Lemna growth inhibition test, freshwater algae, daphnia acute, and mustard germination toxicity test were made and discussed in this paper. Based on the physical and geometrical properties and ecotoxicology of examined waste materials, this work evaluated them as suitable for utilization in concrete as a sand replacement.This article deals with the possibility of utilization of secondary-raw materials as a natural sand replacement in concrete. Four types of waste construction materials were examined—recycled aggregate from four different sources. The natural aggregate was examined as well as used as the reference sample. All the samples were tested to evaluate the water absorption, particle size distribution, and particle density. The basic chemical reactions in the view of ecotoxicology are investigated and measured based on Czech standards. Chemical analysis,  Tendencies toward the use of secondary raw materials have emerged in recent years, given the fact that the most used building materials are completely dependent in the production on primary materials. These sources are decreasing and due to their non-renewable disposition, the use of secondary raw materials is a logical step. However, when replacing, it is always necessary to consider the material being worked with and, with regard to the required properties, to select replaceable components and secondary materials that could be used. Nevertheless, the summarization of the material properties is only the second step, first it is necessary to take into account the properties of the secondary raw materials themselves.Many studies describe the mechanical, physical, and durability properties of recycled aggregate (RA) originated from construction and demolition waste (CDW). The higher content of impurities and also mortar complicates the use of the fine fraction RA as a partial or full replacement of natural sand. For the coarse fraction, this problem is reduced . This faHowever, most studies do not pay sufficient attention to the properties of waste raw materials themselves. The results of experiments on the waste may affect its overall utilization. According to the standards, it is possible to use recycled concrete aggregate (RCA) with a content of concrete particles above 70% as a partial replacement for gross natural aggregate (NA). However, this possibility is limited by the type of application ,4. Never3 [3 and ranges from 1800 to 2700 kg/m3 for fine RMA [Previous research has verified the higher water absorption and lower density of RA. According to the previous studies, the water absorption of fine RCA ranges between 4.3% and 13.1% and the dry density of fine RCA ranges from 1900 to 2360 kg/m3 . The higfine RMA ,11. The Ecotoxicity is one of the indicators that can show the extent to which living organisms, or the entire ecosystem, can be affected . EcotoxiSuch great importance for the basic properties of selected waste raw materials is placed mainly due to their use in concrete. The aim of the research is to analyze the properties of waste materials from an environmental point of view, because in materials such as concrete, it is now desirable to use secondary raw materials that reduce the negative impact on the environment. The production of concrete itself is a great burden for the environment, because of the need to quarry of primary raw materials, often to transport these raw materials and further processing. It is also necessary to consider the amount of concrete produced in the construction industry and subsequently the waste in this sector. Construction waste is often landfilled, which seems to be an inappropriate choice because of the potential for toxicity of some materials. Another possibility is the recycling of waste materials, a suitable choice here also seems to be the overall evaluation of the life cycle to confirm the suitability of this solution .The concrete composition is still developing. Many possibilities to substitute usual components of concrete have been evaluated in the past few years, and the utilization of secondary raw materials such as RA, blast furnace slag, fly ash etc. The use of RA from CDW as a partial or full replacement of aggregate in concrete is solved in terms of mechanical properties in various researches ,15,16,17The properties of recycled aggregate, especially its quality, are the main indicators when used in concrete. Because of the frequent occurrence of impurities, there is a risk of decreasing mechanical properties of the concrete mixture. Generally, the RCA has lower particle density and higher water absorption, which is caused by the porous mortar which is attached in the original aggregate surface and water absorption influences the workability of fresh concrete mixture ,5. It isA study  dealt wiThe properties of RMA may adversely affect the workability of fresh concrete or the mechanical properties of concrete . The comFor all the mixtures containing RMA, the measured compressive strength increased after 90 days. This increase was higher compared to reference concrete sample containing NA. The reason for this phenomenon is given by, for example, the formation of pozzolanic reactions due to the presence of silica and alumina contents in the bricks used or a lower w/c ratio of concrete mixtures containing fine RMA . This asThis article is proceeding the utilization of waste materials—in particular, the CDW. The research is focused on three types of RCA and one type of RMA from different sources. The planned use of these materials is in concrete as a natural sand substitute. However, the properties of CDW materials may be inappropriate, it is necessary to experimentally verify at least the essential properties such as particle size distribution, chemical composition, water absorption, or particle density of the recycled waste materials used in comparison with the reference sample . Among other things, it is necessary to think about the suitability of use due to the possible toxic effects on natural organisms, the possibility of releasing toxic substances, etc.The usual utilization of fine RA as backfilling layers in the Czech Republic leads to the toxic pollution in the environment. In addition, the previous investigation of the research group showed the satisfying results of mechanical properties of concrete with almost full replacement of natural sand by fine RA . The pubThis research work has responded to previous results and dealt with the possible utilization of this waste materials with prediction of the chosen waste materials properties, which will fulfill the requirements for use in concrete especially in the vie w of ecology to show the advantages of this solution. Environmental impacts occur mainly during the integration of the components of concrete with rain or wind. These processes can cause degradation, as various substances can leach out due to the action of water and it is desirable that the waste materials used in concrete do not have a toxic effect on the environment. Therefore, the chemical composition and possible toxic effects must be investigated.There are a number of factors that can affect the overall effect, such as contact time, porosity, or material damage, which only a small number of studies have examined these days . This st(1)RA 1 was prepared from reinforcement concrete in the recycling center by the two-steps recycling process. The crushed and separated recycled aggregate of fraction 16/128 mm from the first step of recycling process was crushed and sieved to the fractions in the second step.(2)RA 2 originated from highway and was partially prepared in the recycling center to fraction 64/128 mm. Afterwards, the fraction 64/128 was crushed and sieved into the fractions in the laboratory.(3)RA 3 originated from the ground floor structures and was partially prepared in the recycling center to fraction 64/128 mm. Afterwards, the fraction 64/128 was crushed and sieved into the fractions in the laboratory.(4)RA 4 originated from the masonry structures and contains mostly red bricks, mortar, and plasters. It was prepared from reinforcement concrete in the recycling center by the two-steps recycling process. The crushed and separated recycled aggregate of fraction 16/128 mm from the first step of recycling process was crushed and sieved to the fractions in the second step.Within the performed experiments, waste materials from CDW were used. The NA, which is widely used in concrete, was tested as a reference. The RA that has been tested should have properties comparable to natural sand, as the aim is to replace these primary raw materials in concrete with the secondary raw materials mentioned below. The RA originated from a recycling centers in the Czech Republic and had different origins:Two types of RA (RA 1 and RA 4) were modified by the two-step recycling process. In the first step, the CDW is crushed and separated to three fractions 0/4, 4/16, and 16/128 mm; the fraction 0/4 and 4/16 mm is separated and used as downcycling material for instance as backfilling material due to the unwanted impurities such as soil and dust. In the second step, the fraction 16/128 mm was crushed again and sieved to the fraction 0/4, 4/8, and 8/16. The content of unwanted impurities is reduced by this process. The recycling process of two other RAs (RA 2 and RA 3) was similar, however, the RA of fraction 64/128 mm from the first step of recycling process was finally crushed and sieved in the laboratory to fractions 0/4, 4/8, and 8/16 mm. Although three fractions of RA were prepared, only fraction 0/4 mm was studied in this investigation.All the samples were used as a fine aggregate and the experiments were performed according to the valid Czech standards. The used samples are shown in All types of aggregates were used in natural humidity conditions. However, for the particle size distribution test the aggregates were dried until stabilization of weight and further tested according to the CSN EN 933-1 . Limits X is the sample of an aggregate retained on each sieve [Fineness modulus (FM) is informative property of aggregate used for determination of the degree of uniformity of the aggregate gradation. It is an empirical number relating to the fineness of the aggregate. The higher the FM is, the coarser the aggregate is CSN EN 12620 + A1 :(1)FM=[.Determination of percentage of fines is based on the sieving test CSN EN 933-1 . The reqf) passing the 0.063 mm sieve in accordance with the following Equation:f is fines content (%), M is the mass of the test portion (kg), and P is the mass of the screened material remaining in the pan (kg) [Calculate the percentage of fines for dry method (pan (kg) .2O and homogenized on an overhead shaker (7 rpm) for 24 h [g, 10 min, 25 °C) and filtered through a membrane paper with pores of 4 μm. pH and electrical conductivity was determined in the filtrated leachates at room temperature. All the leachates were prepared in two replicates. Selected elements  were determined using atomic absorption spectrometry with flame atomizer 280FS AA developed by Agilent Technologies, Inc.  in leachates after acidification by HCl to pH of 2.0.Air-dried samples of 100 g were mixed with 1000 mL of Hfor 24 h . ConsequEcotoxicological bioassays were performed with both untreated leachates and leachates amended with relevant inorganic nutrients according to control media of the given test species. pH adjustment was not included in the leachate’s treatment.Desmodesmus subspicatus, strain Brinkmann 1953/SAG 86.81 which was obtained from CCALA, IBOT, AS CR  partly following the ISO guideline 8692 [Algal growth inhibition test was performed with freshwater green algae ine 8692 . Bold Baine 8692  was usedg, 10 min, 4 °C), 8 mL of pure methanol was added to sediment and homogenized. In case of algae clusters formation, dispersion of the cells was eased by placing into cooled ultrasonic bath for 2 min. After three days of extraction , extracts were centrifuged  and absorbance at 653 and 666 nm was measured using spectrophotometer UV-1900 developed by Shimadzu Corporation . Total chlorophyll content per volume unit was calculated according to [For chlorophyll content determination 9 mL of algae in uspension was isolated by centrifugation , variety Severka C1. Seeds were obtained from Aros company . Petri dishes (diameter of 9 cm) containing membrane paper soaked with 5 mL of medium/sample were prepared in triplicates (samples) or quadruplicates (controls). The control medium consisted of CaCl2·H2O~294 mg·L−1; MgSO4·7H2O~123.25 mg·L−1; NaHCO3~64.75 mg·L−1; and KCl~5.75 mg·L−1; pH of 7.8 ± 0.2 adjusted by 1 M HCl. 17 seeds of approximately 1.5 mm in diameter were placed in a regular net on the membrane paper. Covered dishes were stored under stable temperature (20 ± 1 °C) in dark for 72 h. After exposition, plant root length was