The balance of gut microbiome is essential for maintaining host metabolism homeostasis. Despite widespread antibiotic use, the potential long-term detrimental consequences of antibiotics for host health are getting more and more attention. However, it remains unclear whether diet affects the post-antibiotic recovery of gut microbiome and host metabolism. In this study, through metagenomic sequencing and hepatic transcriptome analysis, we investigated the divergent impacts of short-term vancomycin (Vac), or combination of ciprofloxacin and metronidazole (CM) treatment on gut microbiome and host metabolism, as well as their recovery extent from antibiotic exposure on chow diet (CD) and high-fat diet (HFD). Our results showed that short-term Vac intervention affected insulin signaling, while CM induced more functional changes in the microbiome. However, Vac-induced long-term (45 days) changes of species were more apparent when recovered on CD than HFD. The effects of antibiotic intervention on host metabolism were long-lasting, antibiotic-specific, and diet-dependent. The number of differentially expressed gene was doubled by Vac than CM, but was comparable after recovery on CD as revealed by the hepatic transcriptomic analysis. In contrast, HFD intake during recovery could worsen the extent of post-antibiotic recovery by altering infection, immunity, and cancer-related pathways in short-term Vac-exposed rats and by shifting endocrine system-associated pathways in CM-exposed rats. Together, the presented data demonstrated the long-term recovery extent after different antibiotic exposure was diet-related, highlighting the importance of dietary management during post-antibiotic recovery. The human intestine is colonized by trillions of microbes that participate in many physiological and pathological processes of host including energy metabolism, xenobiotic metabolism, and immune response . A stablClostridium difficile infection-associated diarrhea is a common consequence of antibiotic use  on gut microbiota composition and host metabolism in rats, as well as the recovery from antibiotic exposure on either chow diet (CD) or high-fat diet (HFD) for 45 days. Our results revealed that the impacts of antibiotic exposure on host metabolism were long-lasting, antibiotic-specific, and diet-dependent. Compared to the effect of CD on post-Vac recovery, HFD affected post-Vac recovery of host metabolism and significantly regulated infection, immunity, and cancer-related pathways. In sum, diet plays a critical role in the recovery of host metabolism after antibiotic intervention, which points to the fact that the dietary management should particularly be given more attention at the post-antibiotic recovery stage.Male SD rats of 200-g body weight (BW) were provided by the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine . Rats were orally administrated with vehicle or vancomycin (100 mg/kg per dose) or combination of ciprofloxacin (50 mg/kg per dose) and metronidazole (50 mg/kg per dose) twice daily for 5 days. The dose was doubled at the first and last administration. For the insulin intervention experiment, 8 min after insulin injection (10 U/kg), rats were euthanized and tissues were collected for protein analysis . For theGlucose-tolerance tests were performed on fasted rats  by monitoring glucose levels after a glucose bolus (1 g/kg of BW) by intraperitoneal injection (IP). Data acquisition was carried out at 0, 15, 30, 60, 90, and 120 min after injection. For the diet stimulation experiment, blood glucose was measured on fasted rats (15 h) and re-fed rats (2 h) .Serum biochemical indices such as triglycerides (TG), cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were determined according to the instructions of specific kits . Serum insulin was measured according to the instructions of Elisa kit . The livLiver and muscle protein lysates (20 mg) were subjected to polyacrylamide gel electrophoresis under reducing conditions followed by transfer to polyvinylidene difluoride membranes. The membranes were incubated with 5% skimmed milk followed by antibodies specific for p-AKT , AKT , p-IR , IR , and β-ACTIN . Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The signals were detected using an enhanced chemiluminescence (ECL) system with Pierce SuperSignal West Pico chemiluminescent substrates .Liver tissues were fixed with 10% neutral formalin for 24 h, embedded in paraffin, stained with hematoxylin–eosin staining (H&E), and sections were observed for the degree of hepatic steatosis under the light microscope. The degree of hepatic steatosis was evaluated according to a previous publication in a blinded way . The criPRJNA702866).Fecal DNA was isolated using the Qiagen QIAamp DNA Stool Mini Kit . Illumina sequencing was done based on published methods . The V3–1. Reads with length <50 bp or a quality value <20 or having N bases would be considered as low-quality and be removed by Sickle2. The hit would be removed if it was associated with the reads, which was aligned to the Rattus norvegicus genome by BWA,3 or their mated reads. MEGAHIT, which utilized succinct de Bruijn graphs, was used to assemble data4  (8 (9) with an e-value cutoff of 1e–5. The higher-order functional information is stored in the KEGG PATHWAY database, which contains three different classification levels (PRJNA719272).Total genomic DNA was extracted from fecal samples using the E.Z.N.A. tissue DNA Kit  according to the manufacturer’s instructions. The concentration and purity of extracted DNA were determined with TBS-380 and NanoDrop 2000, respectively. DNA extract quality was checked on 1% agarose gel. Read analysis was performed according to our previous publication . Adapterle data4 . Only codata4 (5 . The preverage7) ; the lonage7)  (8 9 with ann levels . In thisPRJNA713932).Total RNA was extracted from the liver using TRIzol Reagent according to the manufacturer’s instructions (Invitrogen), and genomic DNA was removed using DNase I (TaKara). Then, RNA quality was determined by 2100 Bioanalyzer (Agilent) and quantified using ND-2000 (NanoDrop Technologies). Only the high-quality RNA sample was used to construct the sequencing library. Illumina HiSeq X Ten/NovaSeq 6000 Sequencing and reading were finished based on the published method . Sequencobs = the number of OTUs actually observed; ni = the number of sequences contained in the ith OTU; and N = number of all sequences. Bray–Curtis as the distance algorithm is used to represent β diversity; its calculation is based on the independent taxa , the weighted calculation method is adopted, and the existence and abundance of species are considered at the same time, but without considering the evolutionary relationship or association information among species  unless otherwise noted. Differences between groups at microbiota phylum and genus levels were calculated by the Mann–Whitney U test. Other comparisons were calculated by two-tailed Student’s t-test. p < 0.05 was considered statistically significant.Shannon index and Simpson index are the measures of biodiversity (α diversity). Shannon index is intended to quantify both richness and evenness of the species/individuals in the ecosystem or community . Shannon species . Simpson species . The BraNorank_f_Bacteroidates_S24-7_group (19.4%) and Helicobacter (10.3%) in the Con group to Norank_f_clostridiates_vadinBB60_group (23.8%) and Anaeroplasma (20.5%) in the Vac group (Lactobacillus genus increased to 81% after CM intervention.SD rats were given vancomycin (Vac group), combination of ciprofloxacin and metronidazole (CM group), or water (Con group) twice daily for 5 days on CD . The 16Sac group . Most stp < 0.01, fold change (FC) < 0.5 or >2) compared with Vac-induced changes (130 vs. 48 pathways)  . Among tathways) . In addiathways) , while tathways) . In summSince the influences of antibiotic intervention on glucose tolerance have been previously reported , we expeImpaired insulin signaling is the important cause for dysglycemia . Thus, tGiven the critical roles of gut microbiota in host metabolism , we furtp < 0.05, p-adjust < 0.25), respectively  was employed to identify significantly enriched biological pathways on the basis of normalized enrichment score (NES) ranking. Compared with the Con group, Vac and CM regulated 67 and 44 gene sets , respectively. In particular, four species of top 10 species that changed in the Vac_CD group belonged to Lactobacillus, indicating that the difference of Lactobacillus might account for the difference of the gut microbiota profile between the Vac_CD and CD groups after recovery , respectively  (p < 0.05), four of which were related to sugar and lipid metabolism, such as arachidonic acid metabolism, steroid hormone biosynthesis, fructose and mannose metabolism, and regulation of lipolysis in adipocytes  . Howeveripocytes . Meanwhi disease . Interes disease , 4M. In  disease . AltogetDesulfovibrio, indicating the importance of Desulfovibrio for the difference of gut microbiota structure during recovery on HFD (s_Lactobacillus_johnsonii (the fold change was 2.85) and decreased three species of Lachnospiraceae_bacterium and three species of Desulfovibrio compared to the HFD group , while there were only 13 differential pathways when comparing Vac_CD with CD, suggesting that HFD feeding enlarged short-term Vac treatment-induced changes of liver function at the recovery condition. Among them, 30 pathways were related to infection and immunity and 10 pathways were associated with cancer (p < 0.05) and 7 of them were associated with the endocrine system (The number of differential expressed genes between groups was markedly increased when rats were recovered on HFD than on CD. Specifically, the expressions of 283 and 215 hepatic genes were changed by Vac_HFD and CM_HFD, respectively, in which more genes were downregulated . KEGG enh cancer . Howevere system . GSEA ree system . Regardie system . These rTo investigate how diet affects host recovery after different antibiotic perturbations, we compared the composition and function of gut microbiota as well as the gene expression and biological function of the liver after short-term Vac and CM exposure and at post-antibiotic recovery state when rats were fed with CD or HFD. After short-term antibiotic intervention, compared with the control group, the number of differential genera was higher in Vac than in the CM group. However, the differential genus number was dramatically reduced after recovery on both CD and HFD with a slightly higher number in CM than in Vac . The VenLiver gene expression was affected more significantly by Vac than by CM based on the number of regulated genes . Post-anGut microbiota composition and diversity exert profound effects on host physiology and metabolism. Mounting evidences have shown that exposures to antibiotics in both animals and humans have transient and prolonged effects on host metabolism homeostasis. As summarized in Exposure to antibiotics is closely linked with the changes of glucose metabolism in both