Background: Antimicrobial resistance (AMR) is a great threat to public health. The primary cause of AMR is human antibiotic misuse. Little is known about regional differences of antibiotic misuse behaviours in China.Objectives: To explore the antibiotic misuse behaviours among university students in western and eastern China and find out the regional differences.Methods: Participants were recruited from universities in less developed Guizhou Province and developed Zhejiang Province using a cluster random sampling method. A self-administered questionnaire was designed to collect data, and the χ2 test and logistic regression were adopted to assess the associations between region and antibiotic misuse behaviours.Results: A total of 2073 university students from Guizhou and 1922 from Zhejiang completed questionnaires. Students in Guizhou had lower household income, parents’ education, and urban residence proportion than those in Zhejiang. Compared with those in Zhejiang, students in Guizhou had higher antibiotic use prescribed by doctors (79.8% vs 56.2%) and self-medication with antibiotics (33.0% vs 16.1%). Students in Guizhou were more likely to buy over-the-counter antibiotics without prescriptions (73.9% vs 63.4%), ask for antibiotics from doctors (21.4% vs 15.6%), and use antibiotics prophylactically (29.9% vs 15.7%). Adjusted models showed that the less developed region was significantly associated with higher antibiotic misuse behaviours.Conclusions: Misuse of antibiotics by well-educated young adults was very high in two regions but most serious in the less developed one. Campaigns are urgently needed to promote appropriate antibiotic use especially in less developed regions. The advent of antibiotics is considered one of the most critical advents in medical science, and has saved millions of lives over the past 100 years –4. The rIn China, antibiotics misuse is pervasive ,7, leadiThe massive levels of antibiotic misuse for self-limiting diseases can be attributed to factors on both the supply and demand sides in the Chinese health system. On the supply side, health providers relied heavily on drug sales to make profits, which inevitably resulted in overprescribing of antibiotics by hospitals and community health facilities . Loose rIn order to address this gap, the current study aims to compare antibiotic misuse behaviours among university students in a less developed western province and a more developed eastern province in China and to explore the association between socio-economic factors and antibiotic misuse behaviours.This study is a part of a large national cross-sectional survey regarding antibiotic misuse behaviours among university students in China. Among the six sampled provinces, we selected the western province, Guizhou, and the eastern coastal province, Zhejiang, with greatly differing levels of social and economic development. The GDP per capital of Guizhou was 29,847 RMB  and Zhejiang 77,644 RMB , ranked 29th and 5th, respectively, among the 31 provinces of China in 2015 . In eachself-treatment with antibiotics, antibiotics use prescribed by doctors, asking for antibiotics from doctors in the past month, buying antibiotics without prescriptions, keeping antibiotics in dormitory or home, and taking antibiotics prophylactically in the past year. The questions on antibiotic use behaviours were adapted for the Chinese setting from previous studies [The questionnaire was comprised of two sections: socio-demographic characteristics of participants and their healthcare-seeking behaviours related to antibiotic use. Questions regarding socio-demographics collected information including gender, age, education level, major, household income per month, and parents’ medical background and their education level. The behaviours included  studies ,16. Studhttps://www.sojump.com) was used to conduct the survey. A cluster random sampling method was adopted. Permission was initially obtained from authorities at each university to conduct the survey. The aim was to achieve a sample size of roughly 1800 students across a range of disciplines, including science, social science/the humanities, and medicine, at each university [The data for this study, collected from September to November 2015, included 3995 university students drawn from the two participating universities. The electronic questionnaire tool Wen Juan Xing .Written informed consent was obtained from all respondents who participated in the study. It was clearly explained that participation was not compulsory. The study was approved by the Zhejiang University Research Ethics Committee.A total of 2073 university students in Guizhou and 1922 in Zhejiang completed the questionnaires. Students from the two provinces presented very similar social demographics across age, gender, and education level. Medical students accounted for 22.1% (425) of the respondents from Zhejiang University and 14.6% (303) from Guizhou University. Students in Zhejiang were significantly wealthier than those from Guizhou. One-third of students in Zhejiang and two-thirds in Guizhou originated from a rural area. The parents of students in Zhejiang generally had a higher education level, and almost twice as many had a medical background when compared with Guizhou.In the past month, roughly 30% of students in both Zhejiang and Guizhou experienced a self-limiting illness. Compared with Zhejiang, students in Guizhou were more likely to see a doctor (35.0% (213) vs. 28.3% (162)) when they were ill, less likely to select primary care health facilities (66.7% (142) vs. 77.1% (125)), and more likely to be prescribed with antibiotics by doctors (79.8% (170) vs. 56.2% (91)). Students in Guizhou were twice more likely to self-treat with antibiotics.In the past year, 55.6% (1152) of students in Guizhou and 64.2% (1233) in Zhejiang kept antibiotics in their dormitory or home. Compared with Zhejiang, Students in Guizhou were more likely to buy antibiotics without prescriptions (73.9% (992) vs. 64.7% (578)) and ask for antibiotics when doctors did not initially prescribe them (21.4% (444) vs. 15.6% (300)), and were almost twice more likely to use antibiotics prophylactically (29.9% (620) vs. 15.7% (302)).After adjusting for all covariates, students in Guizhou were more likely to be prescribed with antibiotics by doctors , self-treat with antibiotics , buy antibiotics over the counter without prescriptions , ask for antibiotics when doctors did not initially prescribe them , and take antibiotics prophylactically .Students with a medical background were significantly associated with better antibiotic use behaviours. They were less likely to be prescribed with antibiotics by doctors , ask for antibiotics from doctors , and take antibiotics prophylactically . However, students whose parents had medical backgrounds demonstrated poorer antibiotic use behaviours. They were more likely to self-treat with antibiotics , keep antibiotics in their dorm/home , and take antibiotics prophylactically , but were less likely to buy antibiotics without prescriptions .Female students, those with higher household incomes, from urban areas, and whose parents had a higher education level or medical background were more likely to keep antibiotics in their dorm/home. Students from rural areas were more likely to be prescribed with antibiotics by doctors .This study investigated the antibiotic misuse behaviours of university students from two provinces that represent a wide diversity of social and economic development in China. We identified that university students in the less developed province were more likely to misuse antibiotics, including asking for antibiotics during a hospital visit, using antibiotics for self-medication, buying antibiotics without prescriptions, and taking antibiotics prophylactically. We also demonstrated significant associations between socio-demographic characteristics and antibiotic misuse behaviours. To our knowledge, this represents the first study investigating the disparity in antibiotic misuse behaviours between well-educated young populations in developed and less developed regions with vastly different levels of social and economic development.In China, antibiotic misuse has been attributed to factors at both the supply and demand side of the health system. On the supply side, this study showed that the overprescribing of antiobiotics by doctors was more prevalent in the less developed region. Misaligned economic incentive for health providers is the main contributor to the overprescribing of antibiotics in China. Although China has implemented a zero drug profit policy since 2009, which aimed at removing profits on drug sales of health facilities, the connection between drug companies and individual doctors still contributes to this problem ,19. CompPharmacy also represents a supply-side actor in antibiotic overuse. Globally, most antibiotic use occurs outside hospitals, and non-prescription access to antibiotics is common ,23. AlthOn the demand side, even while participants in both less and more developed regions are well educated, other socio-demographic characteristics were found to affect antibiotic misuse. Urban students were less likely to be prescribed with antibiotics by doctors than were their rural counterparts. A possible reason for this is that students from rural areas were more easily induced by doctors than were urban students. Previous studies have shown that doctors were much less likely to prescribe antibiotics if patients demonstrated their knowledge of appropriate antibiotic use during the consultation ,25. AnotIn this study, we found that having a medical background represents a protective factor for antibiotic use behaviours. The predominant explanation for this result is that, compared with their non-medical counterparts, medical students generally get more professional knowledge and guidance on appropriate antibiotic use through their (at a minimum) five-year medical curriculum, which usually consists of a series of didactic lectures on antibiotics and opportunities to observe the antibiotic prescribing practices of senior physicians during their internship. A study carried out among university students in northeastern China has supported this finding, and similarities can be drawn from previous studies conducted in Jordan that highlight existing gaps in the knowledge between medical and non-medical students regarding antibiotic use ,29. ThusStudents whose parents had a medical background were associated with higher antibiotic misuse behaviours, including self-treatment with antibiotics, keeping leftover antibiotics in their dorm/home, and taking antibiotics prophylactically. A possible reason for this is that students from a family with a medical background have been self-treated by their parents when they were young, and maintained these habits into adulthood. However, a Korean study found that a higher proportion of physicians and pharmacists believed that antibiotics could treat the paediatric common cold or reduce its complications, when compared with parents, which indicates that healthcare providers may possess higher levels of unfounded beliefs regarding the effectiveness of antibiotics than do laypeople .Keeping antibiotics in the dorm/home has been identified as an important contributor to antibiotic misuse . In our Many studies indicated that antimicrobial resistance is increasing in developing countries . As mentAnother notable finding is that university students lacked adequate knowledge on appropriate antibiotic use, and performed a variety of antibiotic misuse behaviours. Greater attention should be paid to improving the awareness and knowledge of appropriate antibiotic use among this population. University students represent a highly educated group of the population, and their knowledge and behaviour concerning public utilization of antibiotics may significantly influence antibiotic-related issues in China in the future. Furthermore, they represent the next generation of parents in China, and paediatric antibiotic overuse is a pressing concern . Most imThere are several limitations to this study. Firstly, this is a cross-sectional study, and as such cannot establish cause–effect relationships between factors and antibiotic misuse behaviours. Secondly, this study mainly focused on antibiotic misuse behaviours of university students, but did not explore the attitudes or knowledge of students on antibiotic use and social habits of self-medication with traditional herbs in Guizhou and Zhejiang, which turned out to be essential factors in analysing determinants of antibiotic use behaviours. Finally, the survey was conducted in Wen Juan Xing, which makes it difficult to validate the behavioural responses. Future studies using experimental, longitudinal, or case-control designs could help provide evidences for causal relationships. Although the data rely on self-reporting, our survey items were adapted from previously tested surveys. In general, we believe the survey is well representative of the university student population in developed and less developed China.Obviously, antibiotic misuse for self-limiting illnesses by well-educated young adults was very high in these two sampled regions but most serious in less developed China. A campaign is urgently needed for appropriate prescribing of antibiotics by doctors, enforcing restrictions on selling antibiotics over the counter without prescriptions, and to educate the general public about the management of self-limiting illness.