determined.Germination test was performed with mustard . Steinberg medium modified by Altenburg (pH 5.5 ± 0.2) [Duckweed assay was proposed by ISO guideline 20079  using Le5 ± 0.2)  served aThe total frond area was determined by image analysis using NIS Elements . Growth rate (GR) was calculated from the values based on repeated measurements during the test exposure, i.e., 0th, 3rd, and 7th day. After the 7-day exposition, fronds were extracted by pure methanol  and the total chlorophyll content was determined spectrophotometrically  according to .Daphnia magna juveniles aged up to 24 h, which were hatched from ephippia obtained from Microbiotests Inc. (Mariakerke (Gent), Belgium). The experimental design was done following ISO guideline 6341 [Acute toxicity assay was performed with ine 6341  with somine 6341  was usedFive juvenile individuals were transferred into 25 mL beakers with 20 mL of leachate or control sample, covered with transparent film and put under stable temperature (20 ± 1 °C) and light cycle . Each sample was represented by four replicates, whereas controls by six replicates. The inhibition of daphnia mobility (viability) was observed after the 48 h exposition.tX0 is value of the parameter in t0 (d), and Xt1—value of the parameter in t1 (d). [In algae and duckweed growth rate (GR) based on cell number and frond area respectively was calculated using Equation. t1 (d). .I is inhibition/stimulation of growth (%), cX0 is average value of control, and ciX—average value of sample i. [All the ecotoxicological data  were consequently expressed as the values of inhibition/stimulation in percentage, where tested organisms in leachates were compared to control organisms using the following Equation.ample i. .EC50 values were calculated from the inhibition data for all ecotoxicity tests using nonlinear regression.3 [3 [In the previous studies, it was found that the dry density of fine RCA (recycled concrete aggregate) ranges between 1900 and 2360 kg/m3  and fine3 [3 ,32,33,343 [3  and RMA 3 [3 ,32,33, wIn this study, granulometry, dry density and water absorption of one type of NA and four types of RA were examined and compared . All typAll tested properties of RA differed from NA, especially the water absorption capacity, which was more than ten times higher and ranged from 2.1 to 8.8% for fine fraction of RA, where the highest water absorption was measured for RA 1 originated from waste concrete from recycling center. On the contrary the lowest water absorption was evaluated for RA 3, which originated from demolished floor structures and was partially prepared in the laboratory. The water absorption of RA 3 was measured for fraction 0.063/4 mm and 1/4 mm to show the influence of fines particles of aggregate. The results of water absorption show the slight relation with the fineness modulus. This evaluation shows slightly lower water absorption of RCA  and sign−1) was approximately 5–10 times higher in comparison with the other samples. However, concentrations below 5 mg·L−1 in freshwaters were found safe for duckweed [−1 [−1) was equal or similar to the Ca content in Steinberg and ADaM media whereas in RA 2 (660.57 5 mg·L−1) the Ca content was 8–13 times higher.Results showed different leaching behavior of the tested samples . All of duckweed  and acutweed [−1  On the cThe response of the model organisms to the tested samples is shown in In RA 2 and RA 3 samples both untreated and nutrients-amended non-diluted leachates the lethal effect was observed for all the test organisms except for mustard e,g. BecaThe most sensitive bioindicator of ecotoxicity was daphnia which was already recommended for ecotoxicological assessment of concrete leachates ,42. Our Based on the ecotoxicity results, the samples RA 2 and RA 3 are suitable to verify the prediction that use of waste materials in concrete can lead to their immobilization. These samples were picked because of their properties, especially higher toxicity level (inhibitory—mild toxic), but other aspects should also be considered. RA 3 with lower water absorption and higher bulk-density also fulfills the limits of current standards in particle size distribution.The water absorption of RA 1–4 is up to ten times higher than NA. The highest absorption was measured on RA 1, the lowest on RA 3.The highest density was measured on RA 3, which corresponds with the lowest water absorption measured on this sample.Samples RA 1 and RA 2 provided higher number of fine particles in the particle size distribution and the limits of the current standards have not been fulfilled.Ecotoxicity of the tested leachates increased from non-toxic effect in NA, RA 1, and RA 4 to inhibitory effect or mild toxicity in RA 2 and RA 3 in following order:This research examined and discussed the experimental verification of physical and geometrical properties of recycled concrete aggregate from four different sources. Tested samples (RA 1–4) were measured and compared with the reference samples (NA). The ecotoxicity tests were made to verify the impact of harmful substances to environment. Based on provided experiments, the final conclusions are summarized in the following points:The novelty of this study is in the effort to create a comprehensive analysis of potential environmental threats in the ecotoxicology point of view considering the physical and geometrical properties of RA. This work presents the results of the effect of waste materials on leachates and ecotoxicity. Because of the higher mortality of the tested organisms, landfilling of these materials is not appropriate. A more suitable variant seems to be the use in concrete because of the new assumption that the use of waste materials in concrete leads to their immobilization. This assumption is verified in a follow-up research.


Resistant strains of bacteria are rapidly emerging with increasing inappropriate use of antibiotics rendering them less efficacious. Self-purchasing of antibiotics particularly for viral infections is a key driver of inappropriate use, especially in lower- and middle-income countries. There is a particular issue in countries such as Pakistan. Consequently, there is a need to assess current rates of self-purchasing especially for reserve antibiotics to guide future policies.Assess the extent of current antibiotic sales without a prescription in urban areas of Pakistan.A multicenter cross-sectional study was conducted in different areas of Punjab, Pakistan using Simulated Client technique. The investigators demanded different predefined antibiotics from WHO AWaRe groups without prescription. Three levels of demand were used to convince the pharmacy staff in order to dispense the antibiotic without a prescription. A data collection form was completed by simulated clients within 15 min of each visit.Overall 353 pharmacies and medical stores were visited out of which 96.9% pharmacies and medical stores dispensed antibiotics without demanding a prescription (82.7% at demand level 1 and 14.2% at demand level 2), with only 3.1% of pharmacies refusing to dispense antibiotics. The most frequently dispensed antibiotic was ciprofloxacin (22.1%). Surprisingly, even the reserve group antibiotics were also dispensed without a prescription. In only 25.2% visits, pharmacy staff guided patients about the use of antibiotics, and in only 11.0% pharmacists enquired about other medication history.Currently, antibiotics are easily acquired without a legitimate prescription in Pakistan. There is a need for strict adherence to regulations combined with a multi-dimensional approach to enhance appropriate dispensing of antibiotics and limit any dispensing of WHO restricted antibiotics without a prescription. Approximately 700,000 people die across the world each year due to drug-resistant infections, with the potential of rising to ten million by 2050 unless actions are taken . The irrThe major cause of resistance comes from the easy availability of antibiotics with pressure on physicians and pharmacists to prescribe and dispense antibiotics for predominantly self-limiting conditions, aided by poor hygiene and living conditions in a number of LMICs fueling infection rates , 20–24. Overall, a lack of diagnostic facilities and poor practices among health care professionals, coupled with public behavior including pressurizing pharmacists to dispense antibiotics for predominantly viral infections, have contributed towards increases in the sale of antibiotics without prescription especially in developing countries , 36, 37.We are aware of high rates of self-prescribing of antimicrobials and other medicines in Pakistan often for economic reasons , 41–48. http://www.raosoft.com/samplesize.html) by keeping 5% margin of error, 95% confidence interval, and 50% response distribution.A multicenter cross-sectional study in pharmacies and medical stores of different cities of Pakistan was conducted using the Simulated Client technique, which is a well-recognized technique to help accurately assess pharmacist and dispenser activities –61. The Pharmacy undergraduates from the University College of Pharmacy, University of Punjab, were recruited as simulated clients and they underwent training. They were advised to select the pharmacy or medical store in their own area and demand any of the access, watch or reserve (AWaRe) group of antibiotics according to predesigned standardized visiting process Fig. . All theSimulated clients visited 353 pharmacies and medical stores out of which 267 were pharmacies and 86 were medical stores. 82.7% pharmacies and medical stores dispensed an antibiotic without demanding any prescription and without investigating the reason for buying the antibiotic (DPR level 1). 14.2% dispensed the requested antibiotic after investigating the disease and condition of the patient (DPR level 2). Overall, 96.9% of all pharmacies and medical stores visited dispensed an antibiotic without a prescription and only 3.1% refused to dispense antibiotics although our simulated clients used all three demands level (Fig. From the access group, amoxicillin was most commonly requested antibiotic in 39 visits. In the watch group, ciprofloxacin was demanded in 46 visits. Interestingly, the reserve group antibiotic linezolid was also requested without prescription in 41 cases. Other antibiotics offered are presented in Table To the best of our knowledge, this is the first study from Pakistan to quantify the extent of non-prescription sales of antibiotics by using a simulated client technique. As mentioned, the simulated client technique is a well-accepted methodology closer to the real-life scenario as compared with other methods which may lead to deceptive results , 62, 63.Out of the total number of antibiotics demanded, ciprofloxacin, linezolid, amoxicillin, metronidazole and co-amoxiclav were the most dispensed. A study conducted in Riyadh, KSA saw higher sales of amoxicillin/clavulanate . Whilst The current status of poor antibiotic dispensing in Pakistan requires that pharmacists must now play a key role in ensuring the rational use of antibiotics  to reducThere is a concern in countries such as Pakistan where patients have limited income alongside high levels of co-payment and ease of access to medicines in community pharmacies. In view of this, self-medication from pharmacies is the preferred option avoiding physicians’ or consultants’ fees , 43, 63,We are aware of a number of limitations with our study. Firstly, we did not distinguish whether the person who dealt with the patient was a pharmacist or another pharmacy worker since both pharmacists and other employees work in community pharmacies. This was because our goal was neither to determine the possible differences between the two nor to investigate the presence of pharmacists. The volunteers didn’t want to appear dubious because, as a result of this, the aim of our study could be compromised. Secondly, our study was directed mostly to pharmacies in a few cities of Pakistan with comparatively better demographic aspects and socioeconomic. Consequently, our findings may not necessarily be generalized to the entire country. Despite this, we believe our findings are robust providing direction for the future.The results of this study reveal that the non-prescription sale of antibiotics is still common practice in Pakistan despite the legislation. In majority of the cases, it was possible to acquire a watch group antibiotic without an official prescription. Moreover, the pharmacy staff typically neither questioned the patient about their history nor encouraged the patient to seek medical advice. The current status of antibiotics sale and dispensing practices in local community setups is a complex issue. A multi-dimensional approach is needed to warrant the appropriate sale of antibiotics in the country.Additional file 1.


