscience has made noticeable progress in cement-based materials. On the one hand, nanomaterials as fillers could effectively fill the pores of the concrete, reduce the number of harmful spores in the cement matrix, and enhance the compactness of the cement matrix. On the other hand, they can react with Ca(OH)2 with low strength in hydration products to produce more hydration calcium silicate gel (C-S-H) and ettringite (AFt), thereby enhancing the strength of cement-based materials and improving their durability. Additionally, nanomaterials have a series of unique properties such as electricity, heat, electromagnetic, and light. The addition of nanomaterials to cement-based materials providesad-based composites have some unique functions, which brings a new approach to realizing multi-functional and high-performance concrete production.In recent decades, nanomaterial science has promptly developed –8. Many Graphene is the thinnest two-dimensional nanomaterial with excellent mechanical, electrical, and thermal properties –15. The In recent years, many experts and investigators in China and abroad have not only limited the mselves to enhancing the mechanical properties of cement-based materials in the research and application of graphene and its derivatives mixed with cement-based composites, but also carried out an in-depth exploration of its durability, conductive and thermal conductivity, pressure sensitivity, electromagnetic shielding, and other aspects –31. Howe3), concentrated sulfuric acid (98%), potassium permanganate (KMnO4), phosphoric acid (H3PO4), hydrogen peroxide (H2O2) and hydrochloric acid (HCl) are all analytically pure, which are provided by Dalian Kexin Chemical Co., Ltd. The DK-PC type polycarboxylate superplasticizer is produced by Dalian Jianke North Chemical Co., Ltd. It is a brownish-yellow liquid with a water reduction rate of 36%, a solids content of 35%, and a content of 1.2% of the cement mass fraction. The test cement is Xiao ye tian PO·42.5 Ordinary Portland cement, and its chemical composition is detailed in The used graphite in the test (particle size <10um) has been produced by Nanjing Xianfeng Nanomaterial Technology Co., Ltd. Sodium nitrate : m(water): m(sand): m(stone): m(water reducing agent) = 420: 168: 782: 1080: 2.1. After molding, the specimen was moulded for 24 h and placed in a standard curing box for 28days for the test.According to the existing research results , 35, theIn order to investigate the distribution of chloride ion content in the GO-modified concrete, this section explores the law of chloride ion erosion in GO concrete by the method of constant voltage electrochemical accelerated corrosion. For this purpose, three levels of the acceleration voltage  are set up, and the specific test groups are presented in Under the relevant provisions of the test method for long-term performance and durability of ordinary concrete(GBJ82-85), the freeze-thaw cycle test was on ducted by the TDR-16 rapid freeze-thaw testing machine, and the dynamic modulus of concrete under the freeze-thaw cycle was measured by DT-12 dynamic modulus tester. The schematic representations of the freeze-thaw cycle device and the temperature change curve are demonstrated in Each freeze-thaw cycle of the freeze-thaw cycle test shall be completed within 2-4h, and the melting time shall not be less than 1/4 of the whole freeze-thaw cycle time. At the end of freezing and melting, the center temperature of the test piece shall be controlled at—17 ± 2°C and 8 ± 2°C, respectively. The time taken for each test piece to drop from 6°C to—15°Cshould not be less than 1/2 of the freezing time, while the required time to increase from—15°C to 6°Cshould not be less than 1/2 of the melting time, and the discrepancy between the inside and outside temperatures of the test piece should not exceed 28°C. The conversion time of freezing and melting should not exceed 10min.After putting the concrete specimen into a freeze-thaw test box, take out one group every 25 cycles. The YAW-YAW2000A type 200t microcomputer-controlled electro-hydraulic servo pressure testing machine is utilized to test the flexural strength and compressive strength of concrete under the current standard condition for Highway Engineering Cement and Cement Concrete Test Procedure(JTGE30-2005). The loading speed is controlled at 2400 ± 200N / s.1cm × 1cm × 1cm was put into anhydrous ethanol to terminate hydration for 48 hours, and the test section of the specimen was not treated. The surface fracture was covered with a thin layer of gold, and SEM micrographs were obtained with a SUPRA 55 SAPPHIRE.The morphology and microstructure of some selected samples of GO cement composites are examined via the scanning electron microscope(SEM) technique. The specimen was cured to the test age and broken. The block of In the current investigation, the mineral composition of samples at various ages was determined by an X-ray diffractometer(XRD). Before the test, the sample was made into a cube with a side length of about 1 cm. In the diffraction test, the test radius was greater than200 mm, the scanning speed was 10° /min, and the scanning angle (2θ) was set as10–90°.In this paper, the content of hydration products  and calcium hydroxide (CH)) in GO-modified cement was determined by TG-DSC STA 409 thermal gravimetric analyzer. First, samples are taken from the core part of the specimen, and the hydration was terminated by acetone for one day. The prepared samples were then dried at 105°C for 4 h in a vacuum drying oven and ground to a fineness of fewer than 150 μm. During the TG analysis, the temperature rate was set equal to15°C /min, the heating atmosphere was nitrogen, and the gas flow rate was set as 50 mL/min.The reference weight calculated by the percentage of CH in the sample is the mass of the sample after burning at 950° C for 30 minutes. TheCH content is could be also calculated by the formula Eqs  and 2)::2):CH=Δm is the mass loss in the temperature range of 420–490°C; m0 is the reference weight, which is the mass of the sample after burning at 950°C for 30 min. MCH and M are the molar mass of Ca(OH)2 and H2O, respectively.Where All the experimental samples are shown in As illustrated in The flexural strength values of different GO cement paste specimens at various curing ages have been illustrated in It can be seen from The compressive strength values of GO cement paste under for various curing ages are illustrated in The change of chloride ion penetration depth of GO concrete in the presence of various applied voltage accelerated corrosion has been illustrated in Under natural infiltration conditions , the DSC curves of GO cement slurry GO-0, GO-1, GO-3, and GO-5 are provided in The contents of CH and chemical binding water in GO-0, GO-1, GO-3, and GO-5 cement slurry are provided in The addition of an appropriate amount of GO could effectively enhance the early mechanical properties of the cement paste. The 7d flexural strength of the cement paste with 0.03% GO was 22.71% higher than that of the ordinary cement paste, and the compressive strength was increased by 22.71%.Under accelerated corrosion conditions, the penetration depth of chloride ions and the ircontent in the same part lessened with the growth of GO content.After freezing and thawing, the compressive strength of GO concrete specimens with different contents was enhanced with the growth of GO contents. The compressive strength was increased by 29.3%. Therefore, incorporating GO could improve the compressive strength of salt-frozen concrete. The stress-strain curves of different GO concrete specimens were slightly different. Compared with ordinary concrete mixed with 0.03% GO, the flattening speed of the curve of the concrete specimen was relatively slow. Therefore, the addition of GO could enhance the ability of concrete to resist freeze-thaw deformation.Microscopic explorations reveal that the GO can promote cement hydration and has the effect of assembling and regulating cement hydration crystals. On the GO sheet, the hydration crystal products are intertwined to form polyhedral hydration products, and the microstructure is regular and orderly denser. Adding a small amount of GO can promote the cement hydration process, and the chemical water binding amounts in the cement slurry with 0.01% and 0.03% GO in order were 2.16% and 2.22% higher than those in the ordinary cement slurry.S1 Data(XLSX)Click here for additional data file.