Antibiotic resistance poses a great threat to global health, especially in low- and middle-income countries with a high infectious disease burden and limited resources. In spite of regulations, antibiotics are sold in many settings as non-prescription medicines, resulting in inappropriate use and resistance.This study aimed to investigate the current status of access and use of antibiotics in rural Bangladesh, by exploring the perspectives and sales practices of antibiotic drug dispensers.We used a mixed methods approach . We mapped and characterized antibiotic purchasing and dispensing sites in the Matlab Health and Demographic Surveillance System catchment area. Furthermore, we investigated the volume of provision of systemic antibiotics in 10 drug outlets. We held 16 in-depth interviews with randomly selected antibiotics dispensers. Interviews explored factors associated with antibiotic selling. Responses were transcribed, coded for themes, and summarized. We used ATLAS.ti 5.2 for conducting a thematic analysis.n = 278) were private and 8% (n = 23) public. 52% (n = 155) operated informally . In order to promote and survive in their business, dispensers sell antibiotics for a range of conditions without a qualified physician’s prescription. Factors that facilitate these inappropriate sales include lack of access to healthcare in the rural community, inadequate doctor: population ratio, limited dispenser knowledge, poor pharmacovigilance concerning safety of self medication, lack of enforcement of policies, financial benefits for both customers and dispensers, and high dependency on pharmaceutical companies’ information.A total of 301 antibiotic dispensers were identified, of whom 92% (Dispensers in rural Bangladesh sell antibiotics inappropriately by ignoring existing national regulations. They operate the antibiotic sales without facing any legal barriers and primarily with a view to sustain their business, resulting in inappropriate sales of antibiotics to the rural community. The influence of the drug industry needs to be replaced with evidence-based, not commercially driven information. Awareness programs for antibiotic providers that promote understanding of antibiotics and antibiotic resistance through tailored interventions may be helpful in changing current antibiotic sales practices. Inappropriate use of antibiotics in the community is common in many low- and middle-income countries (LMICs), where antibiotics are easily available without a prescription from retail drug shops , 2. MajoIt is internationally well recognized that inappropriate and excessive use of antibiotics must be reduced to control antibacterial resistance, while maintaining access to these live saving drugs when they are needed , 14, 15.A significant proportions of antibiotics are sold without registered doctor’s prescription, which violates the National Drug Policy (NDP) 2005 and 2016 , 19, 22.There are several challenges that LMICs faces in their attempts to control antibiotic use. Just restricting the selling of antibiotics may have a negative consequence on the health system if no affordable and adequate alternative is provided. As a means of ensuring that antibiotic sales are only made to those who really need them, it is important to investigate the socio-cultural factors that influence the health care providers to sell these drugs.The aim of this study was to analyze the current status of antibiotics access and selling in rural Bangladesh  and to explore the antibiotic dispensers’ views, their actual practices and dispensing procedures as well as socio-cultural factors that influenced sales of antibiotics in this area. We intend to use the findings as the basis for developing a suitably tailored intervention program to improve the appropriate sales of antibiotics.This study is part of the multi-country ABACUS (Antibiotics Access and Use) study . To undeWe selected this site for the study as the household and population were all prelisted in the HDSS database, and access to the community is logistically straightforward, thereby facilitating the repeated visits that were necessary during the course of the study. We conducted field investigation from November 2016 to April 2017.n = 21) high volume dispensers proportionately 9 and 1 from private and public sectors respectively. This provided context on antibiotic selling practices. Qualitative interviews were also conducted with antibiotic dispensers.As detailed below, a mixed methods study design was followed, including an initial mapping exercise, followed by inventories conducted on the larger antibiotic dispensers. The high volume antibiotic dispensers (≥ 35 customers visit /day) includes 20 (7%) in the private and 1 (4%) in the public sector. However, in this study selected antibiotic inventories were conducted from those  and informal (No drug license) antibiotic purchase and dispensing outlets from the entire Matlab HDSS government service area to discern antibiotic dispensers from different local markets and village shops. At first the study objectives were shared with the drug dispensers and a verbal consent was obtained. This was followed by registering the information on drug dispensing types; sources of financial support to run the business; average number of daily antibiotic encounters; drug outlet geolocation [by means of Global Positioning System (GPS) co-ordinates].We performed inventories at the 10 largest outlets (identified during the mapping exercise as having the highest average daily number of customer encounters for antibiotic purchase). Written informed consent from the antibiotic dispensers was obtained. We assessed the presence of five essential systemic antibiotics  based on availability of antibiotics in 6 countries with different Gross Domestic Product (GDP). A digital thermo-hygrometer was used to assess the antibiotic’s storage temperature and humidity as measured at the actual storage site for each available brand of the particular antibiotic separately. We collected brand name, doses (mg) quantity, formulation, price in local currency, condition of blister pack, package insert, and expiration date. Structured