This study aimed to investigate the protective effect of metformin on trabecular meshwork (TM) and explore its molecular mechanisms in vivo and in vitro. Ocular hypertension (OHT) mouse models were induced with dexamethasone and further treated with metformin to determine the intraocular pressure (IOP)-lowering effect. Cultured human TM cells (HTMCs) were pre-stimulated with tert-butyl hydroperoxide (tBHP) to induce oxidative damage and then supplemented with metformin for another 24 hr. The expression of fibrotic markers and integrin/Rho-associated kinase (ROCK) signals, including α-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), fibronectin, integrin beta 1, ROCK 1/2, AMP-activated protein kinase, myosin light chain 1, and F-actin were determined by western blotting and immunofluorescence. Reactive oxygen species (ROS) content was analysed using flow cytometry. Transmission electron microscopy was performed to observe microfilaments in HTMCs. It showed that metformin administration reduced the elevated IOP and alleviated the fibrotic activity of aqueous humour outflow in OHT models. Additionally, metformin rearranged the disordered cytoskeleton in the TM both in vivo and in vitro and significantly inhibited ROS production and activated integrin/ROCK signalling induced by tBHP in HTMCs. These results indicated that metformin reduced the elevated IOP in steroid-induced OHT mouse models and exerted its protective effects against oxidative injury by regulating cytoskeleton remodelling through the integrin/ROCK pathway. This study provides new insights into metformin use and preclinical evidence for the potential treatment of primary open-angle glaucoma. Intraocular pressure (IOP) elevation, predominantly resulting from increased resistance to aqueous humour outflow (AHO), is a major risk factor for primary open-angle glaucoma (OAG) deterioration . The onlIOP elevation is associated with TM stiffness . TheoretMetformin (MET), an oral biguanide insulin-sensitising drug, is the most widely used treatment for type 2 diabetes mellitus (DM) . It is a+ monocytes, CD3+ T cells, CD19+ B cells, and CD56+ NK cells) (Excessive reactive oxygen species (ROS) in TM play an important role in the disruption of cytoskeletal integrity and apoptosis , leadingK cells) . ConversK cells) , osteosaK cells) , and breK cells) . These sK cells)  and topiK cells) . The resThe experiment included two steps: first, OHT modelling and the subsequent drug’s IOP-lowering effect test were performed, both of which lasted for 28 days. A steroid-induced OHT mouse model was successfully established bilaterally in the first step. The baseline IOP did not differ between dexamethasone (DEX)-treated and phosphate-buffered saline (PBS)-treated vehicle eyes; however, starting at day 5, IOP was significantly elevated in DEX-treated eyes , DEX + PBS , and DEX + 0.3% MET . Meanwhile, a group of mice were treated with solo PBS as control with a duration of 8 weeks .In this step, successfully DEX-induced OHT mouse models were randomly assigned into three groups with the continued prescription of DEX: DEX withdrawal . Western blotting (WB) showed that the expression of myocilin, a glucocorticoid-inducible gene in HTMCs, increased after DEX treatment , confirmHigher doses (≥10 mM) of MET decreased the number of HTMCs, but this inhibitory effect was less evident at lower doses (<10 mM) . To veriFurthermore, cells treated with L-MET showed less intracellular ROS signals compared to the control, and 1 mM MET reduced tBHP-induced ROS production. These results were confirmed by flow cytometry (FCM) analysis  and furtActin filaments play key roles in cortical polarisation and asymmetric spindle localisation during phagocytosis in HTMCs. F-actin was stained to determine whether actin dynamics were involved in tBHP-induced cellular dysfunction. As shown in The ultrastructure of HTMCs was examined by transmission electron microscopy (TEM) . We obseThe major observations of the current study were the three significant phenotypic changes induced by MET in OHT mouse eyes and HTMCs. The first was the IOP-lowering effect in steroid-induced OHT mouse eyes. The second was reversal of the skeletal destruction of TM cells, both in vivo and in vitro. Third, there was a significant decrease in the accumulation of fibrotic markers, namely α-SMA and FN, in TM tissues. These results suggest a protective effect of MET in TM, probably via promoting cytoskeleton recovery through the integrin/ROCK pathway.Steroid-induced OHT in mice was generated  with theTM tissues play an important role in the regulation of AHO function and IOP . TM cellMoreover, as evidenced by immunofluorescence (IF) and WB analyses, L-MET rearranged the disordered cytoskeleton of HTMCs. This finding was supported by a study conducted by As confirmed by WB analysis, complementation with L-MET treatment unregulated integrin, pAMPK, ROCK, and F-actin signals. These in vitro findings indicate that MET might elicit its protective effects through the regulation of the integrin/ROCK pathway in HTMCs. The in vivo observations in the OHT model again support the importance of the integrin/ROCK pathway in TM tissue protection. Generally, these signals mediate cellular biomechanical tension through actomyosin cytoskeletal tension, ECM synthesis, assembly, and degradation .Inconsistent with the previous studies indicating that the AMPK activator suppressed ROCK activity, our study revealed a novel mechanism of MET action within which activation of both AMPK and ROCK signals sequentially upregulated F-actin expression in TM. We hypothesised that ROCK regulators protect TM via different mechanisms, either by inhibiting ROCK signals when increased AHO resistance is attributed to TM stiffness or by activating the ROCK pathway to promote cytoskeletal formation when it is damaged. In the current study, we presumed that MET lowered IOP through short-term changes in TM cell morphology and adhesion by regulating integrin/ROCK signalling. In addition, the steroid-induced OHT recovered its normal IOP with MET treatment, preventing further TM cell loss, whereas MET promoted the maintenance of cytoskeletal morphology and repair after injury; therefore, IOP was maintained at a normal level for an extended period.Generally, these data emphasise the protective properties of MET in the conventional outflow pathway, consistent with a large body of literature on other tissues . In addiWhile experimental data were encouraging, there were limitations in our study. First, the concentration of MET in the anterior chamber was not measured. Additionally, we did not investigate its toxicity on ocular surfaces, such as tear film stability and components, which may change with the application of MET. Second, the sample size of the animals was relatively small, and a larger sample is needed to verify the protective effect of MET on TM. Third, the experimental period was too short to prove the long-term effects of MET and the presented figures were not in the same direction. Thus, additional studies are needed in the future.In summary, promotion of TM cytoskeleton recovery by MET treatment may be an underlying mechanism for IOP reduction and AHO increase. A thorough investigation of the mechanisms by which cytoskeleton recovery is promoted will improve our understanding of the therapeutic mechanism of MET. If MET is confirmed to promote damaged cytoskeleton recovery and maintenance in the human body, it may have clear therapeutic effects on the main sites of glaucoma pathogenesis, which will broaden their application in the field of glaucoma. In conclusion, we revealed that integrin/ROCK activation reprograms metabolism in TM cells by enhancing the cytoskeleton and downregulating excessive ECM proteins.All the animals were treated in accordance with the principles of the Declaration of Helsinki and in compliance with the Association for Research in Vision and Ophthalmology (ARVO) Statement. All experiments were approved by the Institutional Animal Care and Use Committee of the Wenzhou Medical University (wydw2022-0209). Healthy C57BL/6J mice  were used in this study. The mice were purchased from Zhejiang Vital River Experimental Animal Technology Co. Ltd (Charles River Lab China), bred/housed in clear cages, and kept in housing rooms at 21°C with a 12:12 hr light:dark cycle.n = 10), DEX + PBS , and DEX + 0.3% MET . Mice in groups 3 and 4 received an additional 4 weeks of supplemented drug (PBS or 0.3% MET) delivered bilaterally with the continued synchronous use of steroid, leading to an entire 8-week duration of DEX treatment. Meanwhile, PBS-only treatment was administered for 8 weeks in the control . Drugs were prescribed twice daily with at least 15-min interval when administering 2 types of eye drops to ensure effective penetration.The animal experiments were performed in two consecutive steps. In the first step, OHT C57BL/6J mouse model was induced by topical 0.1% DEX phosphate  as described previously , and steBriefly, mice were anaesthetised using gaseous isoflurane (approximately 2 min) and topical alcaine . IOP was measured using a rebound tonometer . Each recorded IOP was the average of five measurements and three IOP readings were recorded for the same eye to calculate the mean value. IOP measurements were conducted every 2–3 days .The anaesthetised mice were gently placed on digital electronic scales  to measure their weight. The effective reading of BW was recorded to an accuracy of 0.01 g. The BW of each mouse was calculated from the average of three test values.2. To identify the characteristics of HTMCs, cells were treated with 500 nM DEX  for 7 days, and the expression of myocilin was evaluated by WB , which were authenticated by STR profiling. We confirmed that no mycoplasma contamination was detected by regular examinations. Cells were cultured in Dulbecco’s Modified EagIe’s Medium: F-12 (DMEM/F12)  containing 10% foetal bovine serum  and antibiotics  at 37°C and 5% COed by WB . For druCell viability was measured using the CCK8 assay kit (APE BIO), following the manufacturer’s instructions. The cells were seeded in 96-well plates and exposed to increasing concentrations of MET  for 24 hr. The cell viability was determined by measuring the optical density at 490 nm using an absorbance microplate reader .Intracellular ROS levels were determined using a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe  by measuring the oxidative conversion of cell-permeable DCFH-DA to fluorescent dichlorofluorescein according to the manufacturer’s instructions and a previous study . BrieflyCells were lysed 24 hr after drug treatment as previously described . After gAt the time of harvest, the mice were re-anaesthetised; the eyes were fixed in 4% paraformaldehyde overnight and embedded in paraffin or optimum-cutting temperature compound in the sagittal axis. The sections were incubated overnight with primary antibodies at 4°C according to the manufacturer’s instructions . The