Lactobacillus harbinensis strain BM-LH14723 were identified in one spoiled beer sample by genome sequencing, with the induction and resuscitation of VBNC state for this strain further investigated. Formation of the VBNC state was triggered by low-temperature storage in beer (175 ± 1.4 days) and beer subculturing (25 ± 0.8 subcultures), respectively, and identified by both traditional staining method and PMA-PCR. Resuscitated cells from the VBNC state were obtained by addition of catalase rather than temperature upshift, changing medium concentration, and adding other chemicals, and both VBNC and resuscitated cells retained similar beer-spoilage capability as exponentially growing cells. In addition to the first identification of both culturable and VBNC cells of an individual L. harbinensis strain from spoiled beer, this study also for the first time reported the VBNC induction and resuscitation, as well as verification of beer-spoilage capability of VBNC and resuscitated cells for the L. harbinensis strain. Genes in association with VBNC state were also identified by the first genome sequencing of beer spoilage L. harbinensis. The results derived from this study suggested the contamination and spoilage of beer products by VBNC and resuscitated L. harbinensis strain BM-LH14723.Occasional beer spoilage incidents caused by false-negative isolation of lactic acid bacteria (LAB) in the viable but non-culturable (VBNC) state, result in significant profit loss and pose a major concern in the brewing industry. In this study, both culturable and VBNC cells of an individual  Bacteria in VBNC state have been considered to be a significant issue in public health and food safety, as on one side they fail to grow and form colonies on the routine bacteriological media, but remain alive and retain metabolic activity, and on the other they could regain culturability after resuscitation3.First reported in 1982, Viable but nonculturable (VBNC) state has been well established and documented to be a survival strategy of nonsporeforming bacteria in response to natural stress, such as starvation, extreme temperature, elevated osmotic pressure, oxygen concentration, or exposure to visible light5. However, none of beer spoilage caused by VBNC bacteria has been verified and confirmed.0Beer is a universally popular beverage and has a large consumption market. Despite high microbiological stability, beer spoilage incidents caused by microorganisms, such as lactic acid bacteria (LAB), have occasionally been reported due to false negative routine detection for microorganismLactobacillus harbinensis, a newly identified beer-spoilage LAB, produces lactic acid as end product, acetic acid and diacetyl as metabolic byproducts during carbohydrate fermentation which significantly and undesirably influence the flavors of beer. Occasional beer spoilage incidents due to false-negative detection of causative microorganisms, result in significantly profit loss and have been considered to be a leading problem in the beer brewing industry8. Failure to detected these spoilage agents, are partially due to the use of traditional culture-based techniques, which are unable to reliably detect the VBNC microorganisms commonly found under high levels of microbial stress11.L. harbinensis strain BM-LH14723 were identified in one spoiled beer sample, with the induction, resuscitation and characteristics of the VBNC state further defined. This study represents the first report of beer spoilage by both culturable and VBNC Lactobacillus cells, and demonstration of L. harbinensis entry into and resuscitation from the VBNC state. The first genome sequence of L. harbinensis is also reported.As occasionally reported, finished beer that previously passed random microorganism detection by “golden standard” routine culturing, are found spoiled after shelf storage. In this study, both culturable and VBNC cells of an individual 2 cells/mL, demonstrating the presence of VBNC cells in the specific spoiled beer sample acquired in Guangzhou of South China in 2014. After 16S rRNA gene sequencing, both bacterial cells in the spoiled beer sample and the isolated L. harbinensis cells were further investigated by genomic sequencing. After assembly and alignment, the genomic sequences showed >99% similarity, highly suggesting both the culturable and VBNC cells in the spoiled beer sample were one distinctive L. harbinensis strain (named BM-LH14723).According to MRS agar growth, AODC and Live/Dead BacLight bacterial viability kit methodologies with fluorescent microscopy and flow cytometer Fig. , the difL. harbinensis strain BM-LH14723 is 3,017,769 bp with a coverage of 99.89%, and the average G + C content is 53.36%  most of which ranged from 100 bp to 1000 bp were acquired  to identify the presence of microbes.Approximately 1013. The most common VBNC-resuscitating factor for low-temperature induced VBNC cells is a simple temperature upshift17. However, culturable cells were not obtained when the VBNC L. harbinensis cells induced by either protocols were subjected to temperature upshift (data not shown), highly suggesting cold stress is not essential for the VBNC state formation of L. harbinensis. An advanced beer-spoiler detection (ABD) medium with the supplementation of a small amount of MRS medium had been proposed and considered to be an effective tool for comprehensive detection of beer-spoilage LAB in breweries18. ABD medium was reported to allow the selective growth of beer-spoilage LAB18. Thus, different concentrations of media were attempted to regain the culturability of VBNC L. harbinensis cells. Unfortunately, change medium concentration was not an effective way to resuscitate VBNC state. Considering the oxidative stress the bacterial cells encountered during the formation of the VBNC state under cold treatment and harsh beer condition, relieving the oxidative stress could be a good way to resuscitate the VBNC state. In this study, VBNC L. harbinensis cells regained culturability on media containing all concentrations of catalase tested. Importantly, heat denaturation of catalase inhibited the resuscitation process. The results demonstrated that the addition of catalase is an effective method for the resuscitation of VBNC L. harbinensis cells.Proving that true resuscitation of cells from the VBNC state occurs has been problematic among bacteriaL. harbinensis strain BM-LH14723 under both the VBNC and resuscitated state. During the 30 days, viable cells were detected in beer samples inoculated with the 3 groups of cells, while culturable cells were only identified in beer samples inoculated with exponentially growing and resuscitated cells. After 30 days of incubation, a high concentration of lactic acid and acetic acid which may eventually lead to beer acidification and high level of diacetyl which imparts a “buttery” off flavors were detected in beer samples inoculated with the exponentially growing, VBNC, and resuscitated state of L. harbinensis strain BM-LH14723 13. It has been reported that the oxidative stress response in bacteria is associated with cold stress32, and addition of catalase could relieve oxidative stress and promote the recovery of nonculturable cells33. Consistently, the addition of catalase effectively resuscitated the VBNC L. harbinensis cells induced by both methods. It has been reported that transfer of cells to nutrient-rich environment initiates a metabolic imbalance, thus leading to a rapid production of superoxide and free radicals34. Furthermore, the antibacterial function of hop compounds has been reported to associate with efficient redox reactivity and cause cellular oxidative damage35. Therefore, it is possible that the VBNC L. harbinensis cells are stressed and sensitized to detoxify superoxide during active phenotypic adaptation to the beer environment containing high concentration of bitter hop compounds. Thus, the antioxidant capacity of catalase may alleviate these stress conditions.The VBNC state has been regard as a significant means of survival since the cells are able to increase metabolic activity and again become culturableL. harbinensis cells remained viable and maintained similar beer-spoilage capacity to exponentially growing cells, which was somewhat in accordance with previous studies43. As beer brewery industry and Lactobacillus were concerned, the VBNC L. paracollinocides, L. actotolerans, L. casei, L. plantarum, and L. lindneri cells also exhibited beer-spoilage ability22. Nevertheless, with its detection in breweries and capacity to enter into VBNC state formation under low temperature in beer, L. harbinensis may represent a significant source of beer spoilage cases.The VBNC and resuscitated L. harbinensis strain BM-LH14723, 6 universal stress proteins UspA, 3 predicted membrane GTPases involved in stress response, 1 putative stress-responsive transcriptional regulator, 2 stress-70 proteins, 1 general stress protein, 6 genes involved in response to oxidative stress, and 3 genes involved in response to stress were identified. Gene encoded oxidative stress response has been reported to related to cold stress response in bacteria32. The 6 genes involved in response to oxidative stress might be functional during the VBNC state formation process of L. harbinensis strain BM-LH14723 under low temperature condition. Sigma factor RpoS has been reported to play key role in enhancing stress resistance44 and associate with the VBNC state45. However, RpoS was absent in the genome of L. harbinensis strain BM-LH14723. Nevertheless, sigma factor RpoD and 6 other RNA polymerase sigma factors appeared in the genome might be alternative sigma factors involved in the formation of VBNC state of L. harbinensis strain BM-LH14723.Concerning the VBNC state is a bacterial survival mechanism under stress conditions, genes associated stress response might play important roles in formation of the VBNC state. In the genome of L. harbinensis strain were identified from one spoiled beer. Based on the ability of L. harbinensis to enter into and resuscitated from the VBNC state, this study strongly suggests that the VBNC L. harbinensis cells in the beer sample likely contributed to this spoilage event. Moreover, addition of catalase to routine detection media may lead to more accurate and rapid diagnosis of spoilage possibility, prevent significant profit loss to the brewing industry, and improve food safety for consumers. The genomic analyses also improved our understanding on the intrinsic characteristics of L. harbinensis strain BM-LH14723 including the VBNC state associated genes. In summary, this study provides further evidence that entry into the VBNC state provides a unique survival strategy for LAB associated with beer spoilage.In this study, both culturable and VBNC cells of an individual S rRNA gene upon PCR amplification and purification) and genomic sequencing using the bacterial genomic DNA extracted from the spoiled beer sample, one L. harbinensis strain was identified, which had been designated BM-LH14723. The culturable, total and viable cell numbers of L. harbinensis were calculated by MRS agar plates , acridine orange direct count (AODC) method and Live/Dead BacLight bacterial viability kit  with fluorescent microscope and flow cytometer, respectively46. The difference between culturable and viable number of L. harbinensis cells was also calculated to quantify the proportion of VBNC cells in spoiled beer.In June 2014, one spoiled lager beer sample  was acquired in Guangzhou of South China. Following routine microorganism identification  and subjected to genomic sequencing by Illumina HiSeq. 2500 platform and paired-end libraries, respectively47. Sequences were quality processed using the FastQC pipeline v.0.10.1 before assembly. Upon de novo assembly using Velvet v1.2.08, both genome sequences were aligned by progressive Mauve genome alignment software with default settings (http://darlinglab.org/mauve/mauve.html)48.To validate the existent bacteria, the genomic DNA of bacterial cells from the spoiled beer sample and the isolated L. harbinensis strain BM-LH14723, genes were predicted through Glimmer 3.049, RNAmmer (v1.2)50 and tRNAscan-SE (v1.21)51, respectively. Predicted genes were further annotated by online BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), KOBAS 2.0  via KEGG pathway database52, COG functional classification system53, and local BLAST via NCBI-NR database and GO database54.To determine the intrinsic characteristics of 19. The low-temperature storage system was set up as follows. Approximately 107 cells of L. harbinensis strain BM-LH14723 were inoculated and anaerobically subcultured at 26 °C in 10 mL of the degassed commercial beer. The exponentially growing cells were harvested by centrifugation at 5,000 rpm for 15 min at 4 °C, and then resuspended in 10 mL of degassed beer using 15-mL sterile polypropylene tube at a final density of 107 cells/mL and maintained at 0 °C without shaking. A total of 50 low-temperature storage systems were prepared.Prior to VBNC state induction, the commercial beer were degassed for 10 min at 20 kHz at room temperatureet al. with some modification19. Approximately 107 exponentially growing L. harbinensis BM-LH14723 cells were inoculated in degassed beer and anaerobically incubated at 26 °C. The exponentially growing cells were harvested by centrifugation at 5,000 rpm for 15 min at 4 °C, and then resuspended by 10 mL of degassed beer in 15-mL sterile polypropylene tube at a final density of 107 cells/mL and anaerobically cultured at 26 °C. The interval of each subculture was 7 days.Beer subculturing was performed as described by Suzuki 47. The number of culturable cells were accessed by a conventional plate culture procedure18. A hundred μL of subcultures was spread on MRS agar and incubated anaerobically at 26 °C. The inoculated agar plate was examined every day for colonies formation, and the day on which colonies were first observed was recorded as the time of positive detection. After 14 days of incubation, cells were counted to determine culturable cells number on agar plates. ‘Nonculturable’ was defined as the inability to grow on MRS agar until 14 days of incubation19. Cell viability was determined by using a Live/Dead BacLight bacterial viability kit  with fluorescent microscopy and flow cytometer12.Total, culturable, and viable cells enumerations were performed every 7 days by taking out single tube of low-temperature storage system and before each subculture, respectively. The total cells number was determined by the AODC method with flow cytometer55. Genomic DNA was extracted using bacterial genomic DNA extraction kit  and used as a template to amplify the 16S rRNA gene  in PCR assay.To verify the existence of VBNC cells, 0.5 μg/mL of PMA was added to 1 mL of the bacterial culture once the nonculturable cells identified. The culture was incubated on ice in dark for 10 min, subsequently exposed to halogen light with a distance of 15 cm for 5 min for covalent binding of PMA to DNA, and further cooled to room temperature after the reactionL. harbinensis cells were added into 1.5-mL sterile polypropylene tubes, respectively. Each sample was initially incubated at 10 °C for 1 h and then the incubation temperature was increased by 5 °C every 1 h until reaching 35 °C. One portion of cells was taken out every hour to determine culturability by plating on MRS agar and incubated anaerobically at 26 °C.Upon entry into the VBNC state, 6 portions (100 μL each) of L. harbinensis cells. A hundred μL of VBNC cells were plating on different concentrations of MRS agar, respectively, and incubated anaerobically at 26 °C to observe colonies formation.MRS agar plates with concentrations at 0%, 25%, 50%, 75%, 100%, 150%, and 200% were prepared to recover culturability of VBNC L. harbinensis cells, MRS agar plates with the addition of 10 μL Tween-20, 10 μL tween-80, 0.05 g vitamin C, 0.05 g vitamin B2, and 800 U catalase  were used, respectively. A hundred μL of VBNC cells were plating on MRS agar with the addition of different chemicals, respectively, and incubated anaerobically at 26 °C to observe colonies formation. In addition, different concentrations of catalase  were added to MRS agar to resuscitate L. harbinensis. Simultaneously, heat-denatured catalase (60 °C for 15 min) served as a control.To determine effects of some chemicals on resuscitation of the VBNC 4. Approximately 105 cells/mL of the strain in exponentially growing, VBNC, and resuscitated state were inoculated into 3 groups of commercial degassed lager beer  under sterile conditions at room temperature, respectively, with uninoculated beer as negative control. With beers incubated at 26 °C for up to 30 days, visible growth were examined every day and cellular culturability and viability were detected every 7 days. Subsequently lactic acid and acetic acid concentration were analyzed by reversed-phase high performance liquid chromatography (RP-HPLC). The diacetyl concentration was measured by Head Space Gas Chromatography. Lactic acid, acetic acid and diacetyl were quantified by the external standard method19.The beer-spoilage ability was investigated using established protocols for “growth in beer test”p-value < 0.05 was considered to be significant.Data are presented as mean ± standard deviation (SD) of three independent biological replicates. Statistical comparisons were made by one-way analysis of variance followed by Tukey’s comparison test (XLstat software). A Table S1Table S2Table S3Table S4


H. pylori infection in Indigenous Arctic communities  show frequent failure of treatment to eliminate the bacterium. Among project participants, treatment effectiveness is reduced by antibiotic resistance of infecting H. pylori strains, which in turn, is associated with frequent exposure to antibiotics used to treat other infections. This analysis compares antibiotic dispensation rates in Canadian Arctic communities to rates in urban and rural populations in Alberta, a southern Canadian province.Community-driven projects that aim to address public concerns about health risks from Project staff collected antibiotic exposure histories for 297 participants enrolled during 2007–2012 in Aklavik, Tuktoyaktuk, and Fort McPherson in the Northwest Territories, and Old Crow, Yukon. Medical chart reviews collected data on systemic antibiotic dispensations for the 5-year period before enrolment for each participant. Antibiotic dispensation data for urban Edmonton, Alberta  and rural northern Alberta  during 2010–2013 were obtained from the Alberta Government Interactive Health Data Application.Antibiotic dispensation rates, estimated as dispensations/person-years  were: in Arctic communities, 0.89 ; in Edmonton, 0.55 ; in rural northern Alberta, 0.63 . Antibiotic dispensation rates were higher in women and older age groups in all regions. In all regions, the highest dispensation rates occurred for β-lactam and macrolide antibiotic classes.These results show more frequent antibiotic dispensation in Arctic communities relative to an urban and rural southern Canadian population. Helicobacter pylori infection [.Antibiotic-resistant bacteria are a major public health concern because they limit the effectiveness of therapeutic options available for the treatment of bacterial infections. Much evidence suggests that exposure to antibiotics leads to antibiotic-resistant bacterial infections. In particular, consistent evidence shows an association between frequent exposure to antibiotics for the treatment of unrelated bacterial infections and the prevalence of antibiotic-resistant nfection –6.Helicobacter pylori (CANHelp) Working Group links northern Canadian communities, health care providers and regional health officials with University of Alberta investigators to conduct community-driven research aimed at addressing concerns about health risks from H. pylori infection; the ultimate goal is to inform public health policy pertaining to control of H. pylori infection [H. pylori infection compared to multi-ethnic populations in southern Canada [The Canadian North nfection . In Canan Canada –10. Avain Canada –13 an ocH. pylori is a bacterium that colonizes the lining of the stomach and/or duodenum, [H. pylori infection has been observed to improve peptic ulcer healing, decrease peptic ulcer recurrence and reduce the risk of stomach cancer [H. pylori infection is a major impediment to the development of effective H. pylori control strategies for such populations [Help community projects collected data on participants’ exposure to antibiotics as part of inquiry aimed at identifying causes of treatment failure.uodenum,  where ituodenum, –18. Treah cancer , 20. Freulations . CANHelpFew studies have collected information on antibiotic exposure in geographically defined communities, even though it is known that the frequency of antibiotic use varies by geographic region, as well as other factors including age and sex –24. GeogThe aims of the current study are to describe antibiotic dispensation rates, as a measure of the frequency of overall exposure to systemic antibiotics, by community, sex, age and antibiotic class, in Canadian Arctic communities and compare these rates to those of urban and rural outpatient populations in the southern Canadian province of Alberta. These results will shed light on whether increased exposure to antibiotics is a potential explanation for reduced effectiveness of antibiotics in Indigenous Arctic populations, thus making them vulnerable to poor infection outcomes.H. pylori projects conducted by the CANHelp Working Group in 4 hamlets in the western Arctic region of Canada: projects launched in Aklavik, Northwest Territories (NT) in 2007, Old Crow, Yukon (YT) in 2010, Tuktoyaktuk (NT) in 2011, and Fort McPherson (NT) in 2012 [H. pylori infection; endoscopy with gastric biopsy for pathological and microbiological assessment; treatment; and ongoing knowledge exchange activities. Diverse outreach strategies sought to encourage all community residents to participate. Project staff obtained informed consent from participants 17 years of age or older, parental consent for children under 17 years of age, and assent from 7 to 16-year-old participants.This analysis includes participants in community  in 2012 –38. Each in 2012 , 40. EacH. pylori infection; [Help community project participants who fulfilled either or both of the following criteria for inclusion in this analysis: 1) available H. pylori isolates cultured from gastric biopsies and tested for antibiotic susceptibility; 2) completed treatment to eliminate H. pylori infection and were tested after treatment to assess post-treatment infection status. We collected each participant’s antibiotic exposure history from their medical chart, housed in community health centres, using a chart review tool to record systemic antibiotics prescribed for any reason during the 5 years before the participant’s enrolment date. This 5-year exposure period spanned different calendar years across communities because community projects were launched sequentially: our chart review recorded medications prescribed primarily during 2002–2007 in Aklavik, 2005–2010 in Old Crow, 2006–2011 in Tuktoyaktuk, and 2007–2012 in Fort McPherson . Data collected from medical charts included: demographic factors; frequency of antibiotic prescriptions; types of antibiotics prescribed; and the reason for each prescription. An antibiotic prescription was defined as a single prescription of at least one systemic antibiotic regardless of the dose, dosing frequency or duration of the prescription.The lead author (KW) collected chart review data for her MSc thesis research on factors associated with antibiotic-resistant fection;  specificAs a measure of antibiotic use frequency, we estimated antibiotic dispensation rates, defining dispensation as the process by which a prescribed medication is given to a patient for whom a prescription was written. We used prescriptions as proxies for dispensations because medical charts noted prescriptions rather than dispensations, and because prescriptions given to patients at health centres in participating northern communities are dispensed routinely from locally stocked medication by health centre staff at the time of prescription; thus, in this study population, it can be assumed that each prescription noted in a medical chart was dispensed.We expressed dispensation rates as the average of the number of antibiotic courses dispensed per person per year during each participant’s 5-year review period. We calculated these rates by dividing the total number of systemic antibiotic courses prescribed for all participants during the 5-year review period by the product of the number of participants and the sum of the number of years reviewed in the medical chart of each participant. We estimated antibiotic dispensation rates by community , sex, age (categorized in 20-year age groups), and antibiotic class .To put the antibiotic dispensation rates observed in the western Arctic communities in perspective, we compared them to rates observed in Alberta, a western Canadian province located directly south of the Northwest Territories. We estimated antibiotic dispensation rates for the outpatient populations of urban Edmonton  and rural northern Alberta using the online Alberta Government Interactive Health Data Application (IHDA),  which inWe estimated the outpatient antibiotic dispensation rates by dividing the number of antibiotic courses dispensed by the sum of the population during each year from 2010 through 2013. For statistical comparison, we used an estimation approach, presenting 95% confidence intervals (CI) for all estimated rates, rather than statistical significance testing, following best practice in statistical methods for epidemiology , 44. To Help community project participants: 164 from Aklavik; 67 from Old Crow; 52 from Fort McPherson; and 14 from Tuktoyaktuk. Table We collected antibiotic exposure histories from medical charts of CANAmong the 297 participants, the median number of systemic antibiotic prescriptions during the 5-year review period was 3 .Help community project participants and the outpatient populations of Edmonton and rural northern Alberta. The overall antibiotic dispensation rate was notably higher in the CANHelp community project study population (8.9 courses per 10 person-years) than in northern rural Alberta (6.3 courses per 10 person-years) or Edmonton (5.5 courses per 10 person-years). The estimated antibiotic dispensation rate in western Arctic communities was 3.3  courses per 10 person-years higher than the Edmonton outpatient population rate and 2.6  courses per 10 person-years higher than the rural northern Alberta outpatient rate.Table Northern Alberta is a large geographic region with two small cities and vast sparsely populated areas. Table Table Evidence from this study suggests that antibiotics are dispensed more frequently to western Arctic Canadians than to either urban or rural residents of the southwestern Canadian province of Alberta, independently of age and sex. This study showed additionally that antibiotics were dispensed more frequently to women relative to men and to the elderly relative to other age groups in each of the 3 settings investigated. Dispensation rates for the most frequently dispensed antibiotic classes, β-lactams and macrolides, was similar in the 3 populations, while fluoroquinolones and tetracyclines were dispensed less frequently and nitrofurans and nitroimidazoles were dispensed more frequently in the Arctic communities.Help Working Group community projects was 8.9 prescriptions per 10 person-years; an even higher antibiotic prescription rate of 15 per 10 person-years was estimated for an Alaska Native population in 2003 [In the present study, the dispensation rate for systemic antibiotic prescriptions among participants in CAN in 2003 . More fr in 2003 , 47.H. pylori infection has been reported to be higher in children than in adults, with evidence suggesting that this contrast is due to more frequent use of macrolide antibiotics in children for the treatment of respiratory tract infections [H. pylori infection has been reported to be higher in women than in men, [H. pylori infection was also higher in women relative to men [H. pylori infection in women relative to men may be due to more frequent use of tetracycline in women for the treatment of urogenital infections [H. pylori infection in women relative to men and in youth relative to adults may be associated with more frequent use of fluoroquinolone antibiotics for the treatment of urogenital and respiratory tract infections, respectively [Variation in the use of antibiotics for the treatment of common bacterial infections may contribute to variation in antibiotic-resistant infection across geographic regions and sociodemographic groups , 26. Forfections , 48. The in men,  perhaps  in men, –54. For fections . Higher ectively .H. pylori projects. Participants’ medical charts only capture antibiotics dispensed at their community health centre. Many residents of the project communities spend time away from home and may receive health care in other locations. Most residents of northern Canadian communities, however, receive most of their health care at their community health centre; it is, therefore, likely that most antibiotics dispensed during the review period for this study were captured by our chart review. The data sources used for the Alberta populations would similarly miss prescriptions dispensed outside Alberta; however, given that Albertans are all entitled to health care provided by the provincial government, they are not likely to seek outpatient care for infections outside the province.An important limitation of the present analysis is the potential underestimation of antibiotic use among participants in the Arctic Canadian community Another limitation pertains to our differential strategy for capturing dispensation events across territorial and provincial populations. In the northern communities included here, prescription events equate with dispensations. In Alberta, however, primary nonadherence is possible: an individual may be prescribed a drug but may not fill the prescription, precluding its dispensation , 57. WhiIn this study, the time periods for which antibiotic dispensation rates were estimated differ in each of the Arctic communities  due to the sequential conduct of these projects. These periods overlap to different degrees with the time period captured by the publicly accessible Alberta data, which was 2010–2013 at the time of data analysis. We would not, however, expect substantial changes in antibiotic dispensation during 2002 to 2013 based on studies of outpatient populations in Ontario, Canada and in the United States, which suggest that antibiotic dispensation rates remained relatively stable across age groups during this time period , 59.H. pylori infection. More generally, Indigenous Arctic populations may be particularly vulnerable to antibiotic-resistant infections associated with frequent exposure to antibiotics. The evidence generated by this analysis will be useful for developing strategies aimed at reducing health disparities arising from inequitable health care in Indigenous Arctic populations relative to other North American populations.This study reveals more frequent dispensation of antibiotics in Arctic Canada relative to urban and rural populations in southern Canada. These results suggest that increased exposure to antibiotics is a potential explanation for reduced effectiveness of antibiotics in Indigenous Arctic populations for treating infections such as


Decreased protein breakdown in pregnant women results in lower concentration of methionine (Met) in plasma, causing pregnancy-related metabolic disturbance. Its dipeptide methionyl-methionine (Met-Met) may exert positive influence in fetal development. This study mainly investigated whether Met-Met can be used as part of free Met to promote reproductive outcomes in mice and the underlying mechanisms. Met-deficient pregnant mice were treated with Met alone or with Met-Met during pregnancy. Daily intraperitoneal injection of 35% dietary Met in pregnant mice was the best dose among the 15–45% doses. Embryo development and newborn birth weight were enhanced when 25% of the Met in the 35% Met group was replaced with Met-Met. Met-Met replacement had higher plasma insulin, glucose, and free amino acids (AA) concentrations. Besides, in the placenta, the AA transporter mRNA abundances and peptide transporters (PhT1 and PepT1) protein levels were higher in Met-Met treatment group. Moreover, Met-Met increased 4E-BP1, S6K1 and AKT/mTOR phosphorylation. These results suggest that Met-Met could be used as a partial source of Met to promote reproductive outcomes in Met-restricted pregnant mice, which might be mediated by promoting nutrient availability and activating AKT/mTOR-mediated signaling pathway. Methionine (Met) is an essential amino acid (EAA) required for protein synthesis and is also a key source of methyl groups for methylation , such asin vitro is clearly established, little is known about its role in fetal development. We hypothesize that partially supplementing pregnant dams with the Met dipeptide methionyl-methionine (Met-Met) is more effective than supplementing them with only free Met in promoting embryo development and improving reproductive outcomes. In this study, pregnant mice with dietary Met restriction were supplemented with Met alone or with Met-Met by intraperitoneal (i.p.) injection to investigate the effect and mechanism of Met-Met on reproductive outcomes in mice.Short peptides have drawn increasing attention as new sources of nutrients. The uptake of short peptides by oligo/dipeptide transporters in cells provides a more effective and energy-saving intracellular source of AAs than the uptake of free AAs (FAAs) ,8. It waBiological fluids, including simulated gastric fluid (SGF) and intestinal fluid (SIF), were prepared and Met-Met stability in biological fluids was measured according to previously described procedures .The use of animals in this study was approved by the Institutional Animal Care and Use Committee of Zhejiang University. ICR and Nu/nu mice were purchased from the Shanghai Laboratory Animal Center. Female virgin mice of approximately 30 g and 9 weeks of age were mated, and the presence of seminal plugs indicated a successful mating. The day that seminal plugs were found was designated embryonic day (E) 0. The mice were housed individually in a controlled environment at 21 ± 3 °C under specific pathogen-free conditions with a 12h light-dark cycle and ad libitum access to food and water.n = 3): blank control, FITC-Met-Met, and Free-FITC, respectively. A total of 100 µL of saline, 23.05 µg/mL FITC-Met-Met , or 15 µg/mL Free-FITC (GL Biochem) was injected into the peritoneal cavity of nu/nu mice in three groups, respectively. At different time intervals , mice were anesthetized with halothane and placed in a fluorescence imaging system  in which the “blue” filter was set for excitation and the tunable filter was automatically increased from 465 to 520 nm in 10 nm stepwise increments. The Maestro software (CRi) was used to unmix the fluorescence signals. Light intensity was indicated by pseudocoloring (red for the highest intensity and blue for the lowest intensity), superimposed over the grayscale body surface.Nine Nu/nu mice aged 6 weeks were used to observe the disappearance of fluorescein isothiocyanate-labeled Met-Met (FITC-Met-Met) in the body, and were randomly divided into the following groups (each group n = 8). The mice were fed a Met-supplemented or Met-deficient diet  to avoid circadian variations in gene expression. Livers were immediately collected, snap frozen in liquid nitrogen, and stored at −80 °C until subsequent analysis for Met metabolites [Experiment I: Forty pregnant ICR mice were randomly assigned to 5 groups . Groups 1 (Control) and 2 (35% Met) were treated as described for the control group and 35% Met group in experiment I, respectively. The other five groups  were treated as described for the 35% Met group, except that the 5, 15, 25, 35, or 45% of free Met in the injected solution was replaced with Met-Met  and received treatments as described for the control, 35% Met, and 25% Met-Met groups in experiment II , leptin (Leptin Mouse ELISA kit) and glucose (Glucose Colorimetric Detection Kit) using kits from Nanjing Jiancheng Bioengineering Institute . The placentas in each horn next to the cervix unit were rapidly removed from these animals, and fetuses were carefully collected and sampled for blood. The remaining 8 dams in the two treatment groups were switched to the control diet without i.p. injections to avoid any abortion. Litters were weighed soon after parturition and before milk consumption.Experiment III: Forty-eight pregnant ICR mice were randomly divided into three groups . Differences between groups were analyzed post hoc using the LSD comparison. Animal litter was used as an observation unit for all offspring analyses. Differences of Met-Met was degraded quickly in SIF and SGF and became undetectable after 1–2 h of incubation A. Howevep < 0.001). Whereas the 35% Met group increased cumulative food intake at E 14 compared with the 15% Met group (p = 0.032), no difference existed among other Met groups , but no differences were found in the dams and fetuses of 25% Met-Met group compared with the control group (p < 0.001) than that in the 25% Met-Met group (p < 0.001) than those in the 35% Met group  in the plasma of the dams, with 45% and 19% reductions in total FAAs in the 35% Met and 25% Met-Met groups, respectively . The ratol group A. Additiol group . Moreoveet group C, so didet group D. Converet group B. Howeveet group . The resp < 0.001, p = 0.02, and p < 0.001 for glucose, insulin, and leptin, respectively, Maternal Met-deficient diet intake decreased plasma glucose, insulin and leptin levels, but the 25% Met-Met group showed higher concentrations than the 35% Met group  family members, SLC38A1, SLC38A2 and SLC38A4 in system A, SLC7A9 in system bment III . HoweverThe mRNA abundance levels of facilitative glucose transporters SLC2A1, SLC2A3, SLC2A4, and SLC2A10 in the placenta at E 17 decreased significantly in the 35% Met group compared with those in the control group , whereasThe protein expression levels of peptide/histidine transporter 1 (PhT1) and peptide transporter 1 (PepT1) were significantly enhanced in the 25% Met-Met group compared with those in the control and 35% Met groups . The pepThe protein expression levels of mTOR, pmTOR (phosphorylated mTOR), S6K1, pS6K1, 4E-BP1, p4E-BP1, AKT and pAKT showed that substitution of 25% of Met with Met-Met resulted in higher levels of p4E-BP1 and pS6K1 A,B. The We first developed a Met-deficient pregnant mouse model in which Met or Met-Met was supplemented by i.p. injection. The reason we used i.p. injection rather than gavage was that Met-Met is not stable in the digestive tract, as shown in stability tests in SGF and SIF. On the other hand, our results show that Met-Met is relatively stable in the serum for a longer period of time. In addition, our in vivo imaging assays show that the i.p.-injected Met-Met conjugated with fluorescein isothiocyanate, an amine-reactive derivative of fluorescein dye extensively used to label peptides , can be To study the role of Met-Met in reproductive performance, the best dose of free Met supplementation by i.p. injection was analyzed first. Met-deficient pregnant mice were i.p. injected daily with 15–45% of the Met in the control diet in experiment I. These doses were chosen based on research indicating that less than half of dietary Met could be absorbed into the blood stream after the digestion and absorption process . AlthougThen, 5–45% of the free Met in the 35% Met group was replaced with Met-Met. The substitution of 15–35% of Met with Met-Met improved the body weight of the dam at day 17 of gestation, with the 25% substitution being the top performer. The 25% substitution also resulted in higher birth weights of the litters born alive. These observations suggest that at certain levels, a large amount of Met-Met can be used not only by the body but also more efficiently than free Met.To address how Met-Met may be used more efficiently than free Met in terms of reproductive performance, the FAAs concentrations in the maternal and fetal blood of the control, 35% Met, and 25% Met-Met groups were measured in experiment III. Dietary Met restriction resulted in 42% and 19% lower EAAs and 48% and 18% lower NEAAs in the maternal blood of the 35% Met and 25% Met-Met groups, respectively. In general, the concentrations of NEAAs were affected more disparate in the 35% Met group. As a consequence, the ratio of EAAs/NEAAs was increased in the 35% Met group compared with in the 25% Met-Met group. In addition, the lower concentrations of EAAs and NEAAs in the fetal blood of the 35% Met and 25% Met-Met groups may have led to slower embryo growth, as AAs are crucial for neonatal growth . The impBoth the 35% Met and 25% Met-Met groups had lower maternal and fetal blood concentrations of Met, indicating that Met supplementation in these two groups could not match the Met supply of the control group. However, although the concentration of Met in the maternal blood was 68% lower in the 25% Met-Met group than in the 35% Met group, resulting from the injection of a lower amount of free Met, the Met concentration in the fetal blood was much higher in the 25% Met-Met group than in the 35% Met group. This difference suggests that at least some Met-Met is more efficiently absorbed as a whole by peptide transporters, such as PhT1 and PepT1, as we measured much higher expression levels of PhT1 and PepT1 in the 25% Met-Met group.+-dependent neutral amino acid uptake by mammalian cells [In general, the concentration of FAAs was substantially enriched in the fetal plasma compared to that in the maternal plasma in the three groups. Specifically, the embryo/maternal ratio was 15.3 and 11.8 for Gly in the 25% Met-Met and 35% Met groups, respectively. In the present study, the abundance of AA transporters in the placenta was consistent with the AA concentrations of the fetal blood of the three groups. Many of the AA transporters were analyzed, including system A , which accounts for most Naan cells , showed Another reason for impaired embryo development in the Met-deficient groups may have been the lower concentrations of insulin, glucose, and leptin in the maternal blood of these dams. The normal metabolism of carbohydrates, lipids, and proteins is critical for pregnancy. Insulin is the central endocrine hormone in the regulation of metabolism. A high physiological level of insulin is required to promote fetal growth and development, whereas low insulin levels, such as in diabetic pregnancy, may lead to an increased risk of congenital abnormalities . The amoThe enhanced reproductive outcomes and nutrient availability in the 25% Met-Met group may have been due to signaling molecule activation in the placenta. A key pathway in the regulation of placental growth and nutrient transport is the PI3K/AKT/mTOR signaling pathway, which is the master regulator of cell growth and protein synthesis, among other functions ,32. ChanIn summary, this study reveals that Met-Met can be used more efficiently than free Met in mice to support fetal growth under Met deficiency. The major reason for the enhanced embryo development was probably due to the higher cumulative food intake in the 25% Met-Met group than the 35% Met group, resulting in higher availability of nutrients and the hormonal/signaling alterations. Met-Met changes the maternal AA profile, increasing the concentration of gluconeogenic AAs and insulin secretion from the dam. Met-Met enters the placental barrier through peptide transporters and is hydrolyzed to Met. Enhanced insulin signaling and Met in the cytosol activate the AKT/mTOR signaling pathway, which increases the expression of nutrient transporters in the placenta and thus nutrient availability in fetal blood to promote fetal growth and development. The current results highlight a new investigation field in human and other mammals to determine whether reproductive outcomes can be optimized by supplementing mothers with small peptides.


The use of plastics has increased over the years, thus resulting in a large volume of plastic waste being generated and accumulated in the environment. Due to its non-biodegradability and persistence, recycling processes have become one of the sustainable solutions for preventing environmental deterioration. Plastic wastes, including high density polyethylene (HDPE), low density polyethylene (LDPE), polypropylene (PP), and polyethylene terephthalate (PET), were collected from industrial sector and used as additional ingredients to improve concrete properties. Prior to concrete processing, an increase in wettability of plastic fibers using nonionic surfactant, Dehydol LS-12, was investigated. At the optimal concentration of 10 times of the critical micelle concentration (CMC), an interfacial tension and a contact angle were reduced to 31–32 mN/m and 65°–68°, respectively. Properties of concrete were determined and compared to those of the mortar samples. Porosity was found to increase with higher volume fraction of plastic fibers, whereas decreases in workability, bulk density, thermal conductivity, splitting tensile strength, and compressive strength were encountered. The lowest thermal conductivity was recorded for concrete samples prepared with 30% by volume of LDPE fibers, and the rest in descending order were HDPE, PP, and PET, respectively. Furthermore, the maximal inclusions of plastic fibers were 5% for HDPE and LDPE, 10% for PP, and 50% for PET so as to satisfy the precast concrete wall requirements. At present, plastic is considered as one of the most important materials of several industries including automobile, electronic appliance, construction, and packaging manufacturers. The rate of plastic consumption has dramatically increased from 200 million tons a year in 2002 to 322 million tons in 2015, and the number is expected to reach 485 million tons in 2030 [Utilization of plastic wastes, or plastic recycling, has become a viable and sustainable solution to avoid environmental impacts caused by plastic wastes. Plastic wastes were utilized through recycling and energy recovery (around 48–69%) in 2006 to 2014 [®) in tunnels [Beneficial properties of recycled plastic are light weight, high chemical resistance, weather durability, and high thermal insulation , therefo tunnels  and conc tunnels  have bee tunnels .Concrete is widely used that contains basic ingredients such as sand, cement, and water. It is more economical, stronger, and more durable in comparison with wood and asphalt. However, tensile strength of concrete is significantly lower ,11,12, rConcrete is used in either load-bearing structures to bear the main building load  or non-load bearing structures . Since supporting structures do not require high-strength concrete like those of main structures, plastic fibers concrete will be possible to apply, especially in wall panels. Moreover, thermal conductivity of plastics, especially high density polyethylene (HDPE), low density polyethylene (LDPE), polypropylene (PP), and polyethylene terephthalate (PET), was found to be lower than that of plain concrete ,30,31. GTo ensure the environmental sustainability and improvement of concrete property, use of plastic waste as reinforcing fibers in the concrete processing is desirable. The objectives of this study are to investigate the possibility of using plastic wastes in construction products such as precast concrete wall. Furthermore, the properties of produced concrete including bulk density, permeable void, thermal conductivity, splitting tensile strength, and compressive strength were also determined.3 for HDPE, 221 ± 22 kg/m3 for LDPE, 179 ± 12 kg/m3 for PP, and 242 ± 5 kg/m3 for PET. The amount of plastic fiber used in the concrete production was calculated according to the different cement volume proportions .Four types of plastic wastes, including high density polyethylene (HDPE), low density polyethylene (LDPE), and polypropylene (PP) collected from a plastic bag manufacturer , as well as polyethylene terephthalate (PET) collected from a PET sheet manufacturer , were utilized as part of ingredients for concrete production in this study. The plastic wastes were cleaned and cut into 3 mm × 50 mm plastic fibers. Thicknesses of the plastic fibers were approximately 0.05 mm for HDPE and LDPE, 0.1 mm for PP and 0.2 mm for PET, respectively. The four plastic fibers were fit as macro synthetic fibers in which their lengths were 12–65 mm  and equi3S), Dicalcium silicate (C2S), Tricalcium aluminate (C3A), and Tetracalcium aluminoferite (C4AF), as well as the trace compounds such as gypsum, free lime, magnesium oxide, and alkaline oxide [3 [3 [Portland cement type I and river sand no. 4 (passing through a 4.75 mm sieve) were used as the two main ingredients in the concrete processing. Portland cement mainly composed of Tricalcium silicate . The ratio of cement to sand was controlled, whereas the water was varied from 15 vol % fraction in order to prevent segregation of reinforced concrete and the plastic fibers were varied from 5% volume fraction to the maximum of 30% volume fraction, as summarized in Prior to the concrete processing, wettabilities of plastic fibers were determined to optimize the nonionic surfactant amount. Two parameters; namely, surface tension (γSL) and contact angle (θc) were used to measure the wettability. Brief procedure includes preparing the plastic fibers of HDPE, LDPE, PP, and PET in a 1 cm × 2 cm size, and measuring the surface tension and contact angle at various LS-12 solution concentrations from 1 to 70 times of its critical micelle concentration (CMC) at 25 °C by DCAT 11  or Dynamic contact angle and tension meter as shown in (1) WorkabilityThe fresh concrete from plastic waste was measured for workability by slump test . The tes(2) Bulk density and porosity The hardened concrete was evaluated for the bulk density and porosity. The testing procedure was as follows; (1) measure the dry weight of produced concrete after heating at 100–110 °C in an oven and cooling down to 20–25 °C in a desiccator, (2) immerse the concrete in the water at 21 °C for 48 h, and then measure the weight, (3) boil the concrete for five hours, and then measure the weight after cooling down, and (4) suspend the concrete in the water, and measure the immersed apparent mass .(3) Thermal conductivity2), k is thermal conductivity (W/m·K), dT is temperature difference between both sides (K or °C), and dx = thickness (m)2·K/W).The cube concrete with a 30 cm × 30 cm × 7.5 cm size was prepared, and the heat flux was measured using NETZSCH HFM 436 equipment  (Departm(4) Splitting tensile strengthThe splitting tensile strength was examined according to American Society for Testing and Materials (ASTM) C496  by moldi(5) Compressive strengthThe compressive strength was evaluated according to ASTM C34  by moldiAll parameters above are explained with mean and standard deviation (SD) values.Prior to concrete processing, the wettabilities of plastic fibers were determined under various nonionic surfactant concentrations (as presented in CMC) using interfacial tension (γSL) and contact angle (θc). From Workability of fresh concrete using HDPE, LDPE, PP, and PET fibers was represented by subsidence values, and the results were shown in 3 on average was obtained in the produced concrete from HDPE fiber, and the bulk densities of the produced concrete with LDPE, PP and PET were about 1853, 1892, and 1960 kg/m3 on average, respectively  of the produced concrete was calculated from the experiments, and the results were shown in li et al  who studli et al ,55 can ali et al ,56. The (1) Splitting Tensile Strength3 of PP hybrid fibers with concrete.Normally, tensile strength of concrete is low when compared with its compressive strength, as a result, high strength materials, such as, steel, plastic, etc., are introduced into the mixture to improve the brittle property of the cementitious composites. According to Hasan et al. , adding Ductile fibers, when added into cementitious composites, can enhance its tensile strength depending on several factors such as fiber toughness, fiber volume fraction, alignment, and bonding between the fibers and the cementitious matrix. In this study, the optimum volume fractions of recycled HDPE, LDPE, and PET plastic to achieve the highest splitting tensile strength of the fiber-reinforced concrete composites were found to be 10%, 10%, and 30%, respectively. Lower and higher volume fraction than these optimum amounts resulted a lower splitting tensile strength. For the case of PP recycled plastic, optimum splitting tensile strength of the composites was found to be at 5% volume fraction, thereafter, at higher volume fractions the tensile strengths of the composites reduced see . In gene(2) Compressive StrengthConcrete is commonly known for its high compressive strength with a rather low tensile strength, typically about one-tenth of its compressive strength. The addition of low fiber volume content  into concrete often does not affect the compressive strength of concrete ,13,24,57Typical compressive strength of concrete used in the USA construction industry varies from 17 MPa for residential buildings to 28 MPa for commercial buildings . As statFrom all experiments and characterization, HDPE, LDPE, and PP have the potential to be used as additional ingredients to hinder concrete’s thermal conductivity and can be used for manufacturing of high insulation material. However, the property was found to be more effective in the produced concrete from LDPE fiber, and to a lesser degree from HDPE and PP fibers. Though PET fiber was found unsuitable for thermal resistance property in concrete because of its inability to show clear improvement over regular mortar, PET fiber compressive strength and splitting tensile strength were higher than other fibers, which was the advantage for utilizing PET waste in green building.3, porosity was 22%, and thermal conductivity was 0.72 W/(m·K) on average. According to the standard of precast concrete wall panel in Thailand, the optimal volume fraction of LDPE fiber should be reduced to 5–10% to maintain the compressive strength of 16 MPa. These alternative renewable materials not only improve lower thermal conductivity (2–31%) of plain concrete but also help solve plastic wastes in our environment. Consequently, they would be recommended for green building in non-load-bearing structure.The industrial plastic wastes  were recycled as reinforcing fiber in concrete processing in this study. The addition of nonionic surfactant solution was required to improve the wettability of plastic fiber surface. At the optimal concentration of 10 CMC, the interfacial tension and contact angle were in the range of 31–32 mN/m and 65°–68° on average, respectively. The results showed that the contact angles of both HDPE and LDPE were distinctively reduced by LS-12 solution at 10 CMC. The properties of fresh and hardened concretes containing plastic fibers were determined and compared with those of the controls (mortar) which contained no plastic fiber. Workability values of all plastic fibers applied in concrete were likely to be decreased with increase fibers content due to complex network structure but 30% PET, which was reversed, might be the cause of fiber agglomeration. Besides, adding fiber contents in concrete could increase the porosity and decrease the bulk density because hydrophobic property of plastics was able to generate air void inside concrete. As a result of porous concrete, the results demonstrated that the HDPE, LDPE, and PP fibers could enhance lowering of thermal conductivity. However, the concrete prepared with PET fibers resulted in the similar property to that of the mortar. Moreover, splitting tensile strength and compressive strength of concrete were not improved by 5–50 vol % plastic contents. The best concrete property was observed when the LDPE fiber was utilized at 30% of volume fraction; subsidence was 4.0 cm, bulk density was 1911 kg/mMore study should be focused on the surfactant role in synthetic fiber reinforced concrete. Moreover, the degradation of plastic fiber in hardened concrete should be considered when applying this composite material as concrete wall panel.


Paulinella micropora is a rhizarian thecate amoeba, belonging to a photosynthetic Paulinella species group that has a unique organelle termed chromatophore, whose cyanobacterial origin is distinct from that of plant and algal chloroplasts. Because acquisition of the chromatophore was quite a recent event compared with that of the chloroplast ancestor, the Paulinella species are thought to be model organisms for studying the early process of primary endosymbiosis. To obtain insight into how endosymbiotically transferred genes acquire expression competence in the host nucleus, here we analyzed the 5′ end sequences of the mRNAs of P. micropora MYN1 strain with the aid of a cap-trapper cDNA library. As a result, we found that mRNAs of 27 genes, including endosymbiotically transferred genes, possessed the common 5′ end sequence of 28–33 bases that were posttranscriptionally added by spliced leader (SL) trans-splicing. We also found two subtypes of SL RNA genes encoded by the P. micropora MYN1 genome. Differing from the other SL trans-splicing organisms that usually possess poly(A)-less SL RNAs, this amoeba has polyadenylated SL RNAs. In this study, we characterize the SL trans-splicing of this unique organism and discuss the putative merits of SL trans-splicing in functional gene transfer and genome evolution. IDs in parentheses are Genbank- and DDBJ-accessions. [1] Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016; 33: 1870–4.Paulinella lineages. BMC Evol. Biol. 2009; 9: 98.[2] Yoon HS, Nakayama T, Reyes-Prieto A, Andersen RA, Boo SM, Ishida K, Bhattacharya D. A single origin of the photosynthetic organelle in different Paulinella chromatophora . J. Eukaryot. Microbiol. 2014; 61: 317–21.[3] Nomura M, Nakayama T, Ishida K. Detailed process of shell construction in the photosynthetic testate amoeba Paulinella Species, with the Description of Paulinella micropora sp. nov. and the Chromatophore Genome Sequence for strain KR01. Protist. 2017; 168: 155–70.[4] Lhee D, Yang EC, Kim JI, Nakayama T, Zuccarello G, Andersen RA, Yoon HS. Diversity of the Photosynthetic (PDF)Click here for additional data file.S4 FigA. Schematic models of the premature and SL trans-sliced forms of the transcripts encoding a serine/threonine kinase. These RNA sequences were searched from the RNA-seq data of P. micropora obtained by Illumina HiSeq 4000. B. Nucleotide sequences covering the trans-splicing site and translation initiation context of the transcripts modeled in A, with the 3’ splicing acceptor motif (AG), termination codon, and initiation codon shown by red characters.(PDF)Click here for additional data file.


For the sake of environmental protection and circular economy, cement reduction and cement substitutes have become popular research topics, and the application of green materials has become an important issue in the development of building materials. This study developed green pervious concrete using water-quenched blast-furnace slag (BFS) and co-fired fly ash (CFFA) to replace cement. The objectives of this study were to gauge the feasibility of using a non-cement binder in pervious concrete and identify the optimal binder mix design in terms of compressive strength, permeability, and durability. For filled percentage of voids by cement paste (FPVs) of 70%, 80%, and 90%, which mixed with CFFA and BFS as the binder  to create pervious concrete with no cement. The results indicate that the complete (100%) replacement of cement with CFFA and BFS with no alkaline activator could induce hydration, setting, and hardening. After a curing period of 28 days, the compressive strength with different FPVs could reach approximately 90% that of the control cement specimens. The cementless pervious concrete specimens with BFS:CFFA = 7:3 and FPV = 90% presented better engineering properties and permeability. Rapid progress in industry and commerce in recent years has brought with it fast and significant growth in inter-city and intra-city transportation. During the process of urbanization, impermeable surfaces such as roads and sidewalks have expanded and taken up a considerable proportion of land area, thereby causing major changes in the regional water cycle. In the past, urban flood control focused on draining away rainwater to prevent overburdening public facilities and flooding low-lying areas in the suburbs during typhoons and torrential rains ,2,3. How2 emissions produced in the process of manufacturing cement and concrete, the most direct approach is to reduce cement consumption by replacing cement with pozzolanic materials that have the binding properties of cement [To reduce the COf cement . The conf cement . The mosf cement . Developf cement . Past stf cement . Experimf cement ,13.3. Furthermore, spherical particles are rare in CFFA because the temperatures in a CFBC furnace (800–900 °C) are generally lower than those in a coal combustion furnace (1300–1500 °C). The high f-CaO content in CFFA causes it to react violently with water and derive self-cementing properties [2 in CFFA are prone to the formation of hydration products such as calcium hydroxide and ettringite. The calcium hydroxide or calcium sulfate that exists within concrete structures may engage in delayed reactions to water and thereby create expansion cracks [3 and f-CaO contents in concrete specimens are controlled at 4.48% and 3.00%, respectively, the volume stability of the cement is acceptable. Furthermore, the primary hydration products of cement and CFFA include C-S-H, ettringite, and other silicate products [The primary industrial product of CFBC is CFFA. Unlike the fly ash by-product of typical coal combustion, CFFA contains large amounts of f-CaO and SOoperties , which mace 1300–00 °C. Thoperties ,17. Baseoperties . The comoperties . CFBC teoperties . As a ren cracks . Relevanproducts .Pervious concrete is a porous composite material that is also referred to as no-fines (no fine aggregates) concrete. It contains a narrow size distribution of coarse aggregates, little or no fine aggregates, and a small amount of cement paste that does not fill the voids among the coarse particles ,23. LighThis study completely replaced cement with a mix of CFFA and water-quenched BFS, and formulated mix designs according to ACI 522 to create pervious concrete with no cement. We then tested the engineering properties of the resulting specimens, including compressive strength and permeability, to determine whether they meet the basic requirements of general pervious concrete. We chose single-size coarse aggregates (19~25 mm). For the filled percentage of voids by cement paste (FPVs), we experimented with 70%, 80%, and 90%. We chose a water-binder ratio of 0.35 and different mixtures of CFFA and water-quenched BFS as the binder  to create green pervious concrete with no cement. We tested its engineering properties  and further observed the microstructures and chemical composition of the binders using X-ray diffraction (XRD) and mercury intrusion porosimetry (MIP).2/g and specific gravity of 3.15. Its primary components were analyzed according to ASTM C114 standards and include tricalcium silicate , dicalcium silicate , tricalcium aluminate , and tetracalcium aluminoferrite , as shown in In this study, we used Type-I Portland cement from the Taiwan Cement Corporation , which has a fineness of 3310 cm2 + Al2O3 + Fe2O3 content >50%. CFFA had a fineness of 2800 cm2/g and specific gravity of 2.73. According to ASTM C618, it meets the designations of Class C fly ash, the differences being that it has a sulfide content greater than 5% and that its activity indices at 7 days and 28 days are higher than the 75% stipulated by ASTM C311. These results demonstrate that the CFFA has pozzolanic effects. The CFFA used in this study is unlike normal fly ash; in appearance, it is a black powder. It is a by-product of burning coal as the fuel with other general industrial waste . With properties similar to original fly ash, CFFA should be able to serve as an auxiliary binder or filler in cementitious composite materials. Chemical analysis shows that CaO content >10% and that SiO2/g and specific gravity of 2.88. Its physical and chemical properties meet the requirements of ASTM C989 as shown in The water-quenched BFS used in this study had a fineness of 5860 cm3, respectively.The coarse aggregates comprised gravel processed from natural pebbles mined from the middle and upper sections of the Lanyang River. Due to the needs of the experiment, we employed single-size aggregates (19~25 mm). The specific gravity, water absorption rate, and dry unit weight of the coarse aggregates were 2.68, 0.69%, and 1619 kg/mTo retain water, decelerate setting, maintain thickness, and increase adhesiveness in the slurry, we added a thickener containing cellulose ether, a natural-thickening modified starch, and a small amount of surfactant. The cellulose ether was 300HYP4 of Methyl Hydroxy Ethyl Cellulose and the weight added was 0.5% of the weight of binder as per ASTM C91 standards.Superplasticizers are a type of polymer that can be adsorbed by the surface of the particles to prevent cement particles from clustering in the water, thereby ensuring that the water achieves its lubricating effect. Using superplasticizers enhances the workability of fresh concrete with low water-cement ratios, reduces the amount of water needed, and increases concrete strength. High-strength superplasticizers can disperse cement particles to maximize flowability. In this study, it used the HP 100 fluidizer, which is a high-performance superplasticizer comprising mainly polycarboxylates. It has good hydrophilicity and is mixed with water before use. Its chlorine content is lower than 1% of its own weight in accordance with the Japanese Industrial Standards (JIS).The mix designs of the concrete specimens in this study were made in accordance with the methods used in ACI 211.1. Absolute volume was used in the calculations, and the specimens were numbered using two letters and numbers to indicate the pozzolanic material ratio (BFS:CFFA) and FPVs. Single-size aggregates (19 mm~25 mm) were used in the mix designs and the aggregates comprised of gravel processed from natural river pebbles. We experimented on three mix ratios for BFS and CFFA: 50% BFS with 50% CFFA, 60% BFS with 40% CFFA, and 70% BFS with 30% CFFA, and there were three settings for FPVs: 70%, 80%, and 90%. The pozzolanic material was either cement, indicated using “C”, or a mix of BFS and CFFA . Finally, the FPV settings of the mix designs were A: 70%, B: 80%, and C: 90%. The water-cementitious ratio was fixed at 0.35. The experiment focused on fresh concrete properties, hardened properties, durability properties, and microscopic analysis. The outer dimensions were divided into three conditions: ϕ10 × 20, fragments, and powders that can pass a #200 mesh (0.075 mm). The measurement results are presented as the mean of the three specimens in each group for all mixtures. The coefficient of variation was controlled to less than 10%.In accordance with ASTM E70, pH measurement was performed on freshly mixed concrete using an electronic pH meter.Unit weight was measured in accordance with ASTM D6023. The concrete was rodded in three layers with 25 times per layer. For the bottom layer, the rod penetrated nearly full depth into the concrete but did not strike the bottom of the measure. The rodding strokes were evenly distributed over the surface of the layer. For the two upper layers, the rod penetrated approximately 25 mm into the layer below with each stroke. After each layer was rodded, the sides of the measure were lightly tapped 10 to 15 times with a rubber mallet to close any voids left by the tamping rod and release any bubbles of air in the concrete. The top surface of the concrete was then layered, and the net weight of the concrete within the measure was weighted. Dividing the net weight by the volume of the measure then produced the unit weight of the concrete.2, and the compressive strengths of the specimens upon failure were recorded.We referred to ASTM C39 when performing the compressive strength test. Freshly mixed concrete was poured into a ϕ10 × 20 mold and cured for 24 ± 0.5 h in the mold before demolding. The specimens were cured in a curing tank containing saturated lime water and then retrieved for compressive strength testing on the designated days . The compressive strength tests were conducted while the specimens were still wet. Any foreign matter on the contact surfaces of the specimens and the testing machine was removed, and the central axes of the specimens had to line up with that of the round base. Uniaxial load tests were performed at a compression velocity of 3.5 kgf/cmFor the splitting strength test, we referred to ASTM C496. The purpose of the splitting strength test is to test the tensile strength of the pervious concrete. Testing was performed once the specimens were cured for the designated number of days . The splitting strength tests were conducted while the specimens were still wet. The specimens were aligned to the fixture and placed in smoothly. Force was then applied along the length of the specimens, and the splitting strengths of the specimens upon failure were recorded.A is weight of oven-dried sample (g), B is weight of saturated specimen (g).Absorption was measured in accordance with ASTM 642. First, ϕ10 × 20 cm cylindrical specimens were cured until the designated 28 days and then dried in an oven at 100 to 110 °C for 24 h. After being removed from the oven, they stood at room temperature to cool. Once their temperatures fell to between 20 and 25 °C, the specimens were weighed and then completely immersed in water for a total of 48 h. At 24 h and 48 h, their surface-dry weights were measured. If the difference between the two measurements was less than 0.5%, then the specimens were considered to be in saturated state. The weights were then recorded and substituted into the formula below to calculate the water absorption of the specimens.For the sulfate soundness test, we referred to ASTM C88. When the concrete is exposed to a harsh environment, such as harmful chemical substances in the soil or water, the concrete can easily expand and crack, which reduces its strength and shortens its service life. The corrosive environments formed by sulfates generally involve multiple types of salts co-existing in groundwater or seawater. These salts do not exist in simple forms and can only infiltrate concrete in solution form. In addition, they must exceed a certain amount to be hazardous to concrete. This sulfate soundness test determines the soundness of concrete aggregates under the effects of weathering and simulates salt infiltrating the pores of the concrete and corroding the interior. When the specimens are again immersed in a saturated sodium sulfate solution, the interior fills with liquid that simulates the influence of freezing and expanding water on concrete. The simulation conditions of the test environment are sufficient to allow aggregate soundness information through repeated immersions in saturated sulfuric acid. The test process involves placing the specimens in a saturated sodium sulfate solution for 24 h and then drying them in an oven at 110 ± 6 °C for 24 h for the specimens to be partially or completely dried. After one round, the specimens are weighed and five rounds were conducted in this study.2), H is height of water head (cm), Δt is the actual time from t1 to t0.The permeability test was conducted based on the Pavement Testing Manual published by the Japan Road Association . The conThe MIP test was conducted in accordance with ASTM D4404. For the testing instrument, we employed the AutoPore IV 9500 mercury porosimeter from the Micromeritics Instrument Corporation  which can measure the diameter of pores within specimens and calculate their cumulative total volume. Pores with a diameter smaller than 10 nm are gel pores, while those with a diameter greater than 10 nm are capillary pores. Based on the diameters and cumulative total volume of the pores, we can calculate the gel pore volume and capillary pore volume in the specimens. Both pore size and volume influence concrete performance. The volume distributions derived from the test can promote understanding on the properties of the concrete. During the tests, test tubes are placed in a high-pressure chamber, and the pressure is increased to 33,000 psi. Pressure, pore diameters, the increase in pore volume, and the cumulative pore volume can be calculated based on the external pressure applied and the amount of mercury forced into the specimen. Once the pressure reaches 33,000 psi, the chamber is depressurized until it reaches atmospheric pressure. Then, the test is completed.XRD is a non-destructive analysis technique that is used to detect the properties of crystalline materials. It enables the analysis of structure, phase, preferred orientation (texture), and other structural parameters such as average particle size, crystallinity, tension, and crystal defects. For XRD, it used the Siemens D5000 X-ray diffractometer , which can analyze the chemical composition of crystalline materials. As cement-based materials are compounds with multiple elements, their powders can be analyzed using XRD to identify their chemical composition.2, CaO, CaSO4, and SiO2, among which CaO and CaSO4 engage in hydration reactions with water:In the 12 mix designs, A, B, and C indicate different FPVs, which merely means different amounts of slurry and does not affect the pH values. Thus, after eliminating the FPV condition, only specimens C, 50% BFS, 60% BFS, and 70% BFS remained, and their pH values were 12.3, 12.1, 12.2, and 12.3, respectively. The three experiment groups differed little from the control groups containing normal cement. However, the experiment groups show that as the proportion of CFFA declined from 50% to 30%, the pH value gradually approached the values of the control group (12.3). On the whole, the pH values of the mortars containing various proportions of CFFA and BFS and no cement were close to that of the mortar containing only cement. From the previous study ,12, we c2 continues to react with SiO2, thereby producing C-S-H gel. As a result, the pH values of the solutions in the pores remain highly alkaline as they do in the control groups, thereby preserving the gelability of cementless composite materials.The Ca(OH)3 to 1900 kg/m3. According to ASTM C330, the unit weight of lightweight concrete must be below 1840 kg/m3. Thus, Specimens 5A, 5B, 5C, 6A, and 6B also meet the requirements for lightweight concrete.Comparison of the specimens with different slurry proportions  and the control groups (C) reveal that the specific weight of the material (cement > BFS > CFFA) exerts significant influence on the unit weight of the concrete as illustrated in 2, and then needed more time to act that the Ca(OH)2 and SiO2 in the CFFA and BFS react with Al2O3 and produce C-S-H or C-A-S-H gel. We speculate that the pozzolanic reactions among the aggregates in the specimens peak at 28 days. At the same time, the FPV influences compressive strength, the two presenting a directly proportional relationship. On the whole, the control groups presented significantly greater compressive strength than the specimens in the experiment groups, regardless of the BFS-CFFA ratio. However, the specimens in the experiment groups still have adequate strength to serve as the materials of waterworks structures or pervious backfill materials that do not need to be compacted, which is 1.5 to 14 MPa [2, CaCO3, and CaO) have poorer crystal strength and density than those of cement . Consequently, replacing more cement with CFFA will inevitably reduce the amount of slurry and the compressive strength of the resulting specimen. Thus, the cementless specimens all had slightly lower compressive strength than the control groups (pervious concrete packed with cement slurry). In contrast, CFFA contains about 7.36% of SO3 and a small amount of gypsum, which react with the calcium aluminate salts in the BFS (such as C3A and C4AF) to form ettringite (AFt). This product improves the crystallinity and density of C-S-H gel and produces viscous hardening behavior in the cementless slurry.o 14 MPa . At 28 dWith the different FPVs in The results for water absorption rate are presented in (a)2SO4), potassium sulfate (K2SO4), magnesium sulfate (MgSO4), and calcium sulfate (CaSO4). Excessive contents of these sulfates mean excessive contents in the soil as well, which results in soil acidification.Groundwater contains natural sulfates such as sodium sulfate (Na(b)Sulfates are one of the main components of seawater. Thus, cement-based materials applied to harbor engineering or marine environments are submerged in seawater for long periods of time and are easily corroded by sulfates in the seawater.The purpose of the sulfate resistance test is to gauge the resistance of cement-based composite materials against sulfates. The two most common types of sulfate corrosion are as follows:In view of the above, sulfate corrosion is a common problem for structures both on land and in the sea, so sulfate resistance evaluations are extremely important.42−) reacts with the monosulfoaluminate in cement-based materials and causes local expansion in slurry, which leads to internal stress and cracking that affects the compressive strength and durability of the concrete. Completely replacing cement with a mix of BFS and CFFA significantly reduces the C3A content. A higher proportion of BFS, in particular, indirectly enhances the sulfate resistance of concrete. As shown in 2 and CaO. When specimens are immersed in the sodium sulfate solution, sulfate ions (SO42−) infiltrate the specimens via pores and cracks and react with Ca(OH)2 and CaO that have not yet been consumed. When the specimens are placed in the oven, the sulfate and high-temperature environment may then produce new crystals within the specimens such as the white crystals in The basic principle of sulfate corrosion is that the sulfate ion  and gypsum (CaSO4). As the primary corrosion target of sulfate is monosulfoaluminate (AFm), these two factors (Ca(OH)2 and SO3) can enhance the sulfate resistance of cementless pervious concrete specimens.Furthermore, as the FPV increases, the specimens become denser and are less susceptible to sulfate corrosion. Based on the figure, we can see that the 6 groups (40% CFFA and 60% BFS) and 7 groups (30% CFFA and 70% BFS) specimens, which contain more BFS, presented the highest chemical resistance. The main reason is that CFFA contains Ca(OH)−2 cm/sec. A look at Pervious concrete does not contain any fine aggregates that can fill in pores, so aggregate stacks are not dense. A higher FPV means that more of the pores have been replaced with slurry, which in turn means lower permeability. Permeable surface standards recommend that the permeability coefficient of pervious concrete be no less than 1 × 10Pore structure is a major factor that influences the permeability and durability of materials. Literature indicates that the pores in concrete can be divided into compaction pores, entrained air, capillary pores, and gel pores, the latter two being smaller and accounting for the majority . The typ2, and sulphoaluminate hydrates (Aft and AFm). XRD analysis of normal cement slurry shows that the hydration products usually exist as phases such as Ca(OH)2, CaCO3, and other types of C-S-H gel. Using XRD, we investigated the influence of different BFS-CFFA ratios on the chemical composition of cementless concrete specimens. XRD analysis presents different peak values versus scattering angle as known chemical compounds (hydrations). 2, CaCO3, and Ca3SiO5 contents of the cementless specimens do not change drastically, but the cementless specimens display stronger CaSO4 peaks than the control groups. In addition, the more BFS and less CFFA a specimen contains, the weaker its CaO and SiO2 peaks are. Peaks for 2CaO-Ai2O3-SiO2 also appeared at 31.5°. This shows that mixing CFFA and BFS together to create cementless concrete can effectively stimulate the potential gel properties of the BFS and generate more calcium silicon oxides, which facilitate the development of C-S-H gel, enhance compactness, and effectively increase concrete strength.Cement-based composite materials comprise a number of elements. XRD analysis can be used to determine the chemical composition of composite materials. Generally speaking, the hydration products of cement include C-S-H gel, Ca(OH)This study examined the influence of completely replacing cement with CFFA and BFS on the fresh concrete properties, physical properties, mechanical properties, microscopic properties, and durability properties of pervious concrete. Two major factors were considered: the ratio of water-quenched BFS and CFFA from CFBC used to replace cement and the FPV of the pervious concrete. The strong alkaline characteristics of the CFFA served as the alkaline activator of the BFS. In the various specimens, we replaced 50%, 60%, and 70% of the weight of the cement with BFA and the remaining 50%, 40%, and 30% with CFFA as the alkaline activator. The performance of the resulting specimens was compared with that of pervious concrete specimens made with pure cement. Furthermore, we also examined the influence of different FPVs on pervious concrete properties. Based on the test results, we arrived at the following conclusions:Replacing cement with CFFA and BFS can improve the bleeding of the specimens and shorten the setting time but it reduces workability.Replacing cement with CFFA and BFS results in lower compressive strength than that in pervious concrete made with cement (only 90% of that after 28 days of curing). However, for normal engineering applications, such concrete still has adequate strength to serve as the materials of waterworks structures or pervious backfill materials that do not need to be compacted, which is 1.5 MPa to 14 MPa.Replacing cement with CFFA and BFS gives pervious concrete greater resistance against chemical substances. However, as the proportion of CFFA increases, the concrete’s resistance to chloride ion penetration declines, which affects the durability of the concrete. Thus, in terms of chemical resistance, a BFS:CFFA ratio of 7:3 is optimal, and the resulting chemical resistance was even better than that of cement specimens in the control groups. What is worth noting is that the chemical resistance remains good even when the FPV is reduced. Whether the FPV can be reduced further for better permeability is an issue worth investigating in the future.From the perspective of sustainable development, cement manufacturing today is accompanied by massive greenhouse gas emissions. Manufacturing 1 ton of cement means releasing 1 ton of carbon dioxide. Fly ash is a by-product of coal burning, and recycling it will help to achieve energy conservation and reduce carbon emissions and air pollution, which has a positive impact on the environment. At present, there are many similar applications employing CFFA in construction materials around the world .Replacing cement with CFFA and BFS in pervious concreteA higher FPV means greater compactness and unit weight in the pervious concrete. Compressive and splitting strength tests indicated slightly poorer strength in specimens with a lower FPV. However, the normal cement specimens and the specimens with BFS:CFFA = 5:5 presented significant differences in permeability and chemical resistance, so more attention will need to be paid to the FPV in pervious concrete in the future.The water absorption rate test revealed higher water-absorbing capabilities in specimens with a higher FPV. Thus, it is possible that the pores in the pervious concrete specimens became filled with gel, which increased the water absorption rate.The cementless pervious concrete specimens with BFS:CFFA = 7:3 and FPV = 90% presented better compressive strength, permeability, and resistance to chemical corrosion.Different FPVs in pervious concrete