human and animal studies. A randomized double-blinded study showed that 7-day Vac treatment reduced peripheral insulin sensitivity in obese males with metabolic syndrome , whereasSince antibiotic treatment dramatically impacted glucose homeostasis and insulin signaling, to our surprise, little is known about how glucose levels change at post-antibiotic recovery state and whether diet will affect the recovery. Lactobacillus genus. Interestingly, when rats recovered under CD, the abundance of three species from Lactobacillus genus, Lactobacillus intestinalis, Lactobacillus reuteri, and Lactobacillus sp. ASF360, was reduced in the Vac_CD group, while there was no difference in the Lactobacillus species between the CM_CD and CD groups. Previous study reported that L. intestinalis was increased in Zucker diabetic fatty rats and can be a potential biomarker for the progression and complications of T2DM (L. reuteri are used as probiotics to improve insulin sensitivity (Lactobacillus might play an important role in post-Vac recovery of glucose homeostasis. In contrast to the findings in the Vac_CD group, when rats were fed with HFD, the most significant long-term effect of Vac intervention was the reduction of the Desulfovibrio genus, which can be boosted by HFD and can produce hydrogen sulfide leading to acute inflammation (Desulfovibrio genus were reduced in the Vac_HFD group compared with the HFD group. Desulfovibrio has been reported to positively correlate with the AUC of OGTT, insulin, and HOMA-IR (Lactobacillus abundance and Desulfovibrio abundance during long-term CD feeding and HFD feeding, respectively.Short-term antibiotic perturbations have a long-term effect on gut microbial composition and function. Based on the KEGG pathway analysis, compared with the effect of CD on post-Vac recovery, HFD intake during post-Vac recovery led to functional changes in bacteria which related to glucose homeostasis. This result was in line with the phenotype changes that HFD reversed the CD-induced decrease in fasting insulin level during post-antibiotic recovery. The most significant change of gut microbiota composition induced by 5-day CM treatment was the 127-fold induction of the abundance of the  of T2DM . Howeversitivity . These fammation . Among t HOMA-IR . TherefoFaecalibacterium genus were reduced in the CM_CD group, while three species that belong to the unclassified Lachnospiraceae genus were reduced in the CM_HFD group. Faecalibacterium prausnitzii, which is a butyrate-producing bacteria, has anti-inflammatory properties and plays a crucial role in maintaining host physiology (L. johnsonii was the only significantly increased species in the CM_HFD group than in the HFD group. Because many studies have shown that L. johnsonii modulates host immune responses (L. johnsonii might lead to better immune response in the CM_HFD group. In sum, different short-term antibiotic exposures have divergent effects on regulating host physiology and metabolism, which depend on the diet used during the recovery state.Early CM intervention also affected gut microbiota recovery based on diet. Compared with their respective control groups, among the top 10 changes species, two species that belong to the ysiology . Certainysiology . Additioysiology . These fesponses , the eleAlthough few studies have reported altered expressions of hepatic genes and shifted functional pathways long after antibiotic exposure , to our In conclusion, long-lasting effects of antibiotic exposure on host metabolism are antibiotic-specific and diet-dependent. Our current study reveals the interplay between antibiotic-driven dysbiosis and biological functions of liver, and more importantly, we demonstrate that HFD intake during recovery could worsen Vac-induced long-term detrimental consequences. Although the role of dietary fiber in post-antibiotic recovery of gut microbiome is reported previously , our stuhttps://www.ncbi.nlm.nih.gov/sra, PRJNA719272, PRJNA713932, and PRJNA702866.The original contributions presented in the study are publicly available. This data can be found here: The animal study was reviewed and approved by Shanghai University of Traditional Chinese Medicine Animal Ethics Committee.in vivo experiments, data analysis, and manuscript writing. HQ, YH, and JM helped in H&E staining of tissues and hepatic steatosis evaluation. NZ, GW, YG, and JZ helped in the animal experiment. WZ helped in the project design. LS and HL co-supervised the study and wrote the manuscript. HL conceptualized the project. All authors contributed to the article and approved the submitted version.BL conducted the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Antibiotic use in pregnant women at the national level has rarely been reported in China. Objectives: We aimed to investigate antibiotic prescriptions during pregnancy in ambulatory care settings in China. Methods: Data of 4,574,961 ambulatory care visits of pregnant women from October 2014 to April 2018 were analyzed. Percentages of Antibiotic prescriptions by different subgroups and various diagnosis categories and proportions of inappropriate antibiotic prescriptions for different subgroups were estimated. Food and Drug Administration (FDA) pregnancy categories were used to describe the antibiotic prescription patterns. The 95% confidence intervals (CIs) were estimated using the Clopper––Pearson method or Goodman method. Results: Among the 4,574,961 outpatient visits during pregnancy, 2.0%  were prescribed at least one antibiotic. The percentage of antibiotic prescriptions for pregnant women aged >40 years was 4.9% , whereas that for pregnant women aged 26–30 years was 1.5% . In addition, percentages of antibiotic prescriptions varied among different trimesters of pregnancy, which were 5.4%  for the visits in the first trimester of pregnancy and 0.5%  in the third trimester of pregnancy. Furthermore, the percentages of antibiotic prescriptions substantially varied among different diagnosis categories and nearly three-quarters of antibiotic prescriptions had no clear indications and thus might be inappropriate. In total, 130,308 individual antibiotics were prescribed; among these, 60.4%  belonged to FDA category B, 2.7%  were classified as FDA category D and 16.8%  were not assigned any FDA pregnancy category. Conclusions: Antibiotic prescriptions in ambulatory care during pregnancy were not highly prevalent in mainland China. However, a substantial proportion of antibiotics might have been prescribed without adequate indications. Antibiotics whose fetal safety has not been sufficiently illustrated were widely used in pregnant women. Antibiotics are among the most commonly prescribed medications in pregnant women for infectious diseases ,2, whichThe appropriate use of antibiotics is a key strategy to ensure maternal health, reduce potential fetal risk and tackle antibiotic resistance ,6. StudiThe national database used in this study has been described in our previous studies ,16. In bAdult pregnant women aged 18–50 years were included in this study and were identified by detecting information on pregnancy in the diagnosis text. We excluded patients with an abnormal pregnancy and those who terminated their pregnancy, for example, induced abortion and ectopic pregnancy. Because for these patients, there was no need to consider the embryo–fetal risk of antibiotics. Pregnancy was divided into three trimesters according to the diagnosis description or gestational week—the first trimester lasted from conception to week 13 of gestation, the second was weeks 14–26 of gestation and the third was from week 27 to the delivery [Fifty-five hospitals that did not submit diagnosis records were excluded from this study . AnotherAntibiotics for systemic use according to ATC code J01 were included in this study. To assess whether potential teratogenic risk and fetal harm were considered when prescribing antibiotics for pregnant women, we classified the antibiotics into categories B, C and D by following the FDA letter categorization system ,17,18. AAntibacterial spectrum was classified according to previous studies ,20. BroaMultiple prescriptions of drugs and diagnoses from the same patient on the same day in the same hospital were considered as one visit in this study . PatientFollowing the approach employed in previous studies ,14,15,16We reported percentages of antibiotic prescriptions overall and by different patient or hospital characteristics including age groups , trimester of pregnancy, department of visit (outpatient or emergency department), diagnosis tier (tier 1–3), year (2014–2018) and season of outpatient visits and China’s economic region. According to the National Bureau of Statistics of China , mainlanThe percentages of antibiotic prescriptions were calculated as the percentages of outpatient visits with antibiotic prescriptions for different diagnosis categories and subgroups. Clopper–Pearson exact method was used to calculate the 95% confidence intervals (CIs) of percentages of antibiotic prescriptions for the entire study population, as well as different subgroups. Predictors for antibiotic prescriptions were assessed using a multi-level binary logistic regression, with random intercept for each hospital. Odds ratios with 95% CIs were reported. The proportion of prescribed antibiotics in each FDA pregnancy category among different diagnosis categories and subgroups was estimated according to the prescription frequency. The simultaneous 95% CIs for multinomial proportions were estimated using Goodman method . We alsoOracle 11gR2 and PLSQL developer 11.0  were used for data extraction and diagnosis classification. Statistical analyses were performed using SAS 9.4 .After excluding the visits of women with the diagnoses of abnormal pregnancy and pregnancy termination, 4,574,961 ambulatory care visits of pregnant women from 90 cities and 28 provinces were included in this study. Among these visits, 2,080,643 (45.5%) involved women aged 26–30 years; 4,502,627 (98.4%) occurred in outpatient clinics, 4,279,852 (93.5%) presented at tertiary-level hospitals. Furthermore, 2,317,388 (50.7%) visits were in the Eastern region and 1,202,141 (26.3%) visits were during winter . In addiOf all ambulatory care visits of pregnant women, at least one antibiotic was prescribed during 92,514 visits  . The perOf the 92,514 visits with antibiotic prescriptions, a total of 130,308 individual antibiotics were prescribed, of which 78,272  were broad-spectrum antibiotics . The thrTo the best of our knowledge, this study is the first in China to investigate antibiotic prescriptions for ambulatory care visits of pregnant women using such a big prescription data covering almost all provinces of mainland China. We found that antibiotics were prescribed during 2.0% of ambulatory care visits in Chinese hospitals for pregnant women. However, nearly three-quarters of the antibiotics might have been prescribed without appropriate indications. Age, pregnancy trimester, department and season of visit should be considered as potential determinants of antibiotic prescriptions. The prescribing of FDA category D antibiotics, which are potentially harmful to fetus, was low in China; nevertheless, the prescriptions of antibiotics with unclear fetal risks were relatively more prevalent.Evidence of antibiotic prescribing for pregnant women at the national level is scarce in China. However, our results that only 2% of ambulatory care visits of pregnant women resulted in antibiotic prescriptions seems obviously lower than the prescription rates in other studies. A survey conducted by Chen et al., in a single hospital indicated that 22.3% of women who delivered in the hospital were prescribed antibiotics during pregnancy . In TaiwWe found that for almost all conditions and diagnosis categories, percentages of outpatient antibiotic prescriptions were considerably lower in pregnant women than in general patients in China. For example, 10.9% of visits with all conditions, 30.6% of visits with tier 2 diagnoses and 7.6% of visits with tier 3 diagnoses were prescribed antibiotics among general outpatients ; howeverOur results suggested that older pregnant women tended to have a higher percentage of antibiotic prescriptions, except pregnant women aged <26 years. Furthermore, pregnant women in their first trimester had the highest risk of receiving antibiotic prescriptions. These results were in agreement with those of previous studies, indicating that pregnant women used more drugs, including antibiotics, in the first trimester ,28,29. HWe found that FDA category B was the most commonly prescribed antibiotic category during pregnancy in mainland China, whereas category D antibiotics with positive evidence of fetal risk only accounted for a very low proportion. Furthermore, our results showed a shift to the prescription of relatively safe antibiotics during pregnancy. In the first trimester category B antibiotics accounted for only 52.4% of all antibiotics and category D antibiotics accounted for 3.3% of all antibiotics; however, the prescription percentage of category B antibiotics increased to 97.6% and category D antibiotics accounted for only 0.2% of all antibiotics in the third trimester. Several studies have suggested that pregnant women in the first trimester are more prone to receiving drugs that are potentially harmful to the fetus ,32,33,34The appropriate use of antibiotics is a key strategy to balance the risk-versus-benefit decisions of drug use in pregnancy and to tackle antibiotic resistance. Available evidence and unknown information regarding the effects of antibiotics on pregnancy and fetus should be carefully considered when prescribing these drugs . AlthougAlthough the majority of the prescribed antibiotics for pregnant women were FDA category B antibiotics, a considerable proportion of antibiotics with little evidence of fetal risk were prescribed during pregnancy in mainland China. Early pregnancy was the period with higher risk of excessive antibiotic prescriptions and unsafe antibiotic use. Physicians should pay attention to the marriage and pregnancy status when prescribing antibiotics to women of childbearing age. An alarm mechanism embedded in medical information systems to indicate fetal risk of drugs may be useful in reducing unsafe antibiotic prescriptions. Insufficient safety evidence may be an important factor leading to the use of drugs that are unsafe during pregnancy and underuse of safer drugs . Hence, Our study has several strengths. We provided the most recent and comprehensive data on antibiotic use during pregnancy in ambulatory care settings in mainland China using a large and nationally representative sample of prescription data. Using a well-established and validated diagnosis classification scheme , our stuThere were some limitations. First, our study was based on cross-sectional data and outpatient visits in different hospitals or on different dates of the same patient could not be linked together; thus, the percentage of antibiotic prescriptions might be underestimated and cannot be regarded as antibiotic use throughout pregnancy. Further studies based on a cohort design and a nationally representative population are needed. Second, some prescriptions that lacked standardization, such as incomplete diagnosis writing, might have been omitted or classified as inappropriate for antibiotic use, resulting in the omission of pregnancy visits and potential bias in the assessment of inappropriate antibiotic prescriptions. Third, as in our previous study , this stIn mainland China, outpatient antibiotic prescriptions during pregnancy were not highly prevalent, with a percentage of 2.0%. Patient- and facility-level characteristics were associated with antibiotic prescriptions during pregnancy. Some conditions that do not justify antibiotic use were prescribed a considerable number of antibiotics and nearly three-quarters of antibiotics might have been prescribed without adequate indications to pregnant women. Antibiotics that fetal safety has not been sufficiently proven were widely used in pregnancy. Antibiotic stewardship efforts to optimize outpatient antibiotic prescriptions and reduce the use of potentially unsafe antibiotics are needed in China. Additional studies are needed to explore antibiotic prescriptions in primary care settings and determine more potential influencing factors, such as physicians’ characteristics, associated with antibiotic prescriptions during pregnancy.


Incorporating recycled plastic waste in concrete manufacturing is one of the most ecologically and economically sustainable solutions for the rapid trends of annual plastic disposal and natural resource depletion worldwide. This paper comprehensively reviews the literature on engineering performance of recycled high-density polyethylene (HDPE) incorporated in concrete in the forms of aggregates or fiber or cementitious material. Optimum 28-days’ compressive and flexural strength of HDPE fine aggregate concrete is observed at HDPE-10 and splitting tensile strength at HDPE-5 whereas for HDPE coarse aggregate concrete, within the range of 10% to 15% of HDPE incorporation and at HDPE-15, respectively. Similarly, 28-days’ flexural and splitting tensile strength of HDPE fiber reinforced concrete is increased to an optimum of 4.9 MPa at HDPE-3 and 4.4 MPa at HDPE-3.5, respectively, and higher than the standard/plain concrete matrix (HDPE-0) in all HDPE inclusion levels. Hydrophobicity, smooth surface texture and non-reactivity of HDPE has resulted in weaker bonds between concrete matrix and HDPE and thereby reducing both mechanical and durability performances of HDPE concrete with the increase of HDPE. Overall, this is the first ever review to present and analyze the current state of the mechanical and durability performance of recycled HDPE as a sustainable construction material, hence, advancing the research into better performance and successful applications of HDPE concrete. About 2.01 billion tons of Municipal Solid Waste (MSW) is generated annually worldwide, and one third of MSW is openly dumped without managing in an environmentally-friendly manner . Only 9%Today, more than 30 types of primary plastics and over thousands of different secondary plastics can be found, produced by using different combinations and proportions of primary plastics . PlasticConcrete is ranked as the topmost man-made resource utilized in the construction industry worldwide. The global aggregate requirement for concrete production accounts for about 4.5 billion tons per year which alarms the necessity of finding alternative aggregate sources required for increasing trends of concrete production . IncorpoHDPE is a thermoplastic synthetic polymer in the subset of the PE macro plastic group. PE polymer consists of neverlasting hydrocarbon chains where each carbon molecule is bound to another two carbon molecules and two hydrogen molecules. HDPE has minimal branches in its polymer chain which results to pack liner molecular chains together regularly during crystallization. As a result, semi-crystalline HDPE polymers become much more dense, rigid, and ductile with a comparatively strong bending strength between 20 to 45 MPa due to the regular packing of polymer chains ,4,5. HDPHDPE is the third most applied plastic type in construction preceded by PE and PET. However, a little research is reported in concrete incorporated with HDPE as a construction material, Throughout the paper, HDPE incorporated concrete is denoted in an abbreviation form, such as 10% of HDPE inclusion as HDPE-10 and 100% of HDPE inclusion as HDPE-100, etc. The 28-days’ splitting tensile strength also shows a decreasing trend with the increase of HDPE content from 0% to 15%, Similar to compressive and splitting tensile strengths, the flexural strength also decreases with the increases of HDPE fine aggregate percentage, 3 to 1825 kg/m3, when HDPE increases from 0% to 15% [The modulus of elasticity is decreased with the increase of HDPE percentage, % to 15%  have obs2/min has been observed in initial rate of adsorption (IRA) from 0.5 kg/m2/min to 6.0 kg/m2/min, with the increase of HDPE from 0% to 15% [Water adsorption of HDPE fine aggregate concrete has increased from 5% to 10.4%, when recycled HDPE content increases from 0% to 15% [% to 15% . For ins% to 15% .After 28 days of curing, the Ultrasonic Pulse Velocity (UPV) test was performed on HDPE fine aggregate concrete and it is observed that the velocity has decreased from 3880 m/s to 2720 m/s, with the increase of HDPE from 0% to 60% in 15% intervals . AdditioThermal conductivity has dropped from 2 W/m·K to 1.14 W/m·K when HDPE increases from 0% to 60% at 7 days [The workability (slump) of HDPE coarse aggregate concrete has reduced from 61 mm to 55 mm, and to 28 mm, when HDPE is increased from 0% to 4% in 2% intervals while keeping the water/cement (W/C) ratio at 0.55, Similar to HDPE fine aggregate concrete, the compressive strength of HDPE coarse aggregate concrete decreases with the increases of HDPE percentage, The splitting tensile strength also shows a decreasing trend with the increment of HDPE content, Flexural strength or bending strength reduces with the increase of HDPE when incorporated as coarse aggregate in concrete 3 and by 675.72 kg/m3 in ¾′′ HDPE coarse aggregate concrete matrix only with the increase of HDPE from 0% to 30% [The density of HDPE coarse aggregate concrete is decreased with the increase of HDPE content, % to 30% . This re% to 30% .Porosity and permeability of HDPE coarse aggregate concrete have increased with the increase of HDPE content . The HDPWater absorption rate of HDPE coarse aggregate concrete is increased by 34.7%, when the HDPE increase from 0% to 20% . In anotAt direct UPV test, the pulse velocity of HDPE coarse aggregate concrete has increased from 4200 m/s to 4650 m/s, when HDPE is added from 0% to 16% . After rWhen considering the sulphate attack test, the compressive strength of HDPE coarse aggregate concrete (with the increase of HDPE from 0% to 32%) is increased when immersed with