secThe cells were fixed with 4% paraformaldehyde for 15 min, permeabilised with 0.25% Triton X-100 for 20 min, and blocked with bovine serum albumin for 30 min at room temperature. After blocking, they were incubated overnight with primary antibodies at 4°C. The next day, the cells were rinsed and incubated with secondary antibodies conjugated to Alexa Fluor (CST) for 1 hr at room temperature. Fluorescent images were obtained using an inverted microscopy  with the luminescence of X-Cite Series 120.For electron microscopy studies, cells were fixed in 2.5% glutaraldehyde  and embedded in Epon resin, and 80-nm sagittal thin sections were cut through iridocorneal tissues using an ultramicrotome . Sections were stained with uranylacetate/lead citrate and examined with a transmission electron microscope .Immunohistochemical images were obtained using a microscope  and analysed using the NIS-Elements Imaging Software . For quantification, high-power fields (×400 magnification) of the AHO from each model were captured. TM region was defined as the area between the ciliary body and the inner wall of Schlemm’s canal, with anterior and posterior end points of Schwalbe line and scleral spur, respectively . For mict-test and analysis of variance were used to test the differences in continuous variables. A paired t-test was used to compare IOP changes from the baseline. Statistical significance was set at p value <0.05.Data analysis was conducted using SPSS (version 22.0) software and GraphPad Prism (version 7.0). Cell- and animal-based experiments included at least three biological and technical replicates. Continuous data are summarised as mean ± standard deviation. Student’s  The claims that metformin reduced elevated intraocular pressure in vivo and regulated cytoskeleton remodeling in vitro are supported by convincing data. This study provides a new direction for further exploration toward the treatment of primary open-angle glaucoma. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, including Mishaela R Rubin as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and Mone Zaidi as the Senior Editor.Thank you for submitting your article "Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Please provide control data2) Please provide information about animal numbers, sexes, biological repeats, and technical repeats3) Please provide information about the duration of dex treatment4) Please consider providing data using dex-treated cells to detect a metformin anti-oxidant effectReviewer #1 (Recommendations for the authors):In the Introduction, consider removing sentences about the cost and availability of other ROCKi, as these are specific to a geographical location and distracting from the scientific rationale.In Figure 2B, it looks like IOP might be rising in the MET groups towards the last few days. If possible, please provide data on the mice beyond D42.There should be mouse control that was not steroid-treated. This would allow comparison of the parameters in the metformin-treated mice with controls.The authors state (line 160) that the inhibitory effect of MET was not evident at lower doses, but in Figure 4C it looks like 3 mM decreased the number of HTMC.What were the ROS signals that were measured by FCM ?In Figure 5, ROCK did not increase statistically in tBHP+MET at any dose, but the text states otherwise (line 192).In Discussion, line 270, the wording is confusing- "recovery to normal IOP prevents further TM cell loss, whereas metformin…." Does this mean that IOP mice recovered in the absence of metformin?Reviewer #2 (Recommendations for the authors):Although the presented results were promising with translational potential in the future, there are some concerns as listed below:1. The animal numbers and sexes in each group and the biological repeats and technical repeats were not clearly listed which must be listed in the manuscript as well as in the figure legends.2. Page 2 Abstract: Methods: Subsequent TEM tests were not mentioned.3. Page 4 lines 64-65: "Theoretically, TM-targeting drugs are potentially effective in lowering IOP caused by diseased TM." This is not clear what the authors meant. Please modify and add references.4. Page 5 Introduction: Please briefly introduce the effect of MET on ROS.5. For all the sections with immunofluorescent staining, it's not clear how the authors defined the TM region. They didn't show any specific marker expression and didn't have the same direction as all the figures.6. Figure 1. How did the authors calculate the a-SMA and TGFb positive cells, especially TGFb is an ECM marker7. How long did Dex treatment last? Was it stopped at 3 weeks or was it continued together with metformin treatment? If continued, how did they give the different treatments? Please describe those in detail. If Dex was not continued, IOP could possibly reduce in 3 weeks. Please compare if IOP in PBS group has statistically changed or not with that when Dex stropped.8. Figure 2, several other control groups should also be compared, like PBS only, PBS+metformin eyedrops, and PBS+metformin oral without Dex treatment.9. Besides the in vitro tBHP induced oxidative cell model, it would be more convincing if the authors could also use Dex-treated cells to detect the metformin anti-oxidation effect since they used Dex induced mouse model.10. Figure 4, how many biological repeats and technical repeats? One of the characteristics of TM cells is that TM cells have increased expression of myocilin after exposure to Dex. Figure 4B didn't show myocilin expression increasing after Dex.11. Fig6, it's not clear what the authors marked. How did they mark the microfilaments? A statistical analysis with graphs would be more clear and convincing.12. The discussion of this study lacks further elaboration. There was no mention of the limitation of the study. Essential revisions:1) Please provide control data.Thank you for this important suggestion. We have conducted a repeated experiment during the submission period and have extended both the OHT modelling process and drugs’ IOP-lowering effect tests from 21 to 28 days to strengthen the power of this study. Metformin oral group was deleted because of the unstable intake amount, which may reduce the reliability of the results. New data, including the control data, have been added to the manuscript. The main points are summarised as follows:Materials and methods “OHT animal model and drug treatmentsLi, et al.,2021), and sterile PBS was used as a vehicle control. The second step was conducted 4 weeks after steroid induction. The successfully created OHT mouse models with elevated IOP were randomly divided into three groups: DEX withdrawal , DEX+PBS , and DEX+0.3% MET . Mice in groups 3 and 4 received an additional 4 weeks of supplemented drug (PBS or 0.3% MET) delivered bilaterally with the continued synchronous use of steroid, leading to an entire 8-week duration of DEX treatment. Meanwhile, PBS-only treatment was administered for 8 weeks in the control . Drugs were prescribed twice daily with at least 15-min interval when administering 2 types of eye drops to ensure effective penetration.”The animal experiments were performed in two consecutive steps. In the first step, OHT C57BL/6J mouse model was induced by topical 0.1% DEX phosphate  as described previously “MET effectively reversed steroid-induced OHT in miceIn this step, successfully DEX-induced OHT mouse models were randomly assigned into three groups with the continued prescription of DEX: DEX withdrawal , DEX+PBS , and DEX+0.3% MET . Meanwhile, a group of mice were treated with solo PBS as control with a duration of 8 weeks .p < 0.05). After 5 days of MET treatment, IOPs significantly reduced in group 4 . In fact, MET almost completely neutralised steroid-induced OHT, returning IOP to near DEX-withdrawal levels on day 21, suggesting a therapeutic role of MET in OAG. Specific IOP values are shown in Figure 2B.”The experimental procedure is illustrated in Figure 2A. The IOPs after 28 days of steroid induction in all three OHT groups were similar . Compared with group 3, DEX-withdrawal mice had significantly reduced IOP after 17–28 days , ## p < 0.01 (comparison between groups 2 and 4), △p < 0.05 and △△p < 0.01 (comparison between groups 1 and 4). DEX: dexamethasone, MET: metformin, PBS: phosphate buffered saline, IOP: intraocular pressure, OHT: ocular hypertension”“MET attenuated the steroid-induced TM cytoskeletal destruction in vivoAs shown in Figure 3, MET improved fibrosis and the intensity of phalloidin labelling of F-actin in the TM tissue of steroid-induced OHT C57BL/6 mice. Quantitative comparison showed a significant difference in the number of α-SMA- and fibronectin (FN)-positive TM cells between MET-treated and control mice . Additionally, MET treatment promoted the cytoskeleton recovery of steroid-induced TM cell damage, as confirmed by the pronounced upregulation of F-actin . We concluded that the IOP-lowering effect of MET in this steroid OHT model can be largely explained by the attenuation of fibrotic alterations and rearrangement of the cell skeleton at sites of TM or trabecular outflow pathways.”p < 0.05, ns: non-significance, DEX: dexamethasone, HE: haematoxylin and eosin; MET: metformin, PBS: phosphate buffered saline. White arrows indicate the representative positive cells.”Figure 3. MET decreased the expression of fibrotic markers in steroid-induced trabecular meshwork stiffening in mice. A-B. Representative images of HE (A) and fibrotic markers (B). C. Quantification of α-SMA of the models. D. Quantification of TGF-β of the models after 4 weeks of DEX withdrawal or MET/PBS treatment. E. Quantification of fibronectin (FN) of the models after 4 weeks of DEX withdrawal or MET/PBS treatment. F. Quantification of F-actin of the models after 4 weeks of DEX withdrawal or MET/PBS treatment. *2) Please provide information about animal numbers, sexes, biological repeats, and technical repeats.Thanks for the suggestion. We have added the animal number in the manuscript . The animal sexes were shown in p. 10, Line 4-5: “ Healthy C57BL/6J mice  were used in this study”. Biological and technical repeats were shown in p. 13, Line 21-22: “Cell-based and animal-based experiments included at least three biological and technical replicates.”3) Please provide information about the duration of dex treatment.Thanks. We have added the information as follows :“Mice in groups 3 and 4 received an additional 4 weeks of supplemented drug (PBS or 0.3% MET) delivered bilaterally with the continued synchronous use of steroid, leading to an entire 8-week duration of DEX treatment.”4) Please consider providing data using dex-treated cells to detect a metformin anti-oxidant effect.Thanks for the suggestion. We have conducted the DEX-treated HTMC to evaluate the anti-oxidant effect of metformin. We found that after DEX treatment, the DCF fluorescence intensity was significantly increased in HTMCs, and this effect was attenuated by the additional treatment