There is increasing concern globally about the enormity of the threats posed by antimicrobial resistance (AMR) to human, animal, plant and environmental health. A proliferation of international, national and institutional reports on the problems posed by AMR and the need for antibiotic stewardship have galvanised attention on the global stage. However, the AMR community increasingly laments a lack of action, often identified as an ‘implementation gap’. At a policy level, the design of internationally salient solutions that are able to address AMR’s interconnected biological and social  dimensions is not straightforward. This multidisciplinary paper responds by asking two basic questions: (A) Is a universal approach to AMR policy and antibiotic stewardship possible? (B) If yes, what hallmarks characterise ‘good’ antibiotic policy? Our multistage analysis revealed four central challenges facing current international antibiotic policy: metrics, prioritisation, implementation and inequality. In response to this diagnosis, we propose three hallmarks that can support robust international antibiotic policy. Emerging hallmarks for good antibiotic policies are: Structural, Equitable and Tracked. We describe these hallmarks and propose their consideration should aid the design and evaluation of international antibiotic policies with maximal benefit at both local and international scales. The global crisis of antimicrobial resistance has led to a proliferation of expert reports and national and international antibiotic action plans.Implementing international antibiotic policy that is meaningful in different social, cultural and economic contexts continues to prove challenging.Our multidisciplinary analysis has identified four critical challenges of metrics, prioritisation, implementation and inequality for international antibiotic policy-making.We propose a corresponding SET of basic hallmarks of good antibiotic policy, which we define as Structural, Equitable and Tracked.Our SET of hallmarks can orientate disciplinary debates and provide a framework for developing robust international interventions.Since their introduction in the 1930s, antibiotics have acquired infrastructural importance in global health and food production.Over the past decade, rising concern about AMR has highlighted the need for collective action to protect our antibiotic ‘commons’.We propose a new wide-angle approach to AMR-focused antibiotic regulation. Two basic questions guide our reflective process: (A) Is a universal approach to AMR policy and antibiotic stewardship possible? (B) If yes, what hallmarks characterise ‘good’ antibiotic policy?et al have proposed three Rs for antimicrobial development).19To answer these questions, we have developed an innovative heuristic evaluation framework, which accounts both for AMR’s biological and social facets. Our approach was informed by the numerous national and international reports proposing various principles for antibiotic policy-makingApplying this approach to AMR and antibiotic policy, our collaborative multidisciplinary analysis employed a three-stage evidence gathering, evaluation and consultation process , which cBox 1Following a 2018 Social Science and antimicrobial resistance (AMR) Research Symposium at the British Academy in London,Identified policy challenges and core hallmarks of successful intervention informed a preliminary paper, which was circulated among correspondents and attendees of a multidisciplinary 2-day workshop in London in March 2019.During presentations, small group breakout sessions, and group discussions, participants reflected on the preliminary paper’s four identified problem areas for antibiotic regulation: (A) metrics ; (B) prioritisation (prioritising specific forms of antibiotic use over others); (C) implementation ; (D) inequality .resilience of health and food systems to diminish the need for antibiotics; (3) be responsive to evolving knowledge regarding AMR and (4) relational in its acknowledgement of differing local challenges and capacities.There was agreement on these interlinked problems but the four provisional hallmarks proved more contentious: (1) antibiotic policymaking should take into account antibiotics’ infrastructural relevance in medicine and food production; (2) should increase the microbial status quo. Key to more effective policymaking has to be an acknowledgement of antibiotics’ primary utility for global health and food production, which consists in their ability to reduce mortality and morbidity resulting from treatable infections. To preserve this utility, the overriding aim of any antibiotic policy must therefore be (1) to maximise and maintain access to effective treatments for infections, which includes the development of new treatments—while (2) reducing the need for antibiotic use by preventing infections, reducing antibiotic dependencies in healthcare and food systems, and minimising the environmental load of antibiotics and resistant bacteria.Participants agreed that there was no single solution to AMR but felt that hallmarks needed to be integrated and go beyond preserving the A refined version of new Structural, Equitable, Tracked Hallmarks was circulated among correspondents and participants, who were invited to be coauthors on the paper.SET can orientate disciplinary debates and provide a framework for robust international interventions.10 20 21We believe that the resulting heuristic compass based first on the identification of central challenges of antibiotic policy-making  and second on the formulation of a corresponding Structural, Equitable and Tracked (SET) of hallmarks of good antibiotic policy, which we define as Despite long-standing regulation attempts, there remains substantial uncertainty about basic metrics surrounding antibiotic usage and AMR as well as about how to correlate measurements in a way that can inform meaningful policy formulation at the national and international levels.22Since the first warnings about ‘drug fastness’ in microorganisms in 1907,25–27Internationally, the establishment of WHONET (est. 1989) and WHO’s Global Antimicrobial Resistance Surveillance System  marked important efforts to standardise AMR reporting in humans and make data comparable.Capturing relevant data on antibiotic use to inform AMR efforts has its own challenges. Starting in the 1990s, various high income countries (HICs) began to compile antibiotic usage data.42Correlating usage and AMR data is even more challenging. In its 2018–2019 report, the English Surveillance Programme for Antimicrobial Utilisation and Resistance (ESPAUR) showed a significant reduction in antibiotic prescriptions but an increase in antimicrobial-resistant infections for the seven priority bacterial pathogens reported.45–47MCR-1 gene.Escherichia coli in China in the mid 1980s.Understanding what the implications of specific forms of antibiotic usage  in different environments are for AMR and health outcomes is similarly crucial for policy formulation. Rising numbers of point prevalence studies and whole-genome sequencing are enhancing our knowledge of drivers and variations of AMR over time and in different areas across the world.Accessed as first-line narrow-spectrum treatment for particular conditions, and those with higher risks of becoming resistant that should be used Watchfully and those that should be Reserved for last-line treatment.66 67While uncertainty remains about the degree to which interventions will be effective, reducing the overall amount of antibiotics used in health and animal and plant production systems is a key component of most AMR action plans.Staphylococcus aureus and E. coli may or may not be causing disease, and therefore, their measurement without corresponding disease burden data might be misleading.Alongside improved usage data, deciding which microbes, resistance genes and AMR reservoirs to monitor in medical, animal and plant production, and environmental settings is similarly important for the formulation of meaningful policy interventions. For humans, WHO published a list of priority pathogens to monitor and target with antibiotic development efforts in 2017.Discussed in more detail below (see Tracked), one recently proposed solution for human health might be to integrate AMR and infection burden measurements by tracking two priority organisms in bloodstream infections as part of United Nations (UN) Sustainable Development Goal (SDG) 3.d.1. This new AMR-specific SDG could help build laboratory capacity in resource-poor settings and spur the development of further integrated AMR-specific metrics. Another approach might be to strengthen point prevalence studies for specific sentinel organisms in LMIC settings.The described temporal and contextual challenges of defining resistance, measuring antibiotic usage in relation to AMR and concerning which microbes to focus on also raise important questions when it comes to defining benchmarks or key performance indicators (KPIs) for policy initiatives: how far should antibiotic sensitivity be restored or preserved for a measure to be considered successful? Should the performance of a policy be linked to its reduction of antibiotic usage or should the KPI be demonstrated impact on human health? Should success be defined as the stabilisation or decline of AMR in specific culturable pathogens? Given the mobility of resistance genes and the One Health dimensions, should a metric of success be the abundance or prevalence of a particular resistance gene in the wider environment, determined through quantitative PCR methodsOveremphasis on surveillance data itself can serve to obscure different regional capabilities, underlying political interests and competing needs in animal and human medicine.Spatial: since the 1940s, regulators have tried to protect important antibiotics by restricting their use,mcr-1 gene on transferable plasmids in bacterial isolates from Chinese pigs that polymyxins were recategorised as highest priority CIAs in 2016, received specific restriction recommendations from OIE, and were banned from use as growth promoters in China and Brazil.57 60 75The same problems of spatial prioritisation hold true in animal and plant production.Temporal: another challenge of prioritisation regards the inevitable temporal conflict between acute healthcare needs and the future-focused dimensions of antibiotic stewardship.104Answers to this temporal dilemma vary and reflect the perceived severity of need, cultural preferences for specific forms of use, and economic considerations. In some cases, the potential longer-term risk to public health of antibiotic resistance has been favoured over immediate clinical needs.34 77 107A third fundamental challenge for antibiotic policy-making concerns the formulation of binding international agreements that can still be implemented flexibly in different settings.82 112 113Using an integrated political, economic, sociological, technological, ecological, legislative and industry framework, reviews of national action plans proposed after the 2015 Global Action Plan on Antimicrobial Resistance have warned of continuing gaps in applying international concepts of stewardship at the national level.34 120However, so far, enactment of proposed measures has been fragmentary34 77One of the most significant challenges facing international antibiotic policy and global health frameworks more widely are significant levels of inequality between and within HICs and LMICs. These inequalities reflect historically uneven social and political opportunity as well as distribution of economic resources and disease burdens. Inequality has large implications for ways priorities are made and implemented in relation to antibiotics. Recognition of these inequalities challenges forms of policymaking that focus on metrics of drug reduction alone (see Metrics and Implementation above) or prioritise protection of HIC hospital antibiotics over LMIC needs for access to antibiotics and protein production.The unevenness of social and political opportunities affects who can make or demand policies. At the international level, the historical dominance of HICs on relevant health bodies and many funders’ and high-level meetings’ location in HICs has led to a relative absence of the voices of some of the most affected LMIC stakeholders.Answering these questions is important. In the case of drug development, historical priority setting has been HIC-centric due to the greater profitability of high-income markets (with a particular emphasis on the most lucrative US market) and differing regional risk priorities.Over time, decontextualised international decision making can result in policies that prioritise HIC-centric stewardship interventions like targeted drug restrictions that may prove deleterious in LMICs where infection risks are markedly higher and different.10 112Similar constraints are true for animal health which is related to issues such as food safety, food security and animal welfare. Many national and international action plans invoke enhanced biosecurity facilities, vaccination or good husbandry practices as a means to avoid unnecessary non-human antibiotic use, but they rarely set these measures as (funded) priorities contrary to raising awareness, developing surveillance or promoting responsible and prudent use, or they risk assuming inappropriate livestock production and disease management approaches in settings where economic and microbiological risks are manifestly different.The four biosocial challenges of metrics, prioritisation, implementation and inequality require a holistic response from international antibiotic policy that is ambitious enough to improve the status quo but concrete and evocative enough to be an effective guide. Because such policies are enacted within a complex ecosystem, a broad perspective needs to inform their design and deployment to maximise effectiveness and minimise unintended consequences. It is this broad heuristic perspective, incorporating One Health, spatial, temporal and ethical dimensions that underlies the three interlinked SET hallmarks proposed here .1. Structural: International antibiotic policies should recognise and respond to the multiple aspects of global antibiotic infrastructures.Historically, there has been a tendency to make AMR manageable by compartmentalising problems and blaming individuals for drug overuse instead of the underlying social, political and economic factors driving antibiotic demand and dependencies (see Metrics and Prioritisation).74 141 142Successful antibiotic stewardship cannot be narrow and divorced from the social and environmental contexts in which use is taking place. More effective international policy requires the abandonment of regulatory silos, as well as the adoption of context-sensitive models. It requires a broad structural approach to reforming not only the international antibiotic supply chain (drug producers) but also wider contributing global and regional consumption patterns  as well as associated socio-structural factors  and environmental factors .As evidenced by recent successes in HIC animal production and health systems, jointly focusing on preventing disease with vaccines and improved welfare, updating the design of surveillance, hospital, animal housing systems and modifying antibiotic-seeking behaviour by patients and animal and plant producers can reduce antibiotic dependencies.20 127 146 1472. Equitable: To be impactful globally, international antibiotic policies must recognise and respond to the unevenness in contributions towards and ability to tackle AMR while aiming for an equitable future for antibiotics regardless of where they are deployed. Historically, inequality and the difficulty of uniform policy implementation across HICs and LMICs have been major obstacles for international antibiotic reform .Because antibiotic effectiveness is a time-limited global common pool resource and even robust national responses offer little protection from the global circulation of AMR genes and organisms, the sustained pooling of international resources is an essential prerequisite to overcome identified challenges. Similar to climate change, some countries have a greater differentiated responsibility to contribute resources to this common pool than others.48 119 149 150Whether action is justified on the basis of historical usage, collective responsibility, obligations towards future generations, or enlightened self-interest, any international policy framework will have to include long-term financial and political HIC and MIC commitments to support antibiotic sensitive interventions in resource-poor communities. In LICs, antibiotic sensitive international support could centre on building human and infrastructural capacity by educating and employing more medical and veterinary professionals,Well-designed structural and equitable international antibiotic policies can generate a global win-win.119 123Because of its structural dimensions, the lack of an easy target and a dysfunctional commercial research and development pipeline for new antibiotics,3. Tracked: Progress towards structural and equitable antibiotic policies has to be tracked to ensure ongoing effectiveness of interventions, promote integration of international efforts and motivate sustained commitment of donors and funding recipients. While a regular independent international stocktake could help ensure that policy interventions remain coordinated, equitable and up to date,Achieving effective tracked interventions requires a unifying global set of multifactorial metrics. To enable the rigorous evaluation of policies, multifactorial metrics need to simultaneously take into account antibiotic access, AMR and stewardship, but avoid rewarding short-termist gaming such as relabelling disease definitions, disincentivising healthcare-seeking behaviour and decontextualising surveillance .155In addition to drawing on multifactorial metrics, policy tracking should aim to integrate metrics across domains: taking into account the legacies of pre-existing interventions, improving knowledge integration across One Health sectors and incorporating knowledge generated in other geographical and social contexts. Enhancing the integration of international knowledge gathering will strengthen regionally nuanced decision making. Since the 1940s, successive generations of regulators have tried to manage the antibiotic commons by focusing on one form of intervention like ‘rational’ antibiotic use, reducing antibiotic use via statutory bans or non-statutory incentives and replacing antibiotics . However, lack of cross-sectoral and international integration meant that benefits were often short-lived and had little effect on global drug usage or AMR.Developing multifactorial and integrated metrics will achieve little if global AMR and usage data continues to be generated and made available unevenly. Equitably boosting laboratory and data analysis capacity is a prerequisite for strengthening the ability of those on the front lines of human, animal and plant health to devise their own solutions and generate accurate data for the global community. For example, in 2010, the Danish Yellow Card Initiative required farmers and veterinarians to track and record on farm antibiotic use. The initiative complemented low-granularity sales data and helped respective communities better understand what they were doing and grow support for stewardship efforts.The past eight decades have seen antibiotic policy-makers struggle to overcome problematic metrics, narrow prioritisations, implementation deficits and global inequalities. The need for integrated architectures that act across existing silos to push evidence into action on AMR is being met through United Nations mandated mechanisms.Policies that take into account antibiotics’ SET have the greatest potential to sustainably adapt the way the global antibiotic commons are managed and accessed. As highlighted in our ideal-type example of multifactorial metrics and equitable capacity building in global food production , SET polIf the ultimate goal of antibiotic policy is to reduce mortality and morbidity resulting from treatable infections, then we need to adapt our food and health systems to provide optimal access to effective antibiotic interventions when they are needed—and simultaneously reduce the need for these interventions. This dual approach requires polices that not only focus on ‘quick fixes’AMR is not a problem to be solved but a phenomenon to be continuously managed. Individual policies may not address all identified problem areas or integrate each of our three intervention hallmarks. However, we believe that our multidisciplinary SET of hallmarks can serve as a compass to critically evaluate and improve antibiotic policy in the present and for decades to come.


Structures and bridges are being designed on the proposed and requested design lifetime of 50 to 100 years. In practice, one can see that the real lifetime of structures and bridges is shorter in many cases, in some special cases extremely shorter. The reasons for the lifetime shortening can be increased of the load cases , using the material of lower quality, implementation of new standards and codes according to Eurocode replacing older ones. During the whole lifetime the structures must be maintained to fulfil the code requests. If the constructions are not able to fulfil the Ultimate Limit States (ULS) and the Serviceability Limit State (SLS), the structures or bridges have to be strengthened (whole or its elements). The purpose of the paper is the presentation of using a layer of the fibre concrete for a columns’ strengthening. Using the fibre reinforced concrete (FRC) of higher tensile strength makes it possible to increase the load-bearing capacity of the cross-section the column. The contact between the old concrete (core of column) and newly added layer (around column) is very important for using that method of strengthening. In the article, there is also a comparison of the surface modification methods. Fibre reinforced concrete (FRC), or concrete with dispersed reinforcement, is a rheological composite material based on silicates with ideally dispersed reinforcement elements in the form of fibres—either steel or synthetic. The dispersion reinforcement significantly affects some shortcomings of conventional concrete ,2,3. TheFibre reinforced concrete belongs to High Performance Concretes (HPC), which are typically used for strengthening linear horizontal members, for instance, beams ,14. The Results from the 3-point a and 4-pmE is the modulus of elasticity of concrete, fE is the modulus of elasticity of the fibre reinforced concrete, εmu is the cracking strain of matrix in composite, Vm is the volume of concrete matrix, fVfuσ is the load-bearing capacity of fibres. The softening or hardening of the fibre reinforced concrete occurs after the appearance of the first crack in the composite. The crack occurs in the brittle concrete matrix, the load is carried by the fibres. If the collapse of the element is to be prevented, the load-bearing capacity of the fibres omposite ,23(1)σfIf Equation (1) was satisfied fR=3 Denotation of the input data, given in Equation (2), is shown in ectively .uw is the ultimate crack opening acceptable in the structural design, Ftsf is the serviceability residual strength, Ftuf is the ultimate residual strength. Residual strength limits must be determined for the input parameters, for the residual serviceability of tensile strength Calculation of the resistance moment, according to the FIB MODEL CODE, is based on the balance of internal forces. The second type of the residual strength calculation of the fibre reinforced concrete is conducted according to the RILEM TC 162—TDF  see Fig. That caFrom the measured values of the crack width and load, it is necessary to determine the stresses according to the following Equations (5)–(7)The paper presents the possibility of using fibre reinforced concrete to strengthen columns taking into account the effect of contact between old concrete and new fibre reinforced concrete on the load-carrying capacity of the cross-section. The practical use of the work can be to strengthen the columns of frame structures or piers of the substructure of bridges with insufficient load-carrying capacity of the cross-section ,29,30. IMost experiments for column strengthening deal with the compressive force and low bending moment only. In this case, the most suitable method is strengthening by the concrete encasement with a layer of reinforced concrete, plain concrete and wrapping with the FRP sheets ,38,39. Ic and friction μ for individual types of surfaces. The bearing capacity for transmission of the shear force along the contact surface was also verified for the individual surface treatment methods, which were used for the following experiments [The load-bearing capacity of the strengthened member (the whole cross-section) depends in part on the shear resistance between the old concrete (core) and a layer of the newly added concrete ,45,46. Teriments .(A)Smooth surface wrapped with fibre reinforced concrete (fibre reinforced concrete with steel wires)—(VH),(B)Indent wrapped with fibre reinforced concrete—(ZZ),(C)Notches wrapped with fibre reinforced concrete—(ZR),(D)Smooth surface wrapped with reinforced concrete—(ZH),(E)Smooth surface wrapped with plain concrete—(PH),(F)Smooth surface wrapped with foil and a layer of fibre reinforced concrete—(FH).Three methods of contact treatment  with different types of reinforcement in the case of smooth surfaces were used for surface treatment :(A)SmootSelection of the surface treatment was performed with regard to the complexity of design in practice. Consideration was also given to the shear strength of individual surface modifications.Dimensions of the column were chosen with the cross-section dimensions of 0.16 m × 0.16 m and a height of 0.35 m. The column (core) was reinforced with 4Ø10 mm, the length of the bars is 0.3 m. The stirrups of diameter 8 mm were placed at an axial distance of 85 mm . ReinforSamples, after concreting, were treated for the next three days to prevent formation of the shrinkage cracks and after 28 days the samples were removed from the forms. Subsequently, the samples´ surfaces were modified according to the above-mentioned surface treatments . All the3 of concrete, Temporary polystyrene adhered to the bottom part of the treated samples. The samples thus prepared were placed in the formwork with centring the spacer screws d. Prior 3 represents a fibre ratio of 0.5%. During the concreting, test cubes with dimensions of 150 × 150 × 150 mm3 were cast in order to determine the strength characteristics of concrete. The compressive strength test of the concrete was conducted on the day of the “push tests”. Samples were concreted in two batches , 3 test samples were cast from each batch. Their cube strengths c,cubef are given in The weight of the fibres Dramix 3D equal to 40 kg/mThe samples were placed in a jack, a steel plate was placed on the upper edge of the sample for better load distribution and then the sensors for distance measurement No. 1 and No. 2 were glued to the samples . The disτ [c is cohesion (kN m−2), μ is the coefficient of friction (-), nσ is the normal stress (kN m−2), nτ is the shear stress (kN m−2), S is the area of contact shear surface (m2). For comparison, the characteristic values of the shear strength τ  were calc = 0.20 for a smooth surface, c = 0.40 for a surface with notches and c = 0.50 for a surface with indent were used to calculate the maximum failure force failureF according to the standard [nσ is considered to be zero (nσ = 0.0 kN m2). Then the coefficient of friction μ does not need to be considered. The calculated values are shown in The standard values of the coefficient of friction standard . In thisSample tests were prepared 55 days after concreting. The main goal was to determine the shear resistance for individual methods of the surface treatment (processing). Seventeen samples were tested and results are shown in From the obtained results , the calFor experimental verification of the strengthened columns, 3 sets (groups) of columns were made for two samples, i.e., a total of 6 experimental samples of columns. The first group consisted of non-strengthened columns (2 specimens), which were used to determine the basic value of load-bearing capacity. The second group of columns (2 specimens) was strengthened with a layer of fibre reinforced concrete (concreting with a layer of fibre reinforced concrete with steel wires) and the last group (2 specimens) was reinforced with the new layer of reinforced concrete.The basic dimensions of the column were determined to be 160 mm × 160 mm and the height 2500 mm, which were based on the experiments in ,50. minρ = 0.002 ≤ ρ = 0.0123 max≤ ρ = 0.04 was observed. The slenderness of the column is equal to the value λ = 54.13 > limλ = 21.95, so the column is considered as slender. The environmental XC1 class was considered. Steel plates were attached to both ends of the columns and mounted before concreting. The samples were treated for 3 days. The lower class C16/20 concrete was used. Steel B 500B was used to reinforce the column. The column was reinforced with 4Ø10 mm and stirrups of a diameter of 8 mm at an axial distance of 100 mm . It was e = 100 mm.According to  and the λ = 37.65 > limλ = 22.94, but the strengthened columns still remained as slender. The chosen thickness of the new layer also took into account the minimum coverage of the main longitudinal reinforcement in the new layer of concrete in the second method of reinforcement [Two strengthening methods were considered in the experimental program. The first alternative was the fibre reinforced concrete reinforcement (steel fibre reinforced concrete concreting—denotation of samples SV). The thickness of the layer was considered to be 35 mm and was chosen with regard to slenderness. The new cross-section dimensions were increased to 230 mm × 230 mm, where the slenderness decreased to orcement .A concrete mix of fibre reinforced concrete, identical to the “push tests”, was used for the cladding . At the The second alternative (method) was to strengthen the column with a layer of reinforced concrete of the same thickness as in the first case, namely 35 mm (layer of concrete with reinforcement—denotation of samples SB). The cross-section was additionally reinforced with longitudinal reinforcement 4Ø10 mm (in the newly added layer) and stirrups with a diameter of 6 mm at axial distances of 150 mm . In thisWithin the laboratory conditions and due to the column height (laboratory options), the test itself was performed horizontally . The assembly consisted of transverse girders and two Dywidag longitudinal bars. A press with a maximum force of 3000 kN was placed on the transverse girders. The columns were supported at both ends of the joints. The measuring devices S1–S5 were arranged along the entire length (column height), . These sAll the members were loaded during the test with an axial force, the increment of the force was 25.0 kN until the deformation stabilised at a particular level. Six samples were tested. Results of the maximum forces during the columns´ failure are shown in It is clear from the results of the experiment that a more effective method of strengthening is reinforcement with the new reinforced concrete layer in terms of a higher increase in resistance than has already been stated . This stHowever, by comparing the load-bearing capacity of the cross-section using these two different methods of strengthening, using the material consumption (concrete reinforcement versus fibres), it is obtained that the use of fibre is more efficient. The total reinforcement consumed  for the strengthening concrete reinforcement was 12.37 kg per column, but the weight of the fibres for the strengthening concrete reinforcement was 3.05 kg per column, which is four times less weight compared to strengthening concrete reinforcement . Taking 3 with a notch were tested. The height of the notch corresponded to 1/6 of the height of the beam. The test was performed according to the standard [In order to obtain the necessary material properties of the fibre reinforced concrete, a test was performed to determine the flexural tensile strength of concrete. The test was performed with a 3-point bending test. Three beams of dimensions 150 × 150 × 700 mmstandard ,53. The To verify the samples from the experimental program, the 3D models were created in the nonlinear program ATENA 3D Červeka Consulting . The proThe input values of materials for the fibre reinforced concrete were not easy to determine. For example, the fracture energy cannot be determined in experiments  ,55. TherNumerical models have been developed to verify the “push tests” (tests to verify the contact between the old part of the column and the new layer). The 3D models consisted of a core, encasement and a steel plate . The entThe numerical model was adjusted  to obtain an average value from the measured experimental values. The results of the numerical analysis (VH_A) indicate that the shear strength was reached at a force of 141.756 kN .nn and Ktt, which indicate normal and shear  stiffnesses, respectively [The setting of the contact is enabled by parameters Kectively . The resNumerical 3D models were also made to verify experimental column samples. The non-strengthening model consisted of a column, vertical and horizontal reinforcement and distribution plates a–c. The Material characteristics for individual elements are given in The resulting model was adapted to the resulting average values from two experimental samples. Deformations at individual locations are shown in For modelling the column with strengthening, the 3D model of the non-strengthened column from the previous section was used. This means that the model was supplemented with a 35 mm thick layer c with maThe resulting values from the path sensors are shown in The results of experimental measurements and numerical modelling have demonstrated the suitability of using new types of materials, such as FRC, for the strengthening of vertical elements, such as columns.Based on results of the experimental program and numerical analysis, it is clear that the surface (contact) treatment on push-test samples results in an increase in the shear resistance. However, in comparison to the experimental values, values calculated according to the standard , in someTwo alternatives were considered for strengthening in the experimental program of column strengthening. The first alternative was strengthening using a layer of fibre concrete. It was a layer 35 mm thick. After the failure of the specimen, the formation of one major crack at the point of greatest stress was seen. Due to the formation of one major crack, it can be established that the composite has been softening. After closer analysis, it was clear that the fibres from the cement matrix were pulled out. Overgrowth of new and original concrete was also observed, during which the individual parts (core and encasement) did not slip. It follows that the strengthening column with a fibre volume of 0.5% carried out 258.19% (235.925 kN) more than the unreinforced column. However, in comparison with the strengthening by reinforcement concrete, there was a brittle failure in the pressure area. Even in this case, there was no shear displacement in the contact. Compared to fibre-reinforced concrete, it carries out an average of 144.71% (172.18 kN) more.


In rice, endogenous SL levels increase in response to deficiency of nitrogen, phosphate, or sulfate . Rice SL mutants show increased lamina joint (LJ) angle as well as dwarfism, delayed leaf senescence, and enhanced shoot branching. The LJ angle is an important trait that determines plant architecture. To evaluate the effect of endogenous SLs on LJ angle in rice, we measured LJ angle and analyzed the expression of SL-biosynthesis genes under macronutrient deficiencies. In the “Shiokari” background, LJ angle was significantly larger in SL mutants than in the wild-type (WT). In WT and SL-biosynthesis mutants, direct treatment with the SL synthetic analog GR24 decreased the LJ angle. In WT, deficiency of N, P, or S, but not of K, Ca, Mg, or Fe decreased LJ angle. In SL mutants, deficiency of N, P, or S had no such effect. We analyzed the time course of SL-related gene expression in the LJ of WT deficient in N, P, or S, and found that expression of SL-biosynthesis genes increased 2 or 3 days after the onset of deficiency. Expression levels of both the SL-biosynthesis and signaling genes was particularly strongly increased under −P. Rice cultivars “Nipponbare”, “Norin 8”, and “Kasalath” had larger LJ angle than “Shiokari”, interestingly with no significant differences between WT and SL mutants. In “Nipponbare”, endogenous SL levels increased and the LJ angle was decreased under −N and −P. These results indicate that SL levels increased in response to nutrient deficiencies, and that elevated endogenous SLs might negatively regulate leaf angle in rice.Strigolactones (SLs) are a class of plant hormones that are synthesized from  Oryza sativa L.), leaf angle is determined by the shape of the lamina joint (LJ), which connects the leaf blade and sheath  stimulate elongation of adaxial parenchyma cells at the LJ and increase LJ angle . BR also pathway . In cont pathway . Recentl pathway . HoweverSL was originally isolated from plant root exudates as a seed germination stimulant of witchweed . Later, d27, d17/high tillering dwarf (htd)1, and d10 are well characterized. D27, D17/HTD1, and D10 encode β-carotene isomerase, carotenoid cleavage dioxygenase (CCD) 7, and CCD8, respectively , and 4DO to orobanchol  at 25°C and 8 h dark at 23°C for 6 days (pre-culture). Seedlings of similar size were then transferred to hydroponic medium containing 1 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 5.7) for a further 3, 6, or 24 days. Seedlings were placed in soil and cultivated in a glass room for 34 days. LJ angle (between leaf blade and sheath) of the 2nd, 3rd, and 4th leaves was measured on leaf images in ImageJ software v. 1.50  plants and ckground . The seeckground . Surfaceckground  solidifi v. 1.50 .rac-GR24, a synthetic SL analog, was purchased from Chiralix  and dissolved in acetone. Mock solution or 20 μM rac-GR24 containing 0.1% Tween-20 were spotted (2 μl) onto the LJ of WT and d mutants on days 0 and 3 of cultivation in hydroponic medium. The following standards were used for SL quantification: 4DO, d1-labeled 4DO, d3-labeled 4DO, orobanchol, d1-labeled orobanchol, and d3-labeled orobanchol; all were kindly provided by Prof. Kohki Akiyama (Osaka Prefecture University).ubiquitin was used as an internal standard. Expression levels of SL-related genes were quantified using the specific primers and probes used in a previous study  following the instructions in the user manual. We used 0.1 μg of the total RNA for cDNA synthesis with a ReverTra Ace qPCR RT kit for quantitative real-time PCR (qRT-PCR) . qRT-PCR was performed in a StepOnePlus thermocycler  with a Thunderbird Probe qPCR mix (Toyobo). Expression of us study .d1-labeled 4DO and orobanchol as internal standards; the homogenates were filtered with Bond Elute reservoirs  and evaporated to dryness under nitrogen gas. The extracts were dissolved in 4 ml water adjusted to pH 2–3 with 1 N HCl and extracted twice with 4 ml ethyl acetate. The ethyl acetate phase was evaporated to dryness under nitrogen gas. The extracts were dissolved in 10% acetone, loaded onto Oasis HLB 3-ml cartridges (Waters), washed with 10% acetone, and eluted with 60% acetone. The eluates were dissolved in ethyl acetate: n-hexane (15:85) and loaded onto Sep-Pak Silica 1-ml cartridges (Waters). After the cartridges were washed with ethyl acetate: n-hexane (15:85), 4DO was eluted with ethyl acetate: n-hexane (35:65) and orobanchol with ethyl acetate: n-hexane (50:50). LJ sample (ca. 0.15 g) were homogenized in 10 ml acetone containing 100 pg each of d3-labeled 4DO and orobanchol; the homogenates were filtered with Bond Elute reservoirs  and evaporated to dryness under nitrogen gas. The extracts were dissolved in 4 ml 50% acetonitrile adjusted to pH 2–3 with 1 N HCl, and 4 ml hexane, mixed gently, centrifuged at 3,400 rpm for 5 min, and the hexane-phase was removed. After the aqueous-phase was extracted with 4 ml ethyl acetate twice, 4DO, and orobanchol in the ethyl acetate-phase were purified according to the method in roots.We measured the levels of 4DO and orobanchol in roots as described previously  with somPurified SL-containing fractions were dissolved in 50% acetonitrile and subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis using a system consisting of a quadrupole tandem mass spectrometer  and a high-performance liquid chromatograph  equipped with a reverse-phase column . Previous papers describe LC and MS parameters for 4DO  and orobt-test (P < 0.05) was used for pairwise comparisons and Tukey's honestly significant difference (HSD) (P < 0.05) for multiple comparisons.Statistical analysis was performed in SPSS 24 software . Student's d3-1 than in other d mutants, but in our experimental conditions, it was smaller in d3-1  and SPX2 expression, which inhibit REGULATOR OF LEAF INCLINATION (RLI1), a positive regulator of leaf inclination, reducing leaf angle via suppression of downstream BR signaling , from Ichimura Foundation for New Technology , and from the Japan Society for the Promotion of Science .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This cohort study analyzes changes in financial risk protection associated with implementation of the Patient Protection and Affordable Care Act (ACA) across income strata and insurance types. We thus sought to analyze changes in financial risk protection associated with ACA implementation across all income strata and insurance types.One decade after passage of the Patient Protection and Affordable Care Act (ACA), despite substantial gains in insurance coverage, health care affordability remains a major concern among US residents.4 . Interrupted time series analysis was used to evaluate changes in the rate of catastrophic expenditures, with an inflection point in January 2014, the start of full ACA implementation.2 Individuals were stratified for analysis by quartile of household income as a percentage of the federal poverty level and by insurance type  reporting guideline for cohort studies.This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (P < .001). Coverage gains were concentrated in the 2 lower income quartiles, in which the uninsured rate decreased from 44.1% to 28.6% (lowest quartile) and 27.0% to 18.7% (P < .001).We identified 159 941 survey respondents , representing 186 million individuals annually after survey weighting. The number of uninsured nonelderly adults declined from 42.9 million (23.5%) in 2010 to 27.9 million (14.8%) in 2017, whereas those with Medicaid coverage increased from 11.0 million (6.0%) to 18.3 million (9.7%) (P < .001) (P < .001).The number of adults experiencing catastrophic expenditures yearly declined from 13.6 million (7.4%) in 2010 to 11.2 million (5.9%) in 2017 ( < .001) . PrivateIn our interrupted time series analysis, individuals in the lowest income quartile experienced a 2.3 percentage point decrease in likelihood of catastrophic expenditures  A, whereaACA implementation was associated with 2 million fewer US adults with catastrophic expenditures each year. Financial protection improved for the lowest income quartile, which was one of the ACA’s principal aims. However, improvements were not observed in higher income quartiles or among the privately insured, who represent an increasing share of those experiencing catastrophic expenditures. Among individuals in the poorest quartile, the privately insured are the most vulnerable, with one-third experiencing catastrophic spending annually. These findings help to explain why so many US residents, including those with insurance, continue to worry about their ability to afford needed care.Limitations include changing patient composition within insurance groups, meaning our analysis evaluates financial protection currently conferred by each insurance type, rather than the effect of gaining coverage. Also, because the Medical Expenditure Panel Survey does not quantify unpaid bills or medical debt, our analysis likely underestimates patients’ true financial hardship. Last, changes in catastrophic spending could have gone undetected in subgroups with small sample size, such as low-income privately insured individuals.Despite large coverage gains, 11 million US adults, including 6 million with private insurance, continue to experience catastrophic health expenditures annually. These figures are likely to increase as millions lose employment or require unexpected medical care because of coronavirus disease 2019. Health reform should move beyond expanding insurance coverage alone to address persistently high out-of-pocket spending among the insured.


Covertly using heroin during methadone maintenance treatment (MMT) is very common among heroin-dependent patients, which has posed threats to the physical health of heroin-dependent patients and social safety. Covertly using heroin may be influenced by many factors, especially social capital. Therefore, we aimed to investigate the relationship between behaviors of covertly using heroin during MMT and social capital heroin-dependent patients in Sichuan Province, China. A cross-sectional study was conducted between October and November 2018, with a total of 581 heroin-dependent patients participating in the study. In addition to socio-demographic characteristics and heroin use related behaviors, the questionnaire also included the measures of social capital: social network (SN), social support (SP), community participation (CP) and social trust (ST). Multivariate logistic regression analyses were used to estimate the association between different measures of social capital and heroin use. The prevalence of covertly using heroin of heroin during MMT was 31.0% among our participants in the 6 months before the study. After adjusting for socio-demographic factors and heroin-use related variables, SN , SP , and ST  were significantly associated with heroin use. Results suggest that social capital may have a protective effect on behavior of covertly using heroin during MMT, which should be consider in the interventions for heroin-dependent patients, in order to reduce the incidence of heroin use during MMT as well as improve the compliance of MMT. Drug dependence not only causes serious damage to individual physical health,,4 mental health and social functions, but also plays a positive role in the increase of crime rate and the spread of AIDS. A multistage systematic review showed, 17.8% of people who inject drugs were living with HIV, 52.3% were HCV-antibody positive, and 9.0% were HBV surface antigen positive in the world.Drug dependence is one of the major problems which the world face today.,11 MMT has achieved remarkable achievements in reducing the use of heroin, reducing the spread of blood-borne diseases such as AIDS, and improving the quality of life during recent years. A study showed that 102 heroin addicts reduced their daily heroin use from baseline to 0.0 after 6 months of MMT. No participant seroconverted to HIV and HCV positivity during the 6 months of MMT.Since 2004, China, one of the countries most affected by the problem of drug dependence, has been implementing methadone maintenance treatment (MMT) for patients with opioid  dependence. Sichuan, as one of the most seriously drug-using provinces in China, had a large number of heroin-dependent patients, which poses challenges for the development of MMT, such as low treatment retention rate and low compliance. Low MMT compliance not only affects the treatment effect of heroin-dependent patients’ MMT, but also increases the risk of relapse of heroin, resulting in damage to the health, increasing the crime rate and pose a threat to social stability.However, repeated client drop-out and re-enrollment cycles is very common in China.,17 Because the psychological craving for heroin is difficult to eliminate in a short time, heroin-dependent patients covertly use heroin during MMT.,19 Meanwhile, low doses of methadone, social and family discrimination can all affect heroin use during MMT.,17–20 Most of these factors are related to social capital.Therefore, it is necessary to improve methadone compliance of heroin-dependent patients. MMT compliance can be measured by covertly using heroin during MMT. Social capital can be divided into micro-social capital which emphasizes the status of individuals in the social structure, medium- social capital which takes the organizational level as the research background, and macro-social capital which pays more attention to. Social network, social support, community participation and social trust are important measurement indexes in social capital research. A multi-level study on harmful alcohol drinking behavior of Chinese residents shows that social capital can influence the occurrence of harmful alcohol drinking behavior of Chinese residents. Under the influence of Chinese alcohol drinking culture, social capital adjustment might help to reduce the occurrence of harmful alcohol drinking, which suggested that social capital can better predict the occurrence of healthy behaviors.The theory of social capital is one of the research hotspots in politics, economy and health in recent years. Social capital is a resource existing in the social structure, which is embodied in personal relationship and social network, meanwhile, these characteristics can improve the efficiency of society by promoting coordinated action. People have obvious sociality, the occurrence of behavior is often closely related to social structure and cognition. Social capital is an important resource existing in the relationship of social structure, which is one of the important predictor of behavior occurrence. In a study of health interventions in Maputo, Mozambique, social capital played a key role in the success of health management decisions, and low levels of social capital were associated with the failure of collective action. Yusuf Ransome et al reported that social capital was closely related to regional differences in AIDS patients’ treatment, and can be used to modify the policy and provide targeted intervention measures for AIDS patients, which would effectively reduce regional differences.Social capital can provide not only basis for making decisions of healthy behaviors, but also ideas for behavioral intervention and health education.–30 especially the behavior of heroin use during MMT. At the same time, as China is a country with a history and tradition of “relational society”, behaviors of heroin-dependent patients are might be closely related to the “relational society”. Having better “popularity” means having more social capital. Therefore, we conducted a cross-sectional study on the relationship between heroin-dependent patients’ behavior during MMT and social capital, and based on the results, provided some ideas on how to use social capital to develop intervention methods for heroin-dependent patients.At present, studies on the application of social capital to MMT are relatively rare. The research did not fully considered the social capital, but only part of it such as social support and social network,22.1All participants were voluntary joined in our study and signed informed consent forms before survey. The design and implementation of this study protocol was approved by the Ethics Committee of the West China School of Public Health and West China Fourth Hospital, Sichuan University.2.2A cross-sectional study was conducted in Sichuan province from October to November in 2018. The inclusion criteria were:1)at least 18 years old at the time of joining MMT,2)meeting the criteria of the national guidelines for the treatment of addictive patients, and3)participated in the community-based MMT clinics where they livedThose who had severe cognitive and mental disorders (schizophrenia and bipolar disorder) were excluded from the study..Finally, in each selected MMT clinics, we invited patients who met the inclusion criteria to join the study.A stratified multi-stage sampling design was adopted. Currently, there are only 49 MMT clinics in 19 districts of Sichuan province. Because of the household registration system, heroin-dependent patients must participate MMT at local MMT clinics. The population mobility of heroin-dependent patients is low and the population is concentrated, so the cumulative number of patients in MMT clinic can reflect the prevalence of heroin dependence in the clinic. Therefore, first of all, we divided 49 MMT clinics according to the cumulative number of patients, and selected 19 clinics with the cumulative number of patients above 1000. Secondly, we grouped MMT clinics into 4 groups according to the geographical location, and then randomly selected 5 MMT clinics as our research sites.This method was also used in the study of Yu, B. et al.  , δ = 0.05, α = 0.05, , and the sample size was calculated to be 350. However, due to the large sampling error of the cluster, the sample size was usually 1.5 times of the sample size of the pure random sample, so the sample size was about 525.The sample size was calculated based on 2.3We conducted an anonymous questionnaire survey on heroin- dependence patients by face-to-face survey. All investigators received two days of investigative training. At the beginning of the survey, the investigators read informed consent forms to the participants and then conducted the survey with their consent. Then the investigators read the questionnaire and recorded the participants’ answers. During the survey, the investigator and the participant sat at the same table, and the investigator's seat was always at a 45° Angle to the participant. The environment for filling in the questionnaire was quiet, closed and alone. A gift (about 15 RMB or 2 USD) was given to participants upon completion of the interview) for their time spent.Of the 634 participants, 581 (91.6%) provided written informed consent and completed the interview, and 53 (8.4%) declined to participate in the study due to lack of time  and/or other reasons . The median time for 581 participants to answer a questionnaire was 10 minutes (6–13 minutes).2.4A panel consisting of 2 epidemiologists, one health psychologist, and 5 methadone replacement therapy patients was formed to design the questionnaire. The questionnaire was tested among four other methadone replacement therapy patients. Based on their feedback, discussion was made by the panel to finalize the questionnaire.2.5A three-part questionnaire was used to perform the investigation. The first part covered socio-demographic characteristics, the second behaviors of heroin use, and the third social capital.2.5.1hukou), highest education level attained, and inhabiting information.Socio-demographic status was considered to include gender, age, nationality, local official registration . The questionnaire also included a survey of the participants’ other drug use. The history of drug dependence among participants was reviewed, including history of forced drug withdrawal and voluntary drug withdrawal. The researchers surveyed participants about their heroin use during the MMT and divided them into those who had covertly used heroin in the past 6 months and those who had not covertly used heroin in the past 6 months.2.5.3 with a total of 17 variables quantifying participants’ relationships with family, relatives, friends, and communities. The first dimension was social networks (SN) with 6 variables, measuring the relationships and networks with family members, friends, and neighbors . The second dimension was social support (SP) with four variables, including available support from family, friends, and neighbors . The third dimension was community participation (CP) with 2 variables, including participation in neighborhood activities and sense of belonging . The last dimension was social trust (ST) with 5 variables, concerning trust and equity . The answers consisted of 5-point Likert scales. The response categories were: 1 = strongly disagree, 5 = strongly agree. Using exploratory factor analysis, 4 factors with characteristic roots greater than 1 were extracted from 17 items, and the cumulative variance contribution rate was 56.81%. The internal reliability  of the scales formed ranged from 0.71 to 0.92  and the behavior of participants used heroin covertly. Therefore, the multivariate analysis model to explain the impact of social capital on the behavior of covertly using heroin was the most important goal of our data analysis. In order to adjust the results of the multivariate analysis model, the results of univariate analysis of covertly using heroin and background factors such as socio-demographic variables and potential risk factors were secondary but necessary.P > .10 in univariate analysis were not included in multivariate analysis model. The same analytical methods have been used in a number of published studies.,33 SPSS version 20.0 for Windows  was used for data analysis, with P values <.05 taken as statistically significant.Descriptive statistics were used to summarize characteristics of the study respondents by expressing the results as mean ± standard deviation (SD) or percentage. The prevalence of covertly using heroin, as well as their respective 95% confidence intervals (CI) were presented. When considering covertly using heroin in the last 6 months before the study as dependent variables, univariate odds ratios (ORu) of background variables were identified first. Then crude odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated with three logistic regression analyses to measure associations between social capital and covertly using heroin in the past 6 months. In the first logistic regression analysis model (model 1), we calculated the crude OR (95% CI) for the relationship of social capital with covertly using heroin. In model 2, the OR (95% CI) was adjusted by controlling for socio-demographic variables. In model 3, the OR (95% CI) was adjusted by controlling for both socio-demographic variables and potential risk factors. The socio-demographic variables and potential risk factors entered into the multivariate analysis model were found to be significant in the respective univariate analysis. Variables with 33.1In this study, 75.6% of participants were male, and 48.9% were between 40 and 50 years old. 35.3% of participants had attended senior high school or above. Of the participants, 64.7% of them were living with their family. Almost all the participants were Han Chinese (98.3%), registered permanent residence in rural areas (90.7%) Table .The gender of male was negatively correlated with heroin use in the past 6 months . However, the relationship between covertly using heroin and age groups, nationality, the type of registered residence, highest education level attained and inhabiting information were not statistically significant.3.2Heroin use behavior can be divided into 2 parts: before MMT and after MMT.Covertly using heroin during the MMT in the past 6 months was used as an indicator of heroin use behavior after participation in the MMT. The 31.0% covertly used Heroin in the last 6 months when they were participating in the MMT , had taken drugs intravenously (59.0%), had not used any drugs other than Heroin (80.4%), had participated in forced drug withdrawal (55.6%) and voluntary drug withdrawal (53.0%). By using univariate analysis, there was a positive correlation between heroin use for 10 to 20 years and heroin use during MMT in the last 6 months , as well as, heroin use for at least 20 years . Had used other drugs  and had joined forced drug withdrawal  were negatively correlated with heroin use in the past 6 months. The relationship between covertly using heroin and had taken drugs intravenously and had joined voluntary detoxification were not statistically significant.3.3The responses of heroin-dependent patients who have covertly used heroin in the past 6 months to the social capital items are summarized in Table 3.4The associations between social capital and covertly using heroin in the past 6 months in the logistic regression models are summarized in Table 4 The prevalence of this behavior was higher than that found in this study, possibly because Guangdong had a higher density of migrant workers and a higher immigration rate than Sichuan. The difficulty of obtaining MMT locally for non-residents made it possible for them to use heroin during MMT.We found that heroin-dependent patients who joined MMT had a higher prevalence of heroin use during the MMT, with 31.0% of the subjects who had covertly used heroin in the past 6 months. A cross-sectional study of 14 MMT clinics in Guangdong province, China, reported that 68.7% of the subjects had used heroin in the past 30 days.The study found that social capital theory may be useful in the design of relevant interventions, in that in the social capital theory module used in this study, social network, social support and social trust were significantly association with heroin use during MMT. A social network is a multi-dimension social connection. The network includes a variety of contacts, including innate network relations (relations with family members) and acquired network relations . It is an inevitable trend for everyone to develop his own resources and make use of social resources. We observed that the social network and the contact scope of heroin-dependent patients were very narrow , which was consistent with the research results of Shen et al. One possible reason was that before MMT, in order to avoid arrest by police, heroin-dependent patients might try to hide their secrets. Another reason might be that heroin-dependent patients have tended to be self-enclosed and hide in order to avoid social discrimination.A negative correlation between the level of social networks and covertly using heroin during MMT, possibly because wider social networks might help people effectively handle life stress. As the origin of social capital, social network plays an important role in a relational society like China. Social support was closely associated with social network,,37 which is also greatly influenced by the supports from family members. Good family support can not only provide material and other objective support, but also improve heroin-dependent patients’ confidence and self-support. Meanwhile, good social support might make heroin-dependent patients feel that they are not abandoned by the society and family, so as to reduce covertly using heroin during MMT and improve MMT compliance. Therefore, the focus of intervention on the families of heroin-dependent patients who joined MMT might effectively improve the treatment compliance of heroin-dependent patients.Heroin-dependent patients’ high level of social support was a protective factor for covertly using heroin during MMT, in that good social support might help heroin-dependent patients achieve higher socio-economic status (SES) driven by Chinese collectivism culture. Social trust was a kind of expectation based on morality, fairness, and interests in the context of certain knowledge and information. Chu et al found that heroin-dependent patients had a higher trust to medical professionals in MMT based on their desire to quit heroin in China. However, because they had been arrested by police before MMT, they were hostile to government, and their trust in the public security department were usually low.Social trust might reflect institutional discrimination against heroin-dependent patients. Because of the risk of being caught while participating in MMT, heroin-dependent patients might be reluctant to go to the MMT clinic for MMT, which might affect their treatment compliance and may also lead to heroin use during the MMT.Heroin-dependent patients’ high level of social trust was protective factor for covertly using heroin in the past 6 months. Social trust was the basis of social capital. China is an interpersonal society, and the relationships among people play an important role in social life, in that SES and social support often depend on the scale of social network. Heroin-dependent patients are encouraged to interact with network members normally and expand their social network actively. Meanwhile, governments can invest in infrastructure , as well as other environment  that provide opportunities for interaction between innate and acquired social networks of heroin-dependent patients. Second, good use of social support may reduce heroin use behavior of heroin-dependent patients during MMT. Through MMT-related knowledge training for family members of heroin-dependent patients, discrimination against heroin dependent patients can be eliminated, mutual trust can be established, and care and support for heroin dependent patients can be strengthened. At the same time, access to social support goes both ways. Heroin-dependent patients should also maintain an optimistic attitude and actively accept support from family members, friends, neighbors and colleagues. Third, we should find the importance of social trust in behavioral intervention. Social trust is an important indicator to connect heroin-dependent patients with society and institutions. To create a comfortable MMT environment for heroin-dependent patients and improve their social trust through establishing partnerships among medical and health institutions, government administration departments and social organizations and groups, and strengthening communication and collaboration.The study provided some insights for reducing covertly using heroin during MMT. First, social networks would improve access to social capital for heroin-dependent patients.Some limitations should be noted in this study. First of all, although the questionnaire is not a standardization questionnaire, it developed according to China's national conditions. In this study, the questionnaire showed good reliability and validity, the Cronbach alpha of the scales of social capital formed ranged from 0.71 to 0.92, as well as, the cumulative variance contribution rate of the items of social capital was 56.81%. Secondly, occurrence of covertly using heroin was self-reported and might subject to social preference desirability. However, we adopted and face-to-face methods to collect behavioral information of heroin dependent patients, and chose quiet places for investigation while ensuring the anonymity of the questionnaire, in order to reduce such bias. Thirdly, we considered including HIV infection as a comorbidity in the questionnaire. However, during the investigation, we could not collect the physical examination data of the participants. HIV infection status was only orally answered by the participants, with bias, so it was not included in the data analysis. Fourth, the study did not find a relationship between community participation and heroin use, possibly because community participation in the population was very low, which may reduce the statistical power to find the differences between groups. Fifth, our study only selected MMT clinics with a cumulative population of more than 1000 as sample objects. As the 49 MMT outpatient clinics in Sichuan province are scattered, and our project fund is limited, it is convenient for us to obtain more information we want by drawing more MMT outpatient clinics. However, such sampling may lose some valuable information and have selection bias. Finally, this is a cross-sectional study, which means it cannot be inferred causal relationships. Therefore, prospective studies are needed to confirm our findings.5The heroin-dependent patients’ social capital has a protective effect on covertly using heroin during MMT. Future interventions should make use of the social capital of heroin-dependent patients, expand their social networks, increase levels of social support, reduce social discrimination, and improve their trust in society.We thanks for the Center for AIDS/STD Control and Prevention, Sichuan Center for Disease Control and Prevention for supporting the data for analysis. We thanks for School of Public Health, Sun Yat-Sen University for providing methodological support. We also thanks for the students from West China School of Public Health for organizing the data for analysis.Conceptualization: Bo Gao, Peng Jia, Shujuan Yang.Data curation: Bin Yu, Jiayu Han.Formal analysis: Shifan Yang, Shujuan Yang.Funding acquisition: Yi Gong.Investigation: Shifan Yang, Bin Yu, Jiayu Han, Peijie Dong.Methodology: Bo Gao, Jing Gu.Project administration: Shujuan Yang.Resources: Yi Gong.Supervision: Jing Gu.Validation: Bo Gao, Shujuan Yang.Writing – original draft: Shifan Yang, Shujuan Yang.Writing – review & editing: Shifan Yang, Bo Gao, Shujuan Yang.