questionnaires were used for both the mapping exercise; inventory and were administered to the formal and informal antibiotic dispensers.A total of 16 in-depth interviews were conducted from 14 randomly assigned participants out of 278 private dispensers  in addition, we interviewed two participants from 23 randomly assigned antibiotic dispensers representing the public sector . Interviewees were sampled to ensure that all the geographical zones of the HDSS were included. We explored perceptions, reported practices, and social norms related to antibiotics selling using an open-ended, semi-structured interview guide. The interviews were classified into five sections: demographics, medicines (knowledge and dispensing practices), customer’s knowledge and awareness of antibiotics, antibiotic resistance, and regulatory issues. The data were collected by an anthropologist along with one sociologist, who had previous experience in qualitative methods of data collection. The anthropologist was trained for this work along with personnel from the other participating ABACUS sites, in order to ensure a consistent approach across all the sites with respect to the data collection process, data coding procedures, and data analysis. Interviews were audio recorded and field notes were also taken for subsequent clarification of any issues that arose, if needed. The duration of the interview was planned for 45–80 min. All questions, clarifications raised by the dispensers were resolved by the interviewer at the time of the interview. We conducted the antibiotics dispensers mapping from 15th November 2016 to 18th January 2017, and conducted the qualitative in-depth interviews from 15th March 2017 to 04th April 2017.In-depth interviews were transcribed verbatim in Bengali, retaining all local terminology. We performed thematic content analysis, based on both a priori and inductively derived codes. A code sheet was developed by researchers based on the core themes of the questions, complemented by additional codes that emerged when reviewing the transcribed interviews to explicit imperative information. The analysis was facilitated by ATLAS.ti 5.2 software, on which we coded the data according to our research objectives. Though we analyzed each in-depth interview separately, in the results section we have drawn inferences collectively.n = 278; retail drug store, diagnostic center, hospital pharmacy, grocery shops) and public facilities . Out of 278 private drug shops 155 (51%) had no formal authorization for selling antibiotics  drug dispensing outlets none had all five essential antibiotics. However, drug inventory conducted on those 10 outlets we found 4 out of 9 (44%) in private and in 1 public sector - 4 and 2 essential antibiotics respectively. Amoxicillin, Amoxicillin+Clavulanic Acid and Ciprofloxacin were widely available while Co-trimoxazole was rarely found in private drug outlets. Chloramphenicol  was not present either in private or public settings. However, Co-trimoxazole and Amoxicillin were frequently present in the public sector, whereas Ciprofloxacin, Amoxicillin and Clavulanic Acid were not.Among the 10 high volume customer visit : amoxicillin (8 brands) and amoxicillin-clavulanic acid (5 brands). Having such high number of brands not only confuses the customers but also for survival in this competitive market the Pharmaceutical companies offer different sorts of promotional activities to doctors as well as the drug dispensers.We conducted in-depth interviews with 16 dispensers, twelve of whom were both owner and seller. Ten of the 16 informants were aged from 20 to 40 years, and the remaining were aged 41 to 60 years old. They had completed secondary (5), higher secondary (8), graduate (1), and post graduate (2) education. Among them there were retailers (13), government health care providers (2), and grocery shop keeper (1). Three informants were female and the rest (13) were male.viii) What steps should be taken to improve use of antibiotics?The findings from the qualitative interviews are presented within the following themes: i) Selling practices; ii) Sources of knowledge about medicine and the influence of promotional activities; iii) Knowledge about antibiotic use and antibiotic resistance; iv) Selling incomplete courses of antibiotics; v) Selling practices of branded antibiotics; vi) Dispensers’ instructions provided with dispensed medicines; vii) Enforcement of rules and regulations; n = 16) the informants narrated that they can run their business without facing any legal barriers. Medicines, including antibiotics, are generally dispensed without a physician prescription from their outlets. According to the respondents, antibiotics are sold based on the health concerns and visible symptom of the customers. Additionally, they also get specific requests for an antibiotic when shown an empty blister pack by the customer or they mention a preferred brand name. Dispensers provide the drugs that they expect the patient wants and needs. If they do not provide medicines according to the customer demand, they will leave the shop and go to another seller and this will adversely affect their business. Informants stated that most of the people ask for antibiotics without prescription. Young people reportedly come to buy medicines without any doctor’s consultation comparatively more often than elderly people.All One respondent mentioned, ““Some customer purchases medicine two times with a prescription, and later they have a tendency to buy medicines without prescriptions based on previous experience.” Another informant noted, Dispensers pointed out that they generally accept returned unused medicines, including antibiotics, from known clients. Customers consider antibiotics are costly medicines compared to others, and they prefer to return leftover antibiotics to dispensers. They are inclined to accept their sold medicines back after checking the blister pack condition and expiry date. In case they are in an acceptable condition, they exchange them for other medicines or refund, as a means to maintain good relationship with the customers. Thus, they resell returned drugs to other customers