of low doses metformin. This part of result was presented as supplemental Figure 1 for Figure 4:p < 0.01, *p < 0.05, ns: non-significance. DEX: dexamethasone, MET: metformin, ROS: reactive oxygen species, HTMCs: human trabecular meshwork cells, L-MET: low doses of metformin (≤ 10 mM)”“Figure S1. L-MET attenuated the ROS production in HTMCs induced by DEX treatment. A. ROS signals of HTMCs were determined by labelling cells with DCFH-DA and photographed by an inverted phase-contrast microscopy. B. Quantification of DCF-positive cells in A. C. The relative ROS levels assayed by flow cytometry showed that 3mM MET reduced the ROS production of HTMC induced by DEX. **Reviewer #1 (Recommendations for the authors):In the Introduction, consider removing sentences about the cost and availability of other ROCKi, as these are specific to a geographical location and distracting from the scientific rationale.Thanks for the comments. We agree with the reviewer’s opinion and have deleted the above sentences from the text.In Figure 2B, it looks like IOP might be rising in the MET groups towards the last few days. If possible, please provide data on the mice beyond D42.Thank you for your advice. We have extended the experimental period of drugs’ IOP-lowering effect from 21 days to 28 days, and the new data have been provided in the manuscript .There should be mouse control that was not steroid-treated. This would allow comparison of the parameters in the metformin-treated mice with controls.Thank you for your advice. We have extended the experimental period of drugs’ IOP-lowering effect from 21 days to 28 days, and the new data have been provided in the manuscript .The authors state (line 160) that the inhibitory effect of MET was not evident at lower doses, but in Figure 4C it looks like 3 mM decreased the number of HTMC.Thanks. We have revised the manuscript accordingly :“Higher doses (≥10 mM) of MET decreased the number of HTMCs, but this inhibitory effect was less evident at lower doses (<10 mM) .”What were the ROS signals that were measured by FCM ?Thanks. Intracellular ROS levels were determined by measuring the oxidative conversion of cell-permeable DCFH-DA to fluorescent DCF. The ROS signals were referred to positive DCF cells analysed by FCM, and the explanation was added in the manuscript :Xu, et al.,2020). Briefly, 10 μM DCFH-DA solution diluted in serum-free DMEM/F12 medium was added and incubated with the cells, then they were kept in the dark for 20 min. Excess DCFH-DA was removed by washing the cells three times with serum-free cell culture medium. ROS was detected by both IF imaging and FCM , and analysed by Flow Jo (Flowjo X 10.0.7r2).”“Intracellular ROS levels were determined using a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe  by measuring the oxidative conversion of cell-permeable DCFH-DA to fluorescent dichlorofluorescein according to the manufacturer’s instructions and a previous study .Thanks for this important comment. We have replicated the experiment, analysed, and modified the text to match the Figure accordingly. Modified texts are shown as follows :“Furthermore, WB results  showed that L-MET significantly activated the integrin/ROCK pathway by upregulating integrin, ROCK, AMPK, and pAMPK in healthy HTMCs, and significantly activated integrin, ROCK, pAMPK, and F-actin in damaged HTMCs, and it was more pronounced in the 3 mM doses.”In Discussion, line 270, the wording is confusing- "recovery to normal IOP prevents further TM cell loss, whereas metformin…." Does this mean that IOP mice recovered in the absence of metformin?Thanks. We have made revision accordingly for a clearer and more accurate expression :“In addition, the steroid-induced OHT recovered its normal IOP with MET treatment, preventing further TM cell loss, whereas MET promoted the maintenance of cytoskeletal morphology and repair after injury; therefore, IOP was maintained at a normal level for an extended period.”Reviewer #2 (Recommendations for the authors):Although the presented results were promising with translational potential in the future, there are some concerns as listed below:1. The animal numbers and sexes in each group and the biological repeats and technical repeats were not clearly listed which must be listed in the manuscript as well as in the figure legends.Thanks. We have added the animal number in the manuscript . The animal sexes were shown in p. 10, Line 4-5: “Healthy C57BL/6J mice  were used in this study”. Biological and technical repeats were shown in p. 13, Line 21-22: “Cell-based and animal-based experiments included at least three biological and technical replicates.”2. Page 2 Abstract: Methods: Subsequent TEM tests were not mentioned.Thanks. We have added the information as follows :“Transmission electron microscopy was performed to observe microfilaments in HTMCs.”3. Page 4 lines 64-65: "Theoretically, TM-targeting drugs are potentially effective in lowering IOP caused by diseased TM." This is not clear what the authors meant. Please modify and add references.Thanks for the suggestion. We have revised the text accordingly :“Theoretically, drugs promoting the recovery of damaged TM are potentially effective in lowering IOP.”4. Page 5 Introduction: Please briefly introduce the effect of MET on ROS.Thanks for this suggestion. We have summarised the effect of MET on ROS as follows :Ota, et al.,2009), vestibular cells , and human immune cells  . Conversely, there is also evidence on cellular ROS level increase in some cancer cells after MET treatment, including AsPC-1 pancreatic , osteosarcoma , and breast cancer cells . These seemingly contradictory results suggested that MET plays different roles under different metabolic environments.”“Reduction of intracellular ROS levels by MET via activating AMPK signal has been reported in primary hepatocytes , SC , CB (ciliary body), and representative positively stained cells in all immunofluoresence figures .Yan, et al.,2022).”“TM region was defined as the area between the ciliary body and the inner wall of Schlemm’s canal, with anterior and posterior end points of Schwalbe line and scleral spur, respectively :“ImageJ  was used to quantify the positively stained cells, which referred to those cells stained by both blue color from DAPI and green or red signals from the secondary antibodies.”7. How long did Dex treatment last? Was it stopped at 3 weeks or was it continued together with metformin treatment? If continued, how did they give the different treatments? Please describe those in detail. If Dex was not continued, IOP could possibly reduce in 3 weeks. Please compare if IOP in PBS group has statistically changed or not with that when Dex stropped.Thanks. Please refer to “Essential revisions 1”.8. Figure 2, several other control groups should also be compared, like PBS only, PBS+metformin eyedrops, and PBS+metformin oral without Dex treatment.We appreciate your comments. We have added the control groups according to your suggestion. Please refer to “Essential revisions 1”.9. Besides the in vitro tBHP induced oxidative cell model, it would be more convincing if the authors could also use Dex-treated cells to detect the metformin anti-oxidation effect since they used Dex induced mouse model.Thanks for this important suggestion. Please refer to “Essential revisions 4”.10. Figure 4, how many biological repeats and technical repeats? One of the characteristics of TM cells is that TM cells have increased expression of myocilin after exposure to Dex. Figure 4B didn't show myocilin expression increasing after Dex.Thanks for the suggestion. We have added the related information  and changed the Figure 4B to a representative one in the manuscript.11. Fig6, it's not clear what the authors marked. How did they mark the microfilaments? A statistical analysis with graphs would be more clear and convincing.Thanks for your comments and suggestions. We have marked representative microfilaments with larger magnified (150000×) figures embedded in Figure 6 and added statistical analysis according to your advice. The related methods and results have been shown in the manuscript :Svitkina,2020) and the remaining three were beyond this area.”“For microfilament quantification, five fields with 80000× magnification of each group were captured with TEM, within which two were from the cell cortex region (defined as within 400 nm beneath the cell membrane) .“While experimental data were encouraging, there were limitations in our study. First, the concentration of MET in the anterior chamber was not measured. Additionally, we did not investigate its toxicity on ocular surfaces, such as tear film stability and components, which may change with the application of MET. Second, the sample size of the animals was relatively small, and a larger sample is needed to verify the protective effect of MET on TM. Third, the experimental period was too short to prove the long-term effects of MET and the presented figures were not in the same direction. Thus, additional studies are needed in the future.”References:Front Immunol 12:581799. doi:10.3389/fimmu.2021.581799Hartwig J, Loebel M, Steiner S, Bauer S, Karadeniz Z, Roeger C, Skurk C, Scheibenbogen C, Sotzny F. 2021. Metformin attenuates ROS via FOXO3 activation in immune cells. Clin Exp Otorhinolaryngol 7:286-294. doi:10.3342/ceo.2014.7.4.286Lee JY, Lee SH, Chang JW, Song JJ, Jung HH, Im GJ. 2014. Protective effect of metformin on gentamicin-induced vestibulotoxicity in rat primary cell culture. JNK signaling pathway in human osteosarcoma. Int J Biol Sci 16:74-84. doi:10.7150/ijbs.33787Li B, Zhou P, Xu K, Chen T, Jiao J, Wei H, Yang X, Xu W, Wan W, Xiao J. 2020. Metformin induces cell cycle arrest, apoptosis and autophagy through ROS/ELife 10doi:10.7554/eLife.60831Li G, Lee C, Read AT, Wang K, Ha J, Kuhn M, Navarro I, Cui J, Young K, Gorijavolu R, Sulchek T, Kopczynski C, Farsiu S, Samples J, Challa P, Ethier CR, Stamer WD. 2021. Anti-fibrotic activity of a rho-kinase inhibitor restores outflow function and intraocular pressure homeostasis. Biochem Biophys Res Commun 388:311-316. doi:10.1016/j.bbrc.2009.07.164Ota S, Horigome K, Ishii T, Nakai M, Hayashi K, Kawamura T, Kishino A, Taiji M, Kimura T. 2009. Metformin suppresses glucose-6-phosphatase expression by a complex I inhibition and AMPK activation-independent mechanism. Trends Cell Biol 30:556-565. doi:10.1016/j.tcb.2020.03.005Svitkina TM. 2020. Actin cell cortex: Structure and molecular organization. Sci Rep 11:14002. doi:10.1038/s41598-021-93270-0Warkad MS, Kim CH, Kang BG, Park SH, Jung JS, Feng JH, Inci G, Kim SC, Suh HW, Lim SS, Lee JY. 2021. Metformin-induced ROS upregulation as amplified by apigenin causes profound anticancer activity while sparing normal cells. J Exp Clin Cancer Res 40:206. doi:10.1186/s13046-021-02012-7Yang J, Zhou Y, Xie S, Wang J, Li Z, Chen L, Mao M, Chen C, Huang A, Chen Y, Zhang X, Khan N, Wang L, Zhou J. 2021. Metformin induces ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer.