Na2SO4 apart from the standard concrete matrix (HDPE-0) . CompresAn acid attack test was carried out through a comparison of compressive strength values obtained after immersing HDPE coarse aggregate concrete mixes in both water and hydrochloric (HCl) acid after curing for 28 days . When thAs illustrated in The 28-days’ splitting tensile strength of HDPE fiber reinforced concrete has increased by 10.4%, when fibers with 0.25 mm diameter are added from 0% to 0.4% and again a 3.9% drop is observed, when fibers are added from 0.4% to 1.25%, A slight increase (1.0 GPa) is observed in the elastic modulus of HDPE fiber reinforced concrete with the increase of 0.25 mm diameter HDPE fibers from 0% to 1.25%, When considering the water permeability after 45 days, it has been identified that, the height of water penetration in HDPE fiber reinforced concrete has reduced from 43 mm to 26 mm, when HDPE fibers with 0.4 mm diameter are added from 0% to 1.25% . SimilarIt has been observed that the overall number of cracks and the widths of cracks were reduced in HDPE fiber reinforced concrete when compared with the standard concrete . AverageAattache et al.  investigHDPE has a comparatively low thermal conductivity of 0.33 W/m·K compared with the concrete . It was On the other hand, Naik et al.  investigThe aggregate shape, size, and surface texture influence the concrete workability and the bond between aggregates and cement/binder matrix, which in turn impact the mechanical performance in concrete . Slump oThere are various techniques used to evaluate the performance of concrete. Scanning Electron Microscopy (SEM) images are used to examine the microstructure development of the HDPE incorporated concrete. Similarly, nanoindentation is used to observe the interfacial transition zone (ITZ) of the concrete matrix and Computed Tomography (CT) to identify the pore-structure of the concrete. Poor bond capacity and the availability of higher air content in the concrete matrix with the inclusion of HDPE aggregates is due to the non-reactive  behavior that leads to reduced mechanical and durability performance of HDPE aggregate concrete . Weaker Increase in water adsorption of HDPE fine aggregate concrete with the increase of HDPE percentage is mainly due to the lack of interface bonding between HDPE and the aggregates, the increase in the porosity of the concrete matrix, and the higher HDPE content which occupies the space in the concrete, which could evaporate water which leads to leaving voids thus increasing the absorption value while creating more spaces to fill up with water ,16. The The addition of HDPE and other plastic fibers to concrete mixes have shown more ductile behavior than conventional concrete . AccordiX-ray Diffract Meter (XDM) spectra analysis  was condOptimum 28-days’ compressive and flexural strength of HDPE fine aggregate concrete is observed at HDPE-10 and splitting tensile strength at HDPE-5 whereas for HDPE coarse aggregate concrete, optimum compressive and flexural strength are recorded in the range of 10% to 15% of HDPE incorporation and splitting tensile strength at HDPE-15.Water absorption of HDPE fine and coarse aggregate concrete is increased by 5.4% and 35% with the increase of HDPE from 0% to 15% and 20%, respectively, while porosity and permeability of HDPE coarse aggregate concrete was increased by 65% and 461% with the increase of HDPE percentage from 0% to 30%.Similarly, other durability properties of HDPE fine and coarse aggregate concrete such as lower thermal conductivity (25% drop) and higher resistance against chloride, sulphate and acid attacks were observed with inclusion of HDPE up to 32% more than the standard/plain concrete matrix (HDPE-0) always.Increasing the HDPE percentage up to 1.25% and maintaining the w/c ratio at 0.62 reduces the workability by 80% and increases the elastic of modulus by 4.1% of HDPE fiber reinforced concrete.The 28-days’ flexural and splitting tensile strength of HDPE fiber reinforced concrete is increased up to an optimum of 4.9 MPa (at HDPE-3) and 4.4 MPa (at HDPE-3.5) with an increase of HDPE from 0% to 6%, and was higher than the standard/plain concrete matrix (HDPE-0) in all inclusion levels of HDPE.HDPE fiber reinforced concrete where HDPE is increased up to 1.25% display better durability properties than the standard/plain concrete matrix (HDPE-0) due to the reduction of water permeability by 39.5% and plastic shrinkage cracking by 83.6% when considering the average crack width reduction.Weaker bonds and lower adhesion strength between the concrete mix and HDPE aggregates are mainly due to the hydrophobic nature, smooth and glossy surface texture and the chemical inert behavior of HDPE which result in reducing both durability and mechanical performances of HDPE fine and coarse aggregate concrete with the increase of HDPE.When ductile fibers are added into HDPE fiber reinforced concrete, it will improve its tensile strength depending on several factors such as fiber toughness, fiber volume fraction, alignment, and the adhesion strength between the fibers and the cementitious matrix.The mechanical and durability properties of concrete incorporated with recycled HDPE as a fine aggregate, a coarse aggregate, a fiber, and a cement binder is comprehensively reviewed. The principal conclusions drawn are listed below:


In concrete structures (concrete), damage from cracks, deterioration, amorphization, and delamination occur in some structures, causing disaggregation (concrete changed to very fine particles) and hollowing out of the concrete. In concrete pavements, damage from large amounts of pop-out of aggregate occurs from the surface of the concrete pavement 4–5 hours after spraying of snow melting agent on the surface of the pavement. The damage from disaggregation, blistering, cracks, and peeling-off of a surface course have also been observed in asphalt runways and highways. The damage from disaggregation, cracks and pop-out of aggregate in asphalt pavements and concrete structures have long been seen as strange and unexpected and have defied explanation. As a result of examinations in various experiments, it was concluded that all of the unexplained kinds of damage of both asphalt pavements and concrete structures were caused by Trace Quantities of Organic Matter (TQOM), Air Entrained (AE) water reducing agent in air and/or cement, and surfactant in snow melting agent. The emission sources of TQOM and these organic substances were also identified by chemical analysis for these unexpected and unexplained phenomena. The TQOM includes phthalate compounds , amine compounds, phosphate compounds, snow melting agent and Sodium Polyoxyethylene Nonyl phenyl Ether Sulfate (SPNES). SPNES is a surfactant in windshield washer fluid for automobiles. We found that the water content and content of organic matter in damaged asphalt pavements and concrete structures are also important indicators for the damage. Further, a new evaluation method for amorphization was proposed in this study and it appears suitable for evaluating the safety of concrete structures along roads which were exposed to TQOM in severely air-polluted environments. However, it seems insufficient to evaluate the damage to concrete by only the pH value observed on the concrete surface. The evaluation method for damage to modern concrete is based on the crack width (0.2 mm or more) on the concrete surface and the application of phenolphthalein. However, little research has been done on the degree of concrete damage and the relationship between cracks and deterioration. For this reason, it is not possible to evaluate the repair time and repair depth of concrete structures that had been thought to be damaged at present.The resolution of causes for damage in cement concrete structures is an important issue for the useful life of structures. To investigate the damage in concrete, a phenolphthalein solution is commonly applied to a cross section of the concrete and the extent of deterioration of the concrete is evaluated by the color corresponding to the reaction products, i.e. the value of pH , amorphization , cracks, and pop-out of aggregate . The disIn this study, we investigated a hypothesis that some of this unexplained damage in concrete could be caused by small amounts of unidentified organic matter. This hypothesis was based on the strange odor emitted by commercially available cement when it is mixed with water. To further elucidate this, this study presumes that TQOM not so far identified in fresh cement and present in the air has an effect on this strange damage to concrete structures and asphalt pavements.To investigate this, many samples were taken from damaged concrete structures and damaged asphalt pavements where various unexplained phenomena had occurred. In order to investigate these causes, also unknown substances that have not yet been determined were sought from these, and the relationship between these causes and the strange phenomena was also investigated.From the results obtained in the various experiments, it was concluded that all of the strange damage suffered by both kinds of structures were caused by TQOM and AE water reducing agent in air and/or cement, and surfactant in snow melting agents.In this study, a new evaluation method was proposed for damage to concrete structures and asphalt pavements. Further, new evaluation methods for damage to concrete structures were also proposed using micro focus CT scanner (CT).It was considered that damages such as disaggregation, delamination, amorphization and cracks in concrete structures and asphalt pavements depend upon the materials , type and content of TQOM in the air and/in the fresh cement, construction method, construction machinery employed, environmental conditions such as temperature and relative humidity, and surfactants in snow melting agents.For the present study, we selected the following 11 types of themes to clarify the causes and mechanisms of damages  in concrete structures and asphalt pavements, and many tests were performed. 1.Determination of details of the strange odor due to TQOM in fresh cement during mortar preparation , 2. Volume expansion of mortar samples due to multiple chemical components of TQOM in cement , 3. Delamination of mortar samples due to separation of TQOM (construction method and vibration processed), 4. Amorphization of mortar due to TQOM in fresh cement  5. Comparison of damage (crack widths and crack lengths) to modern mortar samples and a 120-year-old sample , 6. Deterioration of mortar samples due to multiple chemical components of TQOM in fresh cement  7. Pop-out of aggregate due to TQOM in cement and surfactants in snow melting agent , 8. Organic matter in total suspended matter (TSM)  and emission sources , 9. Content of water penetrated through water proofing sheets in cored samples from a new concrete bridge after transient moisture permeation tests (described in section 2.2.5) , 10. Emission source and chemical components of the organic matter in damaged concrete structures and damaged asphalt pavements , 11. Cause of disaggregation in disaggregated concrete structures and disaggregated asphalt pavements .It was considered that it is necessary to understand these themes in disaggregation in concrete structures and asphalt pavements as will be described in section 3 (results and discussion).The asphalt sample was the penetration grade of asphalt (80/100) which is widely used for asphalt pavements in Hokkaido, Japan. It was used to investigate the emission source of organic matter in damaged concrete structures.Asphalt mixtures on a newly constructed concrete bridge were taken from a two layered-pavement  with a water proofing sheet. These asphalt pavements were placed on a concrete bridge. In the asphalt runway of Nagoya airport, Japan, the asphalt pavement was placed as overlaid-pavements, two times  on an older concrete runway. The first overlaid-pavement was a coarse graded base course (17 cm), dense graded surface course (4 cm) in 1960, and the second overlaid-pavement had a coarse graded base course (17 cm), dense graded surface course (4 cm) added in 1998. Disaggregation had occurred in 2000 at the depth of 15 cm in the asphalt runway in Nagoya. The asphalt cored samples were collected from the disaggregated runway in Nagoya in 2000.The asphalt pavement in the highway in service was formed by two layered-asphalt pavements . The asphalt core sample was collected from the highway in service.Eleven types of cement from seven countries were collected to investigate the quality of the cement to susceptibility to damage of mortar samples and used to investigate the degree of deterioration in new mortar samples. Nittetsu Portland cement (Nittetsu cement) in Japan was used as a standard modern cement, and the contents and chemical components of TQOM in fresh cement were investigated in this study.The 120-year-old sample (120 year-sample in the following) is considered to have had a relatively long useful life as a mortar sample that was made 120 years ago in Otaru, Hokkaido, Japan and maintained indoors at room temperature for the 120 years. The composition and shape of the 120 year-sample are as follows. Asano Ordinary Portland Cement was used for the mortar sample, and the size of the mortar sample was width 4.5 cm, length 8 cm, thickness 2.2 cm, gourd type, and the composition of the sample was cement: volcanic ash: sand = 1: 0.5: 3, ratio of water/cement: 44%. The 120 year-sample was made from old cement  and AE water reducing agent was not used in this sample. This sample used natural sand and volcanic ash.The composition and size of the new mortar samples are 2 x 2 x 2 cm, cement: sand = 1: 2, water cement ratio: 50%. All new mortar samples used Toyoura standard sand  and AE water reducing agent .Mortar samples were used to investigate the degree of deterioration due to TQOM and AE water reducing agent, amorphization, delamination, cracks, and volume expansion in the mortar samples. The TQOM in the crushed 120 year-sample was also investigated.In this study, two types of structures: concrete bridge and runway pavements were used to evaluate the damage to concrete structures. The newly constructed concrete bridge was made with two layered-asphalt pavements with a water proofing layer and concrete slab . The old concrete bridge  was made of old cement in 1934 and demolished in 1980. The cement was manufactured with no waste materials and no AE water reducing agent. The samples of the foundation  of the hand rail and bridge pier in the Tokachi bridge were selected from the surface (0-3cm) of core samples (diameter 10cm). The disaggregated concrete protective wall , where wall and wall bed were held together by reinforced concrete, located at the median strip in a Kobe highway  and it was constructed thirty years ago. Samples of disaggregated concrete  from the Kobe highway were collected for this study.Runway concrete pavement (30 cm) from Chitose Airport, Japan was damaged by snow melting agent. In this runway, pop-out of aggregate and disaggregation of concrete were observed. Composition of the concrete in the concrete runway is as follows. Nittetsu cement, AE water reducing agent , W/C = 42%, Slump: 2.5 cm. Damaged concrete pavement (30 cm) of runway concrete divided into 9 layers, and volume expansion of mortar and the damage to the aggregate due to the surfactant of the snow melting agent were also investigated using these samples.Many samples of damaged concrete were collected in Japan and Europe.These materials were used to identify the emission sources in disaggregated concrete structures and disaggregated asphalt pavements.As a tire, a new radial tire  was used as a standard summer tire in Japan, and the surface of this tire was finely scraped and ground to obtain tire debris. The diesel exhaust particulate (DEP) was obtained with a dilution tunnel  of the Japan Automobile Research Institute (JARI) in Tsukuba, Japan. TSM was collected by a High-volume sampler  in Sapporo in summer (eight days in July 1998). The SPNES was used to examine the damage to concrete structures and asphalt pavements. It is used as a standard surfactant in windshield washer fluid throughout the world.In this study, the following tests were used to investigate the emission sources, water content, content and chemical components of the organic matter in damaged concrete structures and asphalt pavements, chemical components of the TQOM, and three-dimensional CT images were also used to investigate the crack distribution, amorphization, deterioration, delamination, amorphization, disaggregation in the damaged concrete and asphalt pavements.1H NMR: JOEL EX 400) and the device of 1H NMR will show peculiar peaks in the spectrum (1H NMR spectrum) for the organic matter that is part of the chemical components of the organic matter sample, and peculiar peaks of the organic matter appears in the spectrum. The approximate estimation of organic substances can be made from these peak values.To measure the chemical peaks in spectra due to Nuclear Magnetic Resonance for organic matter, organic matter was in the Nuclear Magnetic Resonance device  (2002) 84–88 ).The one-dimensional transient moisture permeation (transient moisture permeation) apparatus is a device newly developed in our laboratory in Hokkaido University, Sapporo, Japan and manufactured for this study to measure the moisture by the weight changes (±0.1g) of samples under respiratory action of concrete structures and asphalt pavements. In this test, a KCL-1000 apparatus  was used .  in the laboratory  like thaThis test was used to identify cracks (crack widths and crack lengths) and amorphous substances in aggregate and concrete samples  267–281 ).The larger the number of pixels in the CT apparatus , the more accurate the shape of the sample can be reconstructed . Here, ahis software was used to identify cracks (crack widths and crack lengths), amorphous substances in aggregate and concrete samples. Software for three-dimensional crack analysis  to examine the crack properties in concrete and aggregate from CT images. The degree of deterioration  of samples was analyzed by two-dimensional CT images and Image-Pro Analyzer 7.1 .The Table of ‘Results and Discussion’ is as follows.3.1 Organic matter in cement 3.2 Expansion of new mortar samples and pop-out of aggregate 3.3 Delamination 3.4 Amorphous substance in mortar samples 3.5 Damage to new mortar samples and 120 year-sample 3.6 Deterioration in mortar samples  3.7 Pop-out of aggregate in concrete pavements 3.8 Trace quantities of organic matter (TQOM) 3.9 Respiratory action of concrete structures and asphalt pavements 3.10 Organic matter in damaged concrete structures and damaged asphalt pavements 3.11 Disaggregation in asphalt pavements and concrete structures.The organic matters in Nittetsu cement and crushed 120 year-sample were extracted 4 times (24 hours) using Soxhlet extractor and chloroform solution. We call it ‘substance extracted with Soxhlet’.The following substances were identified as TQOM in the cement using GC-MS. For the Nittetsu cement in 10 g of cement: 119 μg of Di-butyl phthalate (DBP) and 2.1 μg of Di-(2-ethyhexyl) phthalate (DEHP) were detected. In the 120 year-sample in 10 g of Asano Portland cement: 1 μg of DBP, 0.1 μg of DEHP, and trace amounts of 2, 2, 4-trimethyl-1, 3-pentanediol di-isobutylate (TMPDIB: Texanol) were detected. These substances in 120 year-sample are TQOM in air. It was considered that the organic matter in the 120 year-sample was absorbed from the air. The above finding suggests that the strange odors emitted from cement involved TQOM such as phthalates  when brought in contact with water are possibly due to chemical hydrolysis of phthalates in the cement and that the organic substances contained in cement were hydrolyzed instantaneously and generated the odor of 2-ethyl-1-hexanol (2E1H) gas, and/or butanol. The odor was noted to occur instantaneously during the preparation of the mortar samples using the 9 types of cement but not with the extracted cement (cement extracted organic matter from Nittetsu cement with Soxhlet and chloroform solution), indicating that trace amounts of phthalates were contained in these cements. It has been believed that the TQOM in cement has no effect on the damage to concrete structures and asphalt pavements. A small amount of phosphate compounds (Nittetsu cement), which is not dissolved in chloroform solution and could not be extracted by this method using Soxhlet extractor, was detected at a concentration of 0.12% by ICP . These series of experiments suggest that commercially available Nittetsu cement contains phthalates and phosphate compounds, and that modern concrete structures are also exposed to trace amounts of surfactants added to snow melting agents. The effect of surfactants on the damage induced deterioration of concrete had been also investigated using cement paste samples (mixtures of cement and water) (2011) 267–281 , but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.b) Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.  http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” .Line 199: Kindly write full name for “TSM”, “SPNES”, when they appear for the first time OR separately "Provide a list of abbreviations" and then do not use the whole names throughout the manuscript. At the moment, it is mix-up.Line 218: Kindly Write fully explained term for “1H NMR”. This will be otherwise confusing for the readers. You may alternatively write “Nuclear Magnetic Resonance test”. Similar complete names can be written for 2.2.2, and 2.2.3.Line 233: What is meant by “ “? Kindly omit all such themes throughout the manuscript as they are not understandable at all.As a whole, the test procedures are described in detail. In my humble view, brief description with some reference is sufficient for the interest of the readers.Results and Discussion:It is better to indicate the test methods with reference rather than the brand names of the instrument.Heading 3.1: Can the observations be presented in tabular form rather than paragraphs?Line 391: Kindly elaborate “2E1H” for the first time.Line 395: What is meant by “This gas”?Line 412: Change “investigate” with “investigated”.Kindly elaborate “DD” as “Degree of Deterioration”, when it appears for the first time.Kindly enhance font size in Table 1.Test methods are repeatedly presented: It is suggested to present the names of the tests and their references rather than explaining the tests again and again. This has made the manuscript very lengthy.Conclusions:Conclusions are somewhat dry. Line 1275 to 1286 can be added to the conclusions rather than being the part of the results and discussion.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Paper is very lengthy and readers won’t be able to understand the clear theme of the paper as contents are irregularly arranged.The authors didn’t mention any details regarding 1H NMR, GC-MS (JMS-AX-500), HPLC, High Volume Sampler, One dimensional Transient moisture permeation test and Micro focus CT scanning (CT) in the Introduction section. It’s hard for the reader to pick new terms later in the paper.I believe that this paper would be of interest to highway engineers. Therefore, make it simple to read.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  13 Feb 2021I appreciate with many reviewers.AttachmentPLOS ONE_Response to revierwes.pdfSubmitted filename: Click here for additional data file. 16 Mar 2021PONE-D-20-22508R1Modern cement concrete and asphalt pavement are damaged by respiratory action and trace quantities of organic matter (TQOM): Civil structures are damaged by respiratory action and trace quantities of organic matterPLOS ONEDear Dr. moriyoshi,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Kindly change the title. Presently, it is in sentence format. Change it to the title format. May I suggest the following title."Deterioration of modern concrete and asphalt pavements by respiratory action and trace quantities of organic matter". I also think there is no need for a short title, so that can be omitted.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Apr 30 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. 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For instructions see: We look forward to receiving your revised manuscript.Kind regards,Anwar KhitabAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments (if provided):Kindly change the title. Presently, it is in sentence format. Change it to the title format. May I suggest the following title."Deterioration of modern concrete and asphalt pavements by respiratory action and trace quantities of organic matter". I also think there is no need for a short title, so that can be omitted.[Note: HTML markup is below. Please do not edit.]https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  23 Mar 2021I am very grateful to Dr. Preston for his encouragement and suggestions for the various new discoveries and the structure of the new treatise. We would also like to thank Dr. Khitab and Dr. Magyar for their long-standing advice, proofreading, and support for this treatise.I think this treatise has become very long and difficult to peer review. We would like to thank all the people concerned.AttachmentPLOS ONE_Response to reviewers_23thMarch.docxSubmitted filename: Click here for additional data file. 25 Mar 2021Deterioration of modern concrete structures and asphalt pavements by respiratory action and trace quantities of organic matterPONE-D-20-22508R2Dear Dr. moriyoshi,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. 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Immunotherapy has become a pillar of advanced solid tumors treatment. Patients are more likely to benefit from neoadjuvant immunotherapy compared with traditional neoadjuvant therapy. However, the safety and efficacy of neoadjuvant immunotherapy for the treatment of locally advanced, surgically resectable Esophageal squamous cell carcinoma (ESCC) remain unknown.ESCC patients who received neoadjuvant treatment following minimally invasive esophagogastrostomy were enrolled from June 2020 to September 2021. The characteristics of neoadjuvant treatment and surgery were investigated to determine the safety and efficacy of the neoadjuvant combination of chemotherapy and immunotherapy (NCI).p>0.05). Regarding to the operation, the NCI group had less blood loss , and shorter operation time  than the NCR group. Additionally, the NCI group demonstrated a lower rate of overall perioperative complications (p=0.003) and grade >2 perioperative complications (p=0.042) than the NCR group.A total of 149 patients were included in the study. Patient ratio was 40:109 between NCI and neoadjuvant chemotherapy plus radiotherapy (NCR) groups. No significant difference was found in terms of pathological characteristics, including ypN stage, ypTNM stage, differentiation, lymphovascular invasion, perineural invasion, pathological complete regression and tumor regression score, and these parameters were not correlated with NCI or NCR (all Overall, the findings reported here indicate NCI could result in better outcome and less complications to locally advanced ESCC patients compared with NCR therapy. As a novel therapeutic option, the efficacy and safety of NCI appears to be feasible and safe, while long-term survival data is still needed. Esophageal cancer, a life-threatening disease, has become the 5th leading cause of death worldwide, of which 5-year survival remains approximately 15–25% due to its high malignant potential and poor prognosis . SurgerySince the approval of immune checkpoint inhibitors (ICIs), cancer immunotherapy has made an indelible mark in the field of cancer treatment. With widely application of PD-1/PD-L1 and CTLA-4 inhibitors among various indications, these ICI agents has developed a transformative response to advanced solid tumors and unequivocally shown long-term clinical advantages to certain patients over the last decade –14. DespRadical resection is typically the mainstay of curative treatment for esophageal cancer; however, it is associated with a high rate of postoperative complications, especially in patients receiving neoadjuvant therapy. Neoadjuvant treatment can cause necrosis, oedema, and adhesion of local tissues, which increases operation difficulty as well as the risk of postoperative complications –20. HereConsecutive ESCC patients who received neoadjuvant chemotherapy combined with immunotherapy (NCI) or neoadjuvant chemotherapy combined with radiotherapy (NCR) following minimally invasive esophagogastrostomy (MIE) from January 2020 to May 2021 were retrieved from the esophageal cancer database of West China Hospital. Clinical characteristics, neoadjuvant therapeutic details and operation-related information were exported from our database as well. The study was performed in accordance with the tenets of the Declaration of Helsinki and ethics approval was obtained from the Institutional Review Board and the Ethics Committee of West China Hospital of Sichuan University. Written informed consent were signed from all participants in this study.1N + M0 or T2-4aN0-3M0; and (IV) without severe comorbidities such as active gastrointestinal bleeding, gastrointestinal obstruction, perforation, embolism, shock; (V) noncervical esophagus and (VI) Zubrod-Eastern Cooperative Oncology Group-WHO (ECOG) 0-1  plus cisplatin , fluorouracil  plus cisplatin , etc. Five PD-(L)1 blockades that contained pembrolizumab , tislelizumab , camrelizumab , sintilimab  and toripalimab , were applied to neoadjuvant immunotherapy whereas the total radiation dose was 40–50Gy, which was given in 23 fractions of 1.8–2.0Gy each with 5 fractions per week for radiotherapy. Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 was utilized to evaluate the complications caused by neoadjuvant treatments, with grades 2–5 indicating the existence of complications  0-1 . All patth AJCC/UICC TNM stage  according to formalin-fixed paraffin-embedded (FFPE) specimens. Pathological information, including tumor invasive depth, lymph node status, metastasis, differentiation, lymphovascular invasion (LVI), perineural invasion and tumor regression score (TRS) was recorded as well. The TNM staging method was based on the ypTNM stage referencing to the 8th edition of the AJCC/UICC staging system . TRG wasχ2-test), likehood ratio test Cochran-Armitage trend test and Fisher’s exact test were determined in terms of variable statistics. Statistical significance was determined using the two-tailed test, where p-value of <0.05 was considered statistically significant.All statistical analyses were performed with IBM SPSS Statistics version 25.0 . Methods including Student’s t-test, Chi-square test , and 109 received neoadjuvant chemotherapy plus radiotherapy (NCR). The patient selection process is presented as a flow chart in vs. 2.00 ± 0.67, p=0.33). Paclitaxel + cisplatin + radiotherapy and fluorouracil + cisplatin + radiotherapy were administered to most patients in the NCR group. Among them, 94 patients received a paclitaxel + cisplatin + radiotherapy regimen, 8 received fluorouracil + cisplatin + radiotherapy and 7 received paclitaxel alone + radiotherapy. In the NCI group, the immunotherapy drugs consisted of PD-1 blockades. Eleven patients received pembrolizumab, 9 received tislelizumab, 11 received camrelizumab, 5 received sintilimab, and 4 received toripalimab. Paclitaxel + cisplatin or fluorouracil + cisplatin was also administered as neoadjuvant chemotherapy in the NCI group. There is no significance on the cycles of neoadjuvant treatment between two groups . In addition, two patients experienced grade 2 complications and one experienced grade 3 complications in the NCI group, whereas 4 patients experienced grade 3 adverse effects and 2 experienced grade 2 complications in the NCR group. Additionally, the CTCAE grade distribution was not statistically significant . Other characteristics of adverse effects was presented in The incidence of adverse effects in the NCI group and NCR group was 7.5% (3/40) and 5.5% (6/109), respectively, but this difference was not significant (Chip>0.05).All patients received McKeown MIE and two-field lymph node dissection by experienced surgeons after finishing neoadjuvant regimens. No significant difference was found in terms of the pathological characteristics, including ypT stage, ypN stage, ypTNM stage, differentiation, LVI, perineural invasion TRS grade and pathological complete regression (pCR), and these parameters were not correlated with NCI or NCR . However, there was no significant difference in the other operation-related parameters, including hospital stay after operation, lymph node dissection number, LN resection station, number of positive LNs, interval between neoadjuvant therapy and surgery, chest drainage duration and chest drainage volume . The other details of the operative outcomes are summarized in Blood loss and operation time presented significant differences between the NCI and NCR groups , cases of 30-day mortality and 90-day mortality only occurred in the NCR group. According to Clavien-Dindo grade, not only the overall perioperative complications demonstrated a statistical significance (p=0.003), but also a significant difference existed in the term of grade >2 perioperative complications between the two groups, and grade >2 perioperative complications were correlated with different preoperative treatments (p=0.042)  Table 5.Neoadjuvant therapy is considered as a potential approach to prolong postoperative survival of patients with radical resection. Another presumable advantage is to convert inoperable patients with undistinguished tumor boundaries or unresectable lesions to extent of favorable radical resection condition. Preliminary studies suggested immune therapy combined with chemotherapy exhibited the same objective response rate (ORR) as traditional neoadjuvant chemoradiotherapy –30. In ovia minimally invasive approach. In our research cohort, NCI and NCR shared the same post pathological characteristics including yp stage, TRS grade and lymphovascular/perineural invasion status. However, NCR’s response to neoadjuvant radiotherapy can contribute to both operation difficulty and intraoperative risk regarding to thoracic tissue injury/edema and pleural adhesions caused by 40-50.4Gy  and key projects of Sichuan Provincial Department of science and technology (Grant No. 2021YFS0233).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Immunoglobulin G4-related disease (IgG4-RD) is a rare immune-mediated disease affecting multiple organs and tissues. There is often the presence of elevated serum Ig4 subtype with histological evidence of lymphoplasmacytic infiltration, fibrosis, and phlebitis. The mainstay of treatment is steroids therapy. We report the case of a 66-year-old man presenting with acute on chronic renal failure and pulmonary nodules seen on PET-CT scan. He also had elevated serum IgG4 subclass and histological features in keeping with IgG4-RD. He failed steroid therapy but responded subsequently to rituximab with complete resolution of his symptoms. IgG4-related disease (IgG4-RD) is a chronic systemic inflammatory disorder typically characterized by extensive infiltration of organs by lymphoplasmacytic cells, presence of storiform fibrosis, and obliterative phlebitis. It remains a diagnostic challenge as it is often mischaracterized as a malignant disease especially due to its tumor-like presentation and multi-organ involvement .A 66-year-old Hispanic man presented with a rapid decline in glomerular filtration rate (GFR) of about 50% from baseline in a 2-year period. Initial evaluation revealed elevated serum IgG4 subclass levels of 1340 . He denied any other urinary symptoms; no weight loss or constitutional symptoms was reported. At baseline, he had a history of stage 3 chronic kidney disease secondary to hypertensive nephrosclerosis, essential hypertension, well-controlled type 2 diabetes, asthma, and prior corrective surgery for a deviated nasal septum. His colonoscopy report was up-to-date and unremarkable. Physical examination and review of systems were unremarkable. The patient’s complete blood count result showed a white cell count of 5200 cells/dl with normal differentials. Hemoglobin was 14.4g/dl. He had normal liver enzymes. Serum IgG4 level of 1340 mg/dl, creatinine of 1.5, BUN of 23, EGFR of 45, glucose of 145, and hemoglobin A1c of 6.7. ANCA titers were not obtained. Urine analysis revealed trace blood and trace leukocyte esterase, but was otherwise unremarkable. Renal ultrasound revealed normal-sized kidneys of 12cm and 12.1cm on the right and left, respectively. Urine and serum Immunoelectrophoresis showed no monoclonal immunoglobulins or monoclonal free light chains. The patient had a left kidney biopsy that showed features of chronic interstitial nephritis with increased IgG4 positive plasma cells  is a chronic systemic autoimmune inflammatory disease typically affecting older persons in the age range of 50 to 70 years. Global data on its incidence is limited and prevalence is estimated at 0.82 per 100 persons between 2007 and 2016. This might be underreported given the poor understanding and recognition of this disease [It is typically characterized by extensive organ infiltration with lymphoplasmacytic cells, presence of storiform fibrosis, and obliterative phlebitis. IgG4- RD remains a diagnostic challenge as it is frequently misdiagnosed as malignancies especially due to its mass-forming presentation and multi-organ involvement .The gold standard for the diagnosis of IgG4-RD remains characteristic histopathological findings on tissue biopsy. Ancillary evaluation including clinical, laboratory and imaging evaluations could be helpful especially in the setting of a high index of suspicion. Serum IgG4 levels are typically >135mg/dl , however, this may or may not correlate with the presence of disease and as such is not a required criteria for diagnosis. Albeit, elevated serum IgG levels tend to correspond with the extent of disease ,3.Commonly affected organs include the salivary glands, lacrimal glands, lungs, thyroid, lymph nodes, central nervous system, pituitary body, aorta, pancreas, liver, gallbladder, bile ducts, retroperitoneum, kidney, and prostate .In its severe form, significant symptoms may arise in the form of obstruction or compression of surrounding organs due to tumor effect. It is important to consider IgG4-related disease when evaluating neoplastic conditions .IgG4-related respiratory disease (IgG4-RRD)IgG4-RRD may be detected incidentally. Patients with lung involvement could be asymptomatic or present with vague and non-specific symptoms like dyspnea, cough, chest discomfort. Non-constitutional symptoms have also been reported . Lung inMost pulmonary imaging done in the setting of IgG4 related disease have reported varying pathologic patterns that include solid nodular density, ground-glass opacities, bronchiectasis, honeycombing, and bronchovesicular thickening ,8.Pleomorphic chest radiographic features in an individual patient should heighten suspicion for IgG-related disease. Lung biopsy may be required in some cases to make a definitive diagnosis ,9. This IgG4-related kidney disease (IgG4-RKD)IgG-RKD is the most common prevalent urologic manifestation of IgG4-RD, often present as tubulointerstitial nephritis (TIN) and less often as membranous glomerulonephritis (MGN). Solitary renal mass, though rare, has also been described -11. AbouOur patient presented with elevated IgG4 serum levels. His IgG4/IgG ratio was >40%, therefore meeting the required diagnostic threshold for IgG4-RD. Differential diagnoses like malignant disease, lupus erythematosus, Wegener’s granulomatosis, sarcoidosis, and metastatic carcinoma should be satisfactorily excluded ,14. FurtRecent studies now propose a better understanding of the pathology of a once poorly understood IgG-4 RD. Studies describing oligoclonal cellular expansion of B cells and CD4+ Cells, especially the cytotoxic subsets, is believed to play a central role in disease pathogenesis. The T follicular helper cells (IL-4 secreting subset of CD4+ cells) working in tandem with Interleukin-10 is thought to drive B cell maturation, resulting in IgG-4 class switching. The part played by macrophages in the production of fibrosis through its interplay with the acquired immune system and fibroblasts is currently being studied and provides potential favorable therapeutic advancements .TreatmentThe current recommendation for the initial treatment of IgG4 related disease are systemic corticosteroids . There aIgG-RD is a chronic inflammatory disease that often runs an indolent course and may affect virtually any organ of the body. It could be misdiagnosed clinically as malignancy. Diagnosis remains a challenge. However, the recent EULAR diagnostic criteria are very comprehensive and would serve as a useful guide. The first line of treatment is systemic glucocorticoids. Rituximab may be used as a second line agent if there is intolerance or suboptimal response to steroids. Surgery may also be considered in cases where symptoms are related to mass effect from a pseudotumor.


Introduction: Bacterial biofilms are an important virulence factor inchronic periprosthetic joint infection (PJI) and other orthopedic infectionsince they are highly tolerant to antibiotics and host immunity. Antibioticsare mixed into carriers such as bone cement and calcium sulfate bone voidfillers to achieve sustained high concentrations of antibiotics required tomore effectively manage biofilm infections through local release. The effectof antibiotic diffusion from antibiotic-loaded calcium sulfate beads(ALCS-B) in combination with PMMA bone cement spacers on the spread andkilling of Pseudomonas aeruginosa Xen41 (PA-Xen41) biofilm was investigated using a “large agarplate” model scaled for clinical relevance. Methods: BioluminescentPA-Xen41 biofilms grown on discs of various orthopedic materials were placed within a large agar plate containing a PMMA full-size mock “spacer”unloaded or loaded with vancomycin and tobramycin, with or without ALCS-B.The amount of biofilm spread and log reduction on discs at varying distancesfrom the spacer was assessed by bioluminescent imaging and viable cellcounts. Results: For the unloaded spacer control, PA-Xen41 spread from thebiofilm to cover the entire plate. The loaded spacer generated a 3 cm zone ofinhibition and significantly reduced biofilm bacteria on the discsimmediately adjacent to the spacer but low or zero reductions on those further away. The combination of ALCS-B and a loaded PMMA spacer greatlyreduced bacterial spread and resulted in significantly greater biofilmreductions on discs at all distances from the spacer. Discussion: Theaddition of ALCS-B to an antibiotic-loaded spacer mimic increased the area of antibiotic coverage and efficacy against biofilm, suggesting that acombination of these depots may provide greater physical antibiotic coverageand more effective dead space management, particularly in zones where thespread of antibiotic is limited by diffusion (zones with little or no fluidmotion). While infrequent, PJI has a dramaticeffect on the patient's health, often resulting in joint dysfunction,morbidity, and mortality. Thefinancial burden placed on patients and the healthcare system is staggering. A major complicating factor in treatingPJI is microbiota-produced biofilms. Biofilms are pathogenic communities that adhere to living andnonliving surfaces and exhibit greatly increased antibiotic tolerance andresistance against host immunity. The establishment of biofilms is assistedby the presence of foreign materials of orthopedic implant components, suchas various metals and polymers.Pseudomonas aeruginosa is a Gram-negative opportunistic nosocomial pathogen and is cultured up to20 % of the time in chronic Gram-negative PJI.In previous in vitro studies, P. aeruginosa has shown the ability to display tolerance and resistance to antibiotics commonly used to treat PJIs. Although the Gram-positive Staphylococci are the most commonlyisolated pathogens from PJI, the infecting organism (or organisms) may notbe cultured as much as up to 25 % of the time or treatment isstarted before culture data are available. Thus, in treating PJI,combinations of antibiotics are commonly used to provide broad-spectrumcoverage . Vancomycinand/or aminoglycosides (most commonly gentamicin or tobramycin) can be mixedinto polymethyl methacrylate (PMMA) bone cement and mineral-based absorbable bone fillers such as calcium sulfate dihydrate (CaSOP. aeruginosa Xen41 (PA-Xen41) showed that the number and spacing of ALCS-B were important in the rate and extentof killing of an agar lawn biofilm. Further, we have previously shown in a similar model that theantibiotic loading concentration in ALCS-B did not significantly change theeffective area of antibiotic activity over the observed time frame, fromwhich we concluded that the area of antibiotic potency was limited primarilyby diffusion, not the loading potency. While we expect that increasing the area of spread of antibioticdepots will result in increased spatial coverage of antibiotic activity, wewish to determine the spatial effect of adding ALCS-B to an antibiotic-loaded spacer mimic on the spread of bacteria from biofilm-colonized materials, as well as the killing efficacy of biofilm bacteria on thosematerials in a scaled-up in vitro model. In the present study, we evaluate the effect of ALCS-B and antibiotic-loaded bone cement spacers (ALBC-S) on containing both the spread and killing of P. aeruginosa biofilms using a “large agar plate”model. We used P. aeruginosa Xen41 in part because of its strong bioluminescent signal, allowing for more sensitive and illustrative monitoring of the effect ofantibiotic diffusing from the PMMA or ALSC-B by an in vitro imaging system (IVIS).Previous studies using in vitro biofilms of the bioluminescent 22.1Pseudomonas aeruginosa PAO1 was used. Stock culture was streaked onto 1.5 % tryptic soy agar  and incubated for 24 h. A singlecolony was transferred aseptically to 15 mL of tryptic soy broth  and incubated overnight at37 P. aeruginosa isa relevant Gram-negative PJI pathogen.A bioluminescent derivative strain of 2.2Overnight cultures were diluted to 0.1 % and used to inoculate sterile,circular discs  of hydroxyapatite (HA),ultra-high molecular weight polyethylene (UHMWPE), 316L stainless steel(SS-316), and titanium (Ti). The “as received” roughnesses measured by contact profilometry were 976, 3867, 224, and 300 nm, respectively.The discs had a diameter of 9.5 mm and a thickness of 2 mm. Four milliliters of the diluted culture was added to four different wells of a six-well plate andthree discs of each material were aseptically submerged in the inoculum. Theplate was incubated at 37 2.3® Rapid Cure 10-cc mixing kits. Twenty grams ofCaSOALCS-B were prepared using Stimulan2.4PMMA bone cement spacer mimics were prepared usingSimplex™ P SpeedSet™Radiopaque Bone Cement construction kits , which are commonly used clinically. Antibiotic-loaded spacers were fabricated using 2 g VAN powder, 2 g TOB powder, and 40 g PMMA powder. Unloaded spacers were prepared with onlyPMMA bone cement powder. Twenty milliliters of sterile methyl methacrylate monomer (liquid) was added to the powder mixture, stirred into a dough-like mass, formed into a “hockey puck-like” disc using a 7 cmdiameter circular mold  and allowed to set for 30 min at 20 2.5Fifty milliliters of 1.5 % TSA was added to a 21 cm diameter glass pie baking dish sterilized with 70 % EtOH and allowed to solidify, followed by centralplacement of a PMMA spacer followed by another 100 mL of molten agar. Onceset, three discs of each material  were placed on top of the agar layer radiatingoutwards . For the experiments with ALCS-B, a 10-cc pack of beadswas sprinkled somewhat randomly around the spacer with greater bead densitycloser to the spacer mimic. While sprinkling the ALCS-B without carefulplacement in a specified pattern may cause difficulty with interpretation,we wished to more closely mimic how they might be applied clinically, wherethey might not be spread evenly. Also, small diameter antibiotic depots suchas ALCS-B can fill smaller void spaces than the larger PMMA beads and thuscan be distributed more thoroughly.After adding the beads, another 100 mL of cooled  that semi-quantitatively measures the relative amount of metabolically activebiofilm. Each quadrant of the large plate model was individually imaged andthen stitched together  to show the whole plate. Redrepresented the highest metabolic activity and blue or black low or nometabolic activity. White-light (plain) images of each plate were captured with a cellphone.2.7Colony-forming unit (CFU) counts were performed on mimicked sets of discs, one set on the 3 d biofilms and one set after incubation in the largeplate model. CFUs were performed as previously described.These discs were rinsed and then vortexed with 10 mL of phosphate-bufferedsaline  in 15 mLFalcon tubes ; 10 2.8All experiments were performed in triplicate. The effect of the differentconditions on the log reduction of biofilm at different proximities from thespacer was compared by first performing a log33.1P. aeruginosa Xen41 allowed biofilm on the discs and the spread ofbacteria from these discs to be easily visualized while remaining in situ in the large plate model over the 5 d . The unloaded spacer (US)condition of the large plate, containing no antibiotic, showed substantialbacterial spread from the discs and was confirmed by white-light imaging, showing P. aeruginosa coverage throughout the large plate .The bioluminescence of The antibiotic-loaded spacer (LS) alone exhibited anti-bacterial activity generating a zone of inhibition (ZOI) of approximately 2.4–3.0 cm from theedge of the spacer . After 3 d a small number of colonies were observed growing a few millimeters within the edge ZOI . These were possibly antibiotic tolerant phenotypes andhave been observed previously.The LS 3.2After 3 d of inoculation, the amount of PA-Xen41 biofilm grown onvarious discs was quantified by cell counting (CFU). This analysis wascompleted to confirm biofilm growth on each type of disc beforeimplementation into the large plate model. All four disc materials displayedsizeable biofilm growth of The LS alone reduced biofilm concentrations on the closest discs (1 cm fromthe spacer's edge), with bacterial reduction ranging from 1.5 to 5.5 log4P. aeruginosa Xen41 biofilm growth on materials common to orthopedicimplants in vitro while also increasing the zone of antimicrobial potency beyond that achieved by an antibiotic-loaded PMMA spacer alone. Furthermore, our work demonstrates how such beads sprinkled around the spacer may moreeffectively manage dead space and control the spread of bacteria from suchimplant surfaces, particularly in those locations where the spread ofreleased antibiotics is limited by diffusion, as would be expected in theperiprosthetic tissue, areas of a joint space, or the medullary canal. In awell-mixed system, the specific distribution of antibiotic depots will beless important since the antibiotics will be well distributed by the mixing,but when the rate of spread of antibiotics is limited by diffusion, then thespatial distribution becomes significant.Here we show in our diffusion-based model that ALCS-B significantly reduced. Antibiotics loaded into PMMA cement in the form of both beadsand spacers have been successfully used to treat PJI for many years. However, thereare limitations to the use of PMMA alone with respect to the release oflocal antibiotics at a surgical site. Once an antibiotic has eluted to belowMIC levels, the PMMA itself becomes a surface for bacterial biofilmformation. Further, even though retrieved PMMAspacers are still shown to elute antibiotics and produce a ZOI in laboratorytesting, the infection can persist, suggesting that the zone ofantimicrobial potency may be limited.Antibiotic-loaded PMMA beads have been used to increase the effective area of antibiotic activity, but they have limited penetration into small spacesand require surgical removal. Additionally, their relatively large size discsor fell farther away. This resulted in variability of killing efficacy. Ifbeads fell close to a biofilm-colonized disc, then there was a significant log reduction or eradication to below-detection limits, while discs furtheraway from beads exhibited less or no reduction. This spatial variation isevidenced by some of the large error bars  and illustrates theimportance of adequate spatial coverage within a periprosthetic joint orother infected surgical sites. Nevertheless, our in vitro data as well as those found clinically, and data from other in vitro studies  support theuse of ALCS-B as an effective means of releasing antibiotic to preventbiofilm spread and kill pre-grown bacterial biofilms.We recognize limitations in our study. Our study was conducted with only onebacterial strain. While we have shown similar efficacy with other Gram-negative and -positive species with respect to prevention and killingefficacy by antibiotics released from beads alone, to add rigor to our conclusions, we would need toinclude more pathogenic species and clinical strains. In addition, we used arelatively young biofilm grown for 3 d which may be less antibiotic-tolerant and less clinically relevant than more mature biofilms. Also, ourin vitro model is simplistic compared to the complex mechanical and chemical environment of surrounding bones, particularly that of an articulating humanjoint space. By design our model lacks any fluid flow, which would beexpected to enhance the spread of antibiotic from the spacer mimic and theALCS-B while also removing antibiotic from the system . Apharmacokinetic study by Hayes et al. (2014) found the intra-articular concentration of gentamicin releasedfrom a sponge in a dog dropped rapidly from approximately 2400to 4 The results from our in vitro study may support the adjuvant use of ALCS-B in the management of PJI, particularly for the release of antibiotics to theinterstices of the periprosthetic joint space in light of limited PMMAdiffusion with compromised vasculature. However, further work is required inorder to determine how our in vitro findings relate to the spread of antibiotics in the human joint or may relate to clinical outcomes. Such studies are nottrivial since measuring antibiotic concentrations at different locations intissues and fluids in a joint space requires multiple samplings at differenttimes, since sampling of joint fluid may not represent conditions everywherebacteria or biofilm may be present. We are currently developing a continuousflow reactor system which can accommodate clinically relevant antibiotic-loaded spacer mimics and beads, where we match the flow rate with reportedclinical drainage rates following TKA revision. Such a system may providefurther insight into the complex interplay between antibiotic releasekinetics from various administration methods, including addition ofantibiotic powder.