Antimicrobial stewardship (AMS) practices are well established in acute care, only recently expanding into ambulatory care settings. It has been estimated that 80-90% of antibiotic use occurs in the ambulatory setting with up to 52% of that use being unnecessary. Urinary tract infections (UTIs) are commonly treated in ambulatory settings, and drug selection and duration of therapy vary among different practices. Our study evaluated the impact of a system-wide guideline and prescriber education, two recommended AMS strategies, on antibiotic utilization for UTIs in the ambulatory setting.This retrospective study evaluated female adult patients prescribed an antibiotic for acute uncomplicated cystitis by a primary care provider at a clinic within our organization between January and March 2021. System-wide UTI treatment guidelines were implemented in November 2020, with live education delivered to primary care prescribers in December 2020. The primary objective of this study was to assess prescriber adherence to system-wide guidelines. The secondary objective was to evaluate utilization of antibiotic regimens for treatment of acute uncomplicated cystitis.A total of 100 patients were evaluated. Guideline adherence was met in 49% of patients. The primary reason for guideline non-adherence was duration of therapy, which was inappropriate (too long) in 42% of patients. Nitrofurantoin, the first-line antibiotic recommendation on the system-wide guideline, was the antibiotic prescribed most frequently (48%), but was the antibiotic of choice in 90% of patients. Sulfamethoxazole/trimethoprim was prescribed in 35% of patients and was most often prescribed correctly with respect to appropriate dose and duration of therapy (60%), followed by nitrofurantoin (54%). Recurrent cystitis within 30 days occurred in 11% of patients.Implementation of a system-wide guideline and prescriber education for treatment of UTIs in the ambulatory setting identified areas for future optimization of antibiotic use, namely first line antibiotic selection and duration of therapy. This data will be shared with our prescribers to continue to encourage guideline-adherent antibiotic use.All Authors: No reported disclosures.


This article presents the research on the mechanical characteristics of concrete in the construction of three concrete bridges. A system of recording the internal temperature of concrete and automatic control of laboratory ovens was used for specimen curing. This allowed the specimens to be cured under conditions similar to those occurring in the structure. Before the construction, reference blocks were used to define similar curing conditions. Maximum setting temperatures ranged from 47.6 °C to 62.0 °C and had a favorable effect on the properties of the concrete at an early age. For concretes with the use of CEM I cement, after 3 days of curing, the strength obtained was up to 8.2 MPa (23%) higher than that for specimens cured under standard conditions. The modulus of elasticity was higher up to 4.9 GPa (21%). For concrete with the use of CEM III cement, these differences were 26.9 MPa (174%) and 10.3 GPa (64%), respectively. After 7 days of curing, the results were close to each other and after 14, 28, and 56 days, higher values were obtained for specimens cured under standard conditions. The value of the modulus of elasticity of concrete was determined using the direct method according to Eurocode and the standard A method. A test load of the bridge was carried out to verify the modulus values obtained from laboratory tests. The highest consistency (99%) between the theoretical deflections and those measured in the test load was achieved when using the stabilized modulus values obtained on specimens cured under structure conditions in the FEM model. The research confirms the necessity of determining the mechanical characteristics of concrete with taking into account the curing conditions of concrete in the structure. A procedure for determining the mechanical properties of concrete for the correct construction of a bridge is proposed. These results can also be used in the development of a digital twin for bridge management. In the construction process of prestressed concrete bridges, time is an important factor in determining the final cost of construction. In order to reduce the construction time, contractors are pushing to prestress the superstructure as quickly as possible in order to disassemble the formwork or to proceed to the next segment in the case of incremental launching method or balanced cantilever method . To miniConcrete as a building material is characterized by a very wide variation in deformability ,10. The In Poland, the bridge constructors commonly use granite, granodiorite, porphyry, limestone, dolomite, amphibolite, and basalt aggregates extracted from many mines across the country. The tests carried out by the authors clearly present a wide variation in the modulus of elasticity of concrete depending on the type of aggregate used . The valLaboratory tests of concrete for the construction of bridges have to be performed on specimens cured in water at a controlled temperature of 20 °C ,22,23. MThese curing conditions in the laboratory do not reflect reality. Firstly, the concrete in the superstructure, unlike the specimens in the laboratory, does not have access to an unlimited amount of water. At the construction site, there is only water in the amount specified in the concrete mix recipe. Secondly, the temperature of the concrete in the superstructure during the setting process varies over time and far exceeds the 20 °C that occurs in the laboratory ,5. The aTests of the modulus of elasticity of concrete are performed according to the standard (method A or B) . In the The article presents the results of the research program identifying the mechanical properties of concrete on the example of three bridge structures recently built in Southern Poland: bridge MG-1 over the Oder River, viaduct WG-4 at the grade-separated junction along the provincial road that serves as a bypass for the city of Racibórz and bridge MD-1 over the Kłodnica River along the national road that serves as a bypass for the city of Kędzierzyn-Koźle. The aim of the research was to compare the results of tests on the mechanical properties of concrete carried out using different methods on specimens cured under standard and structure conditions, with the use of a system that allows concrete curing conditions in the superstructure to be replicated in the laboratory. In the case of the WG-4 viaduct, the laboratory test results were verified by in situ tests on its superstructure. Throughout the construction process, the displacements of the measuring points located on the superstructure were measured. The second stage of verification consisted in measuring the span deflections under test loads. The applied numerical models were used to analyze the deformability of the span .The purpose of the system is to replicate in the laboratory the actual concrete curing conditions occurring in the superstructure at the construction site, so that the test specimens are an authoritative representation of the material used in construction. These replicated conditions have been named structure conditions.The system consists of temperature sensors placed in the structure, the LB-480 recorder, the LBX server, and a laboratory oven. The temperature sensors are located inside the girder, which is the main superstructure element for the stiffness of the bridge. In the case of preliminary research, before construction begins, a reference block with dimensions similar to the girder is used .These sensors are connected via cables to a LB-480 recorder located at the construction site. The frequency of measurement is possible to determine; in the case of the described research, a measurement was taken once every 30 min. The recorder is connected to the GSM internet, and on its use sends data to the LBX server located in the laboratory. The laboratory oven is connected to the server, which automatically controls its settings according to the measurements at the construction site. All data and settings can be viewed and possibly changed by the LBX Client, which is also connected to the server. A diagram of the system is shown in In the superstructure, the exchange of water with the environment only occurs over a small area, so it is not an intensive process. To prevent intensive evaporation of water from the specimens in the laboratory oven, they are wrapped in stretch foil after demolding .The research was performed on the construction of three bridges. Three different concrete mixes were used. They all consist of CEM I cement, because it is forbidden in Poland to use cements other than CEM I for prestressed bridge structures. For the purpose of this research, a trial mix with the use of CEM III cement was prepared. The mix recipes are presented in The concrete pouring of the test blocks took place at different times of the year. The different weather conditions had a significant impact on the temperature of the concrete mixture at the construction site and the maximum temperature of the concrete during the setting process .The mechanical properties of concrete were determined on the basis of destructive tests of specimens cured under standard and structure conditions. The first part of the research is the preliminary research that takes place before construction begins. After the concrete mix recipe is approved, a reference block is made. The second part of the research is the tests carried out on the concrete used for the construction of the bridge.d = 150 mm and a height of h = 300 mm were prepared [For the analysis of the concrete mechanical properties, a minimum of 24 cylindrical specimens with a diameter of prepared . Half ofprepared . The chadirect method;method A.The analysis of the concrete modulus of elasticity was carried out using two methods:cmf—compressive strength of concrete;Δε—strain increase in the concrete induced by the stress of 0.4 cmf;Δl—change in specimen length due to stress of 0.4 cmf;l0—base length of concrete specimen.Direct method is proposed by the authors as an exact interpretation of the definition of the modulus of elasticity of concrete described in the Eurocode . The spec,oE and stabilized modulus c,sE. The specimens are subjected to cyclic loading and unloading, as shown in Method A, according to the standard , was usea,iσ—registered stresses at the level of 1/3 cmf;b,iσ—registered stresses at the level of 0.15 cmf;a,iε—registered strain of the concrete induced by the stress of a,Iσ;b,iε—registered strain of the concrete induced by the stress of b,iσ.In each of the three bridges, preliminary research using reference blocks was carried out before construction started. The concrete was made following the approved mix recipes. As a result, the actual mechanical properties of concrete as a function of age were determined. The obtained values of compressive strength and modulus of elasticity allowed to update the technological design. The preliminary tests were used to update the pre-camber and the prestressing program.The results of the laboratory tests are summarized in After 7 days of curing, the results of the concrete properties were similar for both types of specimens. In the following days, higher values of concrete properties were observed for specimens cured under standard conditions. The results indicate that the increase in the mechanical properties of concrete over time does not occur in the structure. Such differences in the results obtained depending on the way in which the specimens were cured confirm the need to take into account the curing conditions of the concrete in the structure in the laboratory tests.In addition, the research has proven that the actual mechanical properties of concrete with the use of CEM III cement used are characterized by a rapid increase in the first few days, similarly to concrete with the use of CEM I cement.The results confirm the relatively low value of modulus of elasticity of concrete with the use of granite aggregate and the relatively high value for concrete with the use of basalt aggregate.Concreting of the superstructure of the WG-4 bridge took place on November 26th, 2020. A mix of S3 consistency, air content 3.8%, and temperature of 12.3 °C was poured. The maximum temperature of 45.8 °C was reached 32 h after concreting the superstructure. During this research, the ambient temperature was approximately 0 °C ± 5 °C.The tests were carried out after 6 days of curing at the moment of prestressing the superstructure and additionally after 28 days. In both cases, the specimens were tested with the use of the direct method and the method A.The results of the laboratory tests are summarized in The results of the modulus of elasticity tests using the direct method are close to the initial modulus values from the tests carried out using method A . The stabilized modulus values, as expected, are higher.On 29 April 2021, 154 days from the concreting of the superstructure, the vertical displacements were measured under a static test load. The load of the bridge was four four-axle trucks with a total weight of 129.04 tons. The trucks were placed on the bridge in order to cause the maximum vertical displacement of the superstructure .Once the trucks were placed on the bridge, measurements of the displacements of both the superstructure and the supports were conducted. Measurements were taken every 10 min until the displacements were stabilized. The test was carried out before laying the wearing surface and utilities placement, which allowed to eliminate its influence on the stiffness of the superstructure .Numerical analysis of the superstructure was performed using the FEM program. The main girders were modeled using beam elements, while the deck slab and cross beams were modeled using shell elements .Numerical analyses were performed using different values of the modulus of elasticity: the standard value and those obtained from laboratory tests. The load of the trucks was modeled as pressure on the wheel-structure contact surface .cmE = 34.0 GPa), higher deflections were obtained than the values measured in the in situ tests. For the modulus of elasticity determined by the laboratory method A on specimens cured under standard conditions (cmE = 42.9 GPa), the theoretical deflections obtained were smaller than the values from in situ tests. A high level of consistency in the results was achieved when the stabilized modulus value from method A obtained for specimens cured under structure conditions (cmE = 38.6 GPa) was used in the numerical model. The deflection ratio ranged from 97.6% to 100.5%.initial value determined with the direct method —prestressing phase;final value determined with method A —useable phase.These values of deflection ratio indicate that concrete in an already prestressed structure has a higher value of modulus of elasticity than at the moment of prestressing. Furthermore, such high deflection consistency clearly presents that the results from tests carried out on specimens cured under structure conditions are close to the actual properties of the material built into the superstructure. The comparative study has confirmed the elastic work of the superstructure after eliminating the initial plastic deformations that occur at the moment of prestressing. Taking into account the prestressing phase and the service phase of prestressed bridges, it is necessary to determine two values of the modulus:The purpose of the research was to propose a procedure for concrete testing that would provide results that would allow a higher consistency between the deflections of the bridge spans measured during the test load and those obtained from the numerical model. The research used a system of recording the internal temperature of the concrete and automatic control of laboratory ovens, which made it possible to cure the specimens under conditions similar to those occurring in the superstructure. The results obtained from the laboratory tests were compared with the results of the test load of the bridge.A comparative study of the mechanical properties of concrete confirmed the beneficial effect of high concrete curing temperature on the early compressive strength and modulus of elasticity of concrete. Similar conclusions were reached in the studies ,7,8,12. A crucial issue in the construction process of prestressed bridges is the deformability of the concrete. This can vary significantly depending on the type of aggregate used ,11,16,17The research confirmed the operation of a prestressed concrete bridge in two phases. The first phase is the prestressing process of the superstructure, in which the initial plastic deformations occur. In this phase, the effectiveness of determining the modulus of elasticity of concrete using the direct method on specimens cured under structure conditions was confirmed. This approach resulted in a structure geometry that was consistent to within 1 cm of the designed grade line.The second phase is the usage phase of the bridge. By performing the test load of the structure without any equipment installed, the deformability of the concrete in the usage phase was determined. On this basis, a reduction in the deformability of the concrete  was confirmed, which is related to the elimination of plastic deformation. The determination of the modulus of elasticity using method A on specimens cured under structure conditions was considered authoritative.In addition, the results of tests on the mechanical properties of concrete with CEM III cement confirmed a rapid increase in the compressive strength and modulus of elasticity at an early age as in the case of concrete with CEM I cement, which allows the use of such concrete for the construction of prestressed bridges.The research confirmed the significant influence of concrete curing conditions on the development of its mechanical properties over time. Tests carried out on specimens cured under the conditions specified by the standard underestimate the values of the compressive strength and modulus of elasticity of the concrete at its early age relative to the actual values and significantly overestimate them in the long term.For the correct design and construction of the bridge, it is necessary to determine the modulus of elasticity using laboratory tests. The specimens for these tests should be cured under structure conditions close to the actual conditions in the structure, which differ significantly from those specified in the standard. In addition, for prestressed bridges, it is necessary to determine two values of the modulus of elasticity—with and without taking into account the initial plastic deformation.The results obtained from laboratory tests and verified by the test load should be the base for the creation of the mechanical twin that is part of the digital twin of the bridge ,29.Tests of concrete specimens with the use of a reference block and monitoring system after approval of the concrete mix recipe;cmE determined with the direct method on specimens cured under structure conditions at the age of scheduled prestressing;Update of the prestressing program and technological design of the superstructure using the modulus of elasticity value Deciding on the prestressing of the bridge based on the results of tests of the strength and modulus of elasticity of concrete on specimens cured under structure conditions;csmE determined with method A on specimens cured under structure conditions.In the analyses of the usage phase, the use of the stabilized modulus of elasticity of concrete Proposed procedure for determining the actual mechanical properties of concrete built into the superstructure of the bridge:


Nowadays, the re-use and recycling of industrial wastes to reduce the environmental impact and landfill problems are the main concerns of researchers. Plastics are one of the main waste materials worldwide, with considerable impacts on health and environmental conditions. Recycling plastic wastes in the concrete industry is one of the adopted ways to reduce such impact and increase the economic recyclability of plastics. In this study, the utilization of recycled polypropylene (rPP) as a fine aggregate in the preparation of cement mortars was evaluated. The river sand was replaced with 10, 20, 30, 40, and 50%, volumes of rPP. The results showed that the inclusion of rPP reduced the mortar’s workability and fresh density. Fresh density dropped from 11% to 35% as the rPP content increased. Furthermore, the compressive strength at early and late age was significantly influenced by the rPP content. At 28 days of curing age, the results showed that the inclusion of 50% of rPP in the mortar matrix led to a drop in the compression strength from 40 MPa to 10 MPa. A similar trend of results was obtained for the flexural (from 8.3 MPa to 2.9 MPa) and tensile strengths (from 3.4 MPa to 1.21 MPa). The chloride ion penetration went through a maximum of 5000 Coulombs between 10% and 50 % of rPP. Therefore, it can be concluded that the use of 10% of rPP as a river sand replacement can achieve acceptable strength (25 MPa) for several applications in the construction industry.  A significant increase in the use of plastic products globally has been witnessed in the past few decades. According to a report, the production of plastic materials in 2019 was approximated 3.7 × 10he ocean ,6.The re-use of plastics, particularly in construction, is gaining momentum, because it can potentially provide a solution to minimize environmental pollution by preventing plastic waste being dumped in landfills and incinerated. Studies suggest that plastic waste can be used in concrete as a partial substitute for sand and coarse aggregates, such as polyethylene terephthalate (PET) ,10,11,12Different researchers have studied different factors affecting the addition of polymer-filled mortars, including polymer content ,21,24,27The addition of plastic waste as a replacement produces different outcomes depending on the percentage of the replacement. In all the studies, it has been concluded that, with the addition of plastic waste, the unit weight declines significantly when increasing the replacement ,34,35,36In the application of polymer concrete, Yu et al.  incorporRecently the use of recycled polymers in geopolymer concrete has been under investigation . AbousniThe overall decrease in mechanical properties of plastic-filled mortar can be attributed to plastic properties, such as low elasticity and large molecules compared to sand aggregate. It also could be due to weak adhesion between plastics and cement, as plastics are hydrophobic ,43. AnotAlthough some properties of mortars are generally affected by plastic addition, studies emphasized that adding recycled plastics to mortars or concrete could assist some properties at certain levels to be used in non-engineering applications, while reducing plastic disposal and helping its sustainability ,27,31,45Most literature studies deal with PET and PE, with less focus on PP aggregate replacements; this paper aims to contribute to the studies of PP replacement. Thus, this work’s goal is to evaluate the effect of rPP as a fine aggregate replacement on fresh and hardened mortar properties, and obtain the optimum level of replacement to achieve the acceptable strength value. For this purpose, several tests are adopted, such as flow diameter, fresh density, compressive, splitting tensile, and flexural strengths, and chloride penetration. The next step is to extend this work to examine the effect of the local weather on concrete mixed with PP. This should benefit the local building and construction industry. It also should encourage the utilization of plastic waste.In this study, ordinary Portland cement (OPC), satisfying the ASTM C150 requirement for type I cement, was used to prepare the mortar specimens. The OPC was obtained from a local cement producer in Saudi Arabia and utilized as the primary source of calcium oxide. The rPP was purchased from the local market, with a particle size of no more than 4 mm and used as a fine aggregate to replace river sand.In preparing the mortar specimens, the mix design was very important in order to produce a batch of mortar with desirable properties. For the purpose of this study, mortar specimens as control samples were prepared using a cement to fine aggregate ratio (c/a) of 1:3 and cement to water ratio (c/w) of 2:1. As shown in In this work, the effect of rPP content on fresh and hardened performance of proposed mortars was evaluated in terms of flowability, density, compressive, flexural, and splitting tensile strength, and chloride ion penetration. The flowability of the proposed mortars was measured using a flow table method modified from ASTM C230 (Standard Specification for Flow Table for Use in Tests of Hydraulic Cement). The flow table provides an efficient means of determining the flow of cement pastes and hydraulic cement mortars. A specific volume of material was melted on the table using the included flow mold. The mold was then removed, and the table was subjected to a specific number of 1/2 in. (12.7 mm) drops using the crank handle. The increase in the average diameter of the sample indicated the flow. The compressive strengths (CSs) of the mortars were measured using ASTM C109/C109M-20b. Three mortar cubes for each category were prepared using steel molds of 50.8 mm a side (2-inch sides). The CSs of mortars mainly depended on a number of factors, which included the properties and proportions of the constituent materials, degree of hydration, rate of loading, method of testing, and specimen geometry. The properties of the constituent materials mainly affected the strength. The CS test method consisted of applying a compressive axial load to molded mortar cubes at a continuous rate without shock and within a prescribed range until failure occurred. Testing variables had a considerable influence on the measured compressive strength. Following the test procedures, the specimens were left to cure for 7, 28, and 56 days prior to the test. The cubes were placed in the testing machine to ensure that they were directly under the center of both the top and bottom plates. They were then loaded at a rate of 1.4 MPa per minute until failure. The failure load was recorded, and the cube was removed in preparation for the next test. The compressive strength of each cube was calculated as follows:The tensile strength of mortar is a fundamental material characteristic used to predict crack formation. This property is generally expressed as a function of the compressive strength of a material. The splitting tensile strength of mortar is typically determined with a split cylinder test in accordance with ASTM C348-21. The tensile strength affects other properties, such as stiffness, damping action, bond to embedded steel, and the durability of the mortar. The tensile strength is determined either with a direct tensile test or with an indirect tensile test, such as a flexural or split-cylinder test. It is often assumed that the direct tensile strength of a mortar is approximately 10% of its compressive strength. The diameters of the specimens were first marked using a pencil prior to the test. Then, the specimen was placed on its side on top of a bearing strip of plywood with a nominal thickness of 1/8 inches and an approximate width of 1 inch. The length of the bearing strip had to be the length of the specimen or slightly longer. Another bearing strip was placed on top of the cylinder. The cylinder was then loaded at a constant rate that ranged from 0.68 to 1.3 MPa per minute. The cylinder was tested to failure and the ultimate load was recorded. For each category, three cylinders were tested. The splitting tensile strength of a cylinder was calculated using the following equation:Flexural strength is the ability of a beam to resist failure in bending. The flexural strength is expressed as the modulus of rupture in MPa. The flexural strength is approximately 12 to 20% of the compressive strength. In the test, the three specimens for each category were subjected to concentrated loads at a third point in order to produce a pure bending movement. The test was conducted in accordance with ASTM C78. In the central-point loading, a beam of span L was loaded with a concentrated load P at the mid-span of the beam. The beam was supported as a simple beam. The deflection at the center of the span was:The chloride ion penetration test was conducted to study the effect of chloride ions in mortar specimens. This test was conducted at 28 days of curing age. The test for measuring the chloride diffusion was carried out using a cylindrical mortar specimen with a diameter of 100 and 200 mm thickness. This test was conducted following ASTM C 1202-97. Three samples were tested for each category.The optical microscopy technique was used to investigate the structure of the rPP- filled mortar. An optical microscope  was used, with magnification of ×8. Three-centimeter cube samples were cut by a special cutter to be used under the microscope.3. The replacement of aggregates with 10%, 20%, 30%, 40%, and 50% of rPP resulted in a decrease in unit weight, which was found to be 1916 kg/m3 (11%), 1786 kg/m3 (17%), 1608 kg/m3 (25%), 1478 kg/m3 (31%), and 1408 kg/m3 (35%), respectively. This drop in unit weight was because of the addition of rPP, which has a low bulk density and was thereby responsible for a decrease in the overall weight, which was in accordance with the results published by other researchers [The replacement of aggregate with rPP tended to lower the overall unit weight, as shown in 78 kg/m3 %, and 14earchers .3, while the unit weight of the sample with 30%, 40%, and 50% rPP replacement was found to be 1446 kg/m3 (27.30%), 1390 kg/m3 (30.11%), and 1297 kg/m3 (34.79%), respectively. The decrease in the unit weight of the same sample at intervals of 7 and 28 days was not significant. The decreasing weight with increasing rPP percentage was because of the lighter density of rPP plastics in comparison to the natural aggregates used in the reference sample. The results were in accordance with the findings of other researchers [earchers ,19,33.With the addition of rPP in various percentages, the compressive strength of the prepared concrete was measured at the age of 7 days and 28 days, as shown in The flexural strength for the prepared concrete with different percentages of rPP is shown in Splitting tensile strength was used to determine the tensile strength of concrete using a cylindrical shape specimen, which split across the vertical diameter. At the age of 28 days, the prepared concrete samples were subjected to a splitting tensile test. The results are shown in In order to analyze the mixing of rPP with concrete, an optical microscopy analysis was performed on samples with 20% and 50% rPP, as shown in Samples with 20% rPP possessed clear voids or air pockets around the rPP pellets a. With aThe penetration of chloride ions in concrete is influenced by factors such as the absorption of water, the degree of porosity, and concrete’s water permeability, as a more compact structure results in a lesser chloride ion penetration. The addition of plastic waste to concrete caused it to be prone to void formation due to the lack of adhesion between the concrete and plastic waste. The graphical presentation of the results obtained from concrete prepared with different percentages of plastic waste is shown in The replacement of natural fine aggregates with rPP led to a reduction in the workability of the prepared mortars.The fresh and hard unit weight of the proposed mortars tended to decrease with an increasing rPP content in the mortar matrix as a fine aggregate replacement. At a 50% replacement, the loss in the unit weight was approximately 35%.Compressive strength of the prepared mortar was negatively influenced by the rPP content. The results showed that the mortar prepared with 50% of rPP aggregates lost 75% of its strength. However, the specimens containing 10% rPP showed acceptable compressive strength for the construction industry.The reduction in the flexural strength was up to 65% at a 50% replacement.A similar trend of results was observed for flexural and splitting tensile strengths, and the strength values tended to decrease with an increasing rPP content. Due to a 50% of replacement, the reduction in the tensile strength was 67.6%.The durability of the mortar was significantly influenced by the rPP content. The chloride ion penetration value tended to increase with an increasing rPP content.Overall, the recycled-plastic-incorporated mortars showed a good potential for specific non-bearing lightweight construction applications while reducing the environmental impacts of plastic wastes. For example, they could be used in pavements and non-bearing loading walls.In this work, the possibility of producing sustainable construction materials using waste plastics, such as rPP, as a river sand replacement was explored. From the obtained results, it can be concluded that replacing more aggregates with rPP led to a higher reduction in mechanical properties. The following conclusions were derived:


No consensus exists whether to administer prophylactic antibiotics in conjunction with manual placenta removal. This study aimed to investigate the postpartum risk of a new prescription of antibiotic treatment, a possible indirect variable for infection, after manual placenta removal.n = 13 877) at Helsingborg Hospital, Helsingborg, Sweden, from January 1st, 2014 until June 13th, 2019 were included. Diagnosis codes for infection can be lacking, while the Anti-Infection Tool is complete as it is unavoidable in the computerized prescription system. Logistic regression analyses were performed. The risk of a prescription of antibiotics 24 h to 7 days postpartum was analyzed in the entire study population, and in a subgroup of women not having received any antibiotics 48 h prior to delivery until 24 h after delivery, referred to as “antibiotic-naïve.”Obstetric data were merged with data from the Anti-Infection Tool (Swedish antibiotic registry). All vaginal deliveries  OR = 2.9 (95%CI 1.9–4.3). In the antibiotic-naïve subgroup, manual placenta removal was associated with an increased risk of antibiotic prescription, in general, aOR = 2.2 (95%CI 1.2–4.0), endometritis-specific antibiotics, aOR = 2.7 (95%CI 1.5–4.9), and intravenous antibiotics, aOR = 4.0 (95%CI 2.0–7.9).Manual placenta removal is associated with an increased risk of antibiotic treatment postpartum. An antibiotic-naïve population might benefit from prophylactic antibiotics to reduce the risk of infection, and prospective studies are needed. Retained placenta occurs after approximately 2% of deliveries. Risk factors include previous placenta retention, prolonged labor, preterm delivery, and multiple pregnancies –4. PlaceThe aim of the study was to assess the postpartum risk of a new prescription of antibiotics, as a indirect variable of infection and unavoidable to be registered in the computerized prescription system, in patients having undergone MPR compared with spontaneous placental delivery.n = 13 877) giving birth vaginally at Helsingborg Hospital from January 1st, 2014 until June 13th, 2019 were included in the study. Women with multiple gestation were only included once.All women  and the nationwide database recording all antibiotic prescriptions (Anti-Infection Tool) , 12. RegGestational age was calculated using estimated date of delivery at the first ultrasound and date of delivery.ICD-10 procedure codes [Antibiotics were classified according to the Anatomical Therapeutic Chemical (ATC) classification system  : AntibiProphylactic antibiotics in conjunction with MPR are not recommended. Postpartum endometritis is treated with a combination of cephalosporins and metronidazole, oral or intravenous. Mild cases are treated with amoxicillin and enzyme inhibitors.The main end point of the study was a new prescription of antibiotics 24 h to 7 days postpartum. Multiple antibiotic prescriptions to one and the same patient within this time slot were recorded as one prescription. Analyses were also performed focusing on endometritis-antibiotics and intravenous antibiotics.n = 300, 3.2% in the MPR group, 2.1% in the reference group). Erythromycin is routinely prescribed in cases of preterm premature rupture of membranes prior to 35 weeks of gestation and used as an indirect variable. Multiple gestation (n = 102) was not included in the multivariate analyses as none of these women received postpartum antibiotics.Fourteen potential confounding variables for postpartum antibiotic prescription were identified: Maternal age (years), body mass index , primiparity (yes/no), multiple gestation (yes/no), gestational age (weeks + days), smoking during pregnancy (yes/no), induction of labor (yes/no), epidural anesthesia (yes/no), prelabor rupture of membranes , vacuum extraction (yes/no), third grade perineal tear (yes/no), fourth grade perineal tear (yes/no), prelabor erythromycin prescription (yes/no), peripartum antibiotics (defined as having received a new antibiotic prescription in the time period 48 h prior to until 24 h after delivery). PROM was defined using ICD-diagnoses O420, O421, O422 and O429  was calculated from rates and adjusted odds ratios (OR) [P < 0.05 was regarded as statistically significant.Statistical analyses were performed using IBM SPSS Statistics for Macintosh, Version 25.0 . ios (OR) . P < 0.0n = 349) of vaginal births. MPR was significantly associated with increased maternal age, primiparity, induced labor, third and fourth grade perineal tear, an increased use of epidural anesthesia, vacuum extraction, and a longer hospital-stay. Women having undergone MPR lost more blood and blood transfusions were more common.Manual placenta removal was performed in 2.5% (n = 6) of women were diagnosed with endometritis, compared to 0.2% (n = 22) of women with a spontaneous placenta delivery. Endometritis (P < 0.001), puerperal fever (P < 0.05) and unspecified postpartum infection (P < 0.001) were more common in women having undergone MPR.In the MPR group, 1.7% , MPR was associated with an increased risk of antibiotic prescription 24 h to seven days postpartum; crude OR = 2.6 (95% CI 1.5–4.5); adjusted OR = 2.2 (95% CI 1.2–4.0) (Table In the antibiotic-naive subgroup (n = 86) were prescribed peripartum antibiotics (48 h prior to until 24 h postpartum). In this group, a greater share of women, who were prescribed peripartum antibiotics, were prescribed antibiotics the first week postpartum as well (16.3%), compared to women not having received peripartum antibiotics (5.3%).Approximately 25% in the MPR group , nor in the antibiotic-naïve subgroup, adjusted OR = 1.9 (95% CI 0.8–4.5).In the antibiotic-naïve subgroup, for every 34 women undergoing MPR, one additional woman was prescribed intravenous antibiotics 24 h to seven days postpartum, compared with the reference population (NNH = 34). When calculated based on adjusted OR, NNH was 42.Our study including 13,877 deliveries showed that MPR was associated with an increased risk of postpartum antibiotic prescription, a proxy variable of infection. The incidence of MPR was 2.5% and MPR was linked with a high-risk pregnancy. This is in line with previous findings [The high frequency of peripartum antibiotics prescribed in the MPR group would be a complicating factor in a randomized controlled trial (RCT). The antibiotic-naïve subgroup represents the women who could gain from prophylactics in conjunction with MPR. In this group, MPR was associated with a fourfold increased risk of being prescribed intravenous antibiotics, suggesting more serious infections.As many as 25% undergoing MPR received peripartum antibiotics, and it was more common with a postpartum antibiotic prescription among women receiving peripartum antibiotics. This suggests that women receiving peripartum antibiotics were a high-risk group, receiving peripartum antibiotics as well as a new prescription of antibiotics postpartum.n = 390) than received an ICD-diagnosis of infection (n = 289), suggesting that ICD-codes could underestimate the rate of infection. With the aim of performing research based on everyday clinical judgment, we hypothesized that a prescription of antibiotics was a valid end point for infection. A similar design was applied in the ANODE study, an RCT assessing the effect of prophylactic antibiotics after operative vaginal delivery [Previous research has defined postpartum endometritis based on documented symptoms and/or ICD-diagnoses , 7–9. Hodelivery .Difficulties exist in distinguishing different indications for antibiotic treatment near delivery. Underlying reasons could be preexisting infection, prophylaxis , or treatment of endometritis which usually develops within 48 h postpartum . FurtherIf antibiotic prescription is to be a successful tool to measure infection, it is essential that antibiotics are not prescribed in a facile manner. Due to increasing antibiotic resistance, a restrictive policy of antibiotic use has been evolved in Sweden . AccordiBlood loss was not included in the multivariate analyses as it is not a confounding variable, being on the pathway between MPR and infection, and a direct effect of the exposure. In a sub-analysis in which blood loss was adjusted for, the risk of intravenous antibiotics after MPR was not significant. Our results support previous studies showing an association between anemia and postpartum infection , 8, 20. The WHO recommends prophylactic antibiotics after MPR, although concludes that the level of evidence is low . SwedishOur results support findings that manual placenta removal is associated with an increased risk of postpartum infection , 8. Furt


Desulfurization slag (DS) is the solid waste discharged from the bottom of the circulating fluidized bed (CFB) boiler. It has good pozzolanic activity, self-hardening property and large expansibility. In this paper, ground desulfurization slag (GDS) is used as mineral admixture to replace cement to prepare self-compacting concrete (SCC). In order to find out the influence laws of different factors on the axial compressive properties of the self-compacting concrete filled steel tube (CFST), seven types of SCC are prepared and nine groups of CFST short column are fabricated. Filling ability test, compressive strength test and axial compressive test are performed. The filling ability and the compressive strength of the SCCs are investigated, and the axial compressive properties of the CFSTs are researched. The results show that the amount of polycarboxylate superplasticizer (PS) increases with the amount of GDS, and the addition of GDS decreases the 3d, 7d and 28d compressive strength of the SCCs. The optimum amount of GDS for SCCs and CFSTs is 30%. When the amount of GDS is 30%, the ultimate bearing capacity of CFST short column (GP3) is the highest, which is 33.6% higher than that of GP1 without GDS. The influence law of the GDS’s amount on the CFSTs’ ultimate bearing capacity is quite different from that of the GDS’s amount on the SCCs’ compressive strength. The ultimate bearing capacity of CFSTs can be significantly improved by adding GDS. Sodium sulfate can improve both the compressive strength of the SCC and the bearing capacity of the CFST. Self-compacting concrete (SCC) refers to the concrete with great fluidity, good uniformity and stability, which can flow by virtue of its own weight and fill the whole formwork space without vibration during pouring. Compared with ordinary concrete, fresh SCC has the properties of good filling, clearance passing and segregation resistance . After hConcrete filled steel tube (CFST) refers to the composite component formed by pouring concrete into the steel tube. It is a new type of composite structure developed by combining the advantages of steel tube and concrete . PouringCirculating fluidized bed (CFB) combustion technology is a new generation of efficient and low pollution combustion technology, which developed rapidly in the world in recent years . However2 and Al2O3, which forms the foundation as supplementary cementitious material. They can be used to replace cement for preparing green, low-carbon building materials, which has low energy consumption in cement production and low CO2 emissions [3 in desulfurization ashes can lead to expansion even crack, which cause difficulties for large-scale resource utilization. Desulfurization ashes should be modified before use to eliminate the destruction caused by expansion. Physical grinding can not only raise the desulfurization ashes activity, but also decrease the expansive destruction. By grinding, the dense structure of Ⅱ-CaSO4-wrapped CaO is broken, and CaO is released [4 can dissolve quickly to form ettringite, which enables the expansion to be released at an early stage [4 and improve the early strength of the cementitious system [Studies showed that desulfurization ashes have good pozzolanic activity, self-hardening property and large expansibility . Desulfumissions . Howeverreleased ,24. In aly stage . As a chs system . Researcs system ,28.2 emissions but also solve many environmental problems caused by the large amount of CFBDS stockpiles [The use of circulating fluidized bed desulfurization slag (CFBDS) to replace cement can not only reduce COockpiles . The use2/kg, its density is 2.61 g/cm3, its color is dark grey, and its main chemical composition is shown in The raw desulfurization slag (RDS) was from Shanxi Pingshuo Coal Gangue Power Plant . The ground desulfurization slag (GDS) was prepared by grinding the RDS in a 5 kg mill for 39 min, which was used to prepare the SCCs in the experiment. The digital images of RDS and GDS are shown in 2/kg, its density is 3.1 g/cm3, and its main chemical composition is also shown in Grade P.O 42.5 Portland cement (PC)  was used to prepare the SCCs in the experiment. Its specific surface area is 370 m3, the bulk density is 1472 kg/m3, the mud content is 0.7%, and the fineness modulus is 2.8. The crushed limestone (CLS) was used as coarse aggregate, which was from Jiancaoping District . The particle size range is 5–20 mm, the mud content is 0.3%, the apparent density is 2786 kg/m3, and the bulk density is 1510 kg/m3. The polycarboxylate superplasticizer (PS)  was used to improve the fluidity and the strength of the SCC, the water reduction rate of which was 34%. Sodium sulfate was used as activator to improve the GDS’s performance. The circular steel tubes with a straight slit were used to fabricate CFSTs, which were from Tangshan Iron & Steel Group .The natural river sand (S) was used as fine aggregate, which was from Douluo Town . The apparent density is 2654 kg/mThe mix design and preparation of the SCCs were carried out according to JGJ/T 283-2012 “Technical Specification for Application of Self-compacted Concrete”. The GDS replaced cement in equal quantity. The water–binder ratio (W/B) of 0.3, 0.34 and 0.38, and the GDS’s amount of 0, 10%, 30% and 50% were used in the mix design. After trial and adjustment, the amount of each material is shown in The parameters of the CFST specimens are shown in According to JGJ/T 283-2012 “Technical Specification for Application of Self-compacted Concrete”, the self-compacting properties of SCC include filling ability, passing ability and segregation resistance. Filling ability is measured by slump flow or slump-flow time, which is the control index. Passing ability is measured by the difference of slump flow value and J-ring flow value. Segregation resistance is measured by segregation percent and vibration segregation percent of coarse aggregate .In this paper, the SCCs were used to fill steel tube as core concretes, which belong to concrete without steel bar. According to JGJ/T 283-2012, the filling ability of the used concrete needs to be tested, while the passing ability and segregation resistance do not require a test. Slump flow was tested to evaluate the filling ability of fresh SCCs in the test . By adjuThe concrete test blocks of compressive strength were produced at the same time as the CFST specimens, the size of which is 100 mm × 100 mm × 100 mm. After casting, they were put into the standard curing room along with the molds for curing, the mold was removed mold 1 day later, then the test blocks were wrapped with plastic film and put into an air-conditioned room with 20 ± 2 °C and 50% relative humidity for curing. The blocks were tested at 3d, 7d and 28d ages, respectively.The test method and result evaluation were performed according to Chinese Standard GB/T 50107-2010 “Standard for evaluation of concrete compressive strength”.A total of 9 groups of specimens mentioned in 2.3 were subjected to axial compressive test after 28d curing age. Before the test, four pairs of strain gauges were glued on the middle of the steel tube’s surface, which were used to obtain the axial strain and circumferential strain values .The axial compressive test was carried out on the 3000 KN hydraulic test machine in Engineering Structure Laboratory of North University of China. The test schematic diagram is shown in −1. In the formal load stage, the load rate was 1.0 kN·s−1 initially. After loading to 80% of the estimated ultimate bearing capacity, the load rate reduced slowly to 1.0 kN·s−1 until the specimen was destroyed [The load process of CFST specimens included two stages, which are the preload stage and the formal load stage. Before loading, the ultimate bearing capacity of each specimen was estimated according to CECS 28 “Technical specification for concrete-filled steel tubular structures”. In order to ensure the measuring instruments working normally, the preload was operated. In the preload stage, the load of 10% estimated ultimate bearing capacity was applied with a load rate of 1.0 kN·sestroyed .In order to facilitate the comparative analysis of concrete properties, the slump flow values were limited to a small range, which is 550 ~ 600 mm. Different influence factors on different amounts of PS and slump flow of SCCs are shown in From Different influence factors on the compressive strength at different ages are shown in 2 and Al2O3. The GDS has good activity after grinding. There is also Ⅱ-CaSO4 in the GDS, which hydrates slowly and influences the increase rate of SCC’s strength [From strength . From Fistrength . The typical failure form of specimens is shown in The axial load–deformation curves of CFSTs are shown in It can be seen from The axial load–strain curves of CFSTs are shown in The influence laws of different influence factors on the ultimate bearing capacity of CFSTs are shown in 2) and aluminum oxide (Al2O3). The desulfurization slag also contains free calcium oxide (f-CaO) and anhydrite (II -CaSO4), which is different from the fly ash. The f-CaO reacts with H2O to form calcium hydroxide. The II-CaSO4 first dissolves to gypsum, then the gypsum reacts with calcium aluminate hydrate to form high sulfur calcium sulphoaluminate hydrate, commonly known as ettringite (AFt) [The influence law of the GDS’s amount on the CFSTs’ ultimate bearing capacity is quite different from that of the GDS’s amount on the SCCs’ compressive strength when comparing te (AFt) . Both ofte (AFt) .As shown in As shown in As shown in While the slump flow of the SCCs is almost constant, the PS’s amount increases with the GDS’s amount but decreases with W/B. The addition of sodium sulfate has little effect on the amount of PS and the slump flow of the SCC.The addition of GDS decreases the 3d, 7d and 28d compressive strength of the SCCs. The decrease rate of the 28d compressive strength of SCC3 is the minimum, at only 2.8%, which means that the optimum amount of GDS for SCCs is 30%. With the increase in W/B, the compressive strength of the SCCs decreases. The addition of sodium sulfate can improve the SCC’s compressive strength. The compressive strength of 3d, 7d and 28d increase by 17.6%, 3.5% and 2.9%, respectively. Relative to the late compressive strength, sodium sulfate improved the early compressive strength of SCC to be more pronounced.The failure mode of the CFST short column is ductility failure. The failure form of the specimens is shear multi-fold waist drum failure. The axial load–deformation relation curve of all CFSTs can be divided into three stages, which are the elastic stage, the elastoplastic stage and the strengthening stage.Although the amount of GDS increases, the ultimate bearing capacity of CFSTs first increases and then decreases. When the amount of GDS is 30%, the ultimate bearing capacity of CFST short column (GP3) is the highest, which is 33.6% higher than that of GP1 without GDS. When the amount of GDS is 10%, the ultimate bearing capacity of CFST short column (GP2) is 12.8% higher than that of GP1. When the amount of GDS is 50%, the ultimate bearing capacity of CFST short column (GP4) is 2.1% lower than that of GP1. The optimum amount of GDS for CFSTs is also 30%.The influence law of the GDS’s amount on the CFSTs’ ultimate bearing capacity is quite different from that of the GDS’s amount on the SCCs’ compressive strength. The ultimate bearing capacity of CFSTs can be significantly improved by adding GDS. With the increase in wall thickness, the ultimate bearing capacity also increases. The ultimate bearing capacity of CFSTs decreases with the increase in W/B. Compared with GP3, the ultimate bearing capacity of GP9 increases by 15.3%, which contains 1.5% sodium sulfate. Sodium sulfate can improve the bearing capacity of the CFST.In this paper, the ground desulfurization slag is used to replace cement to prepare SCCs. Then, the SCCs are used as the core concrete of the CFSTs. Seven types of SCC are prepared and nine groups of CFST short column are fabricated. The filling ability and the compressive strength of the SCCs are researched, and the axial compressive properties of the CFSTs are analyzed. There are several conclusions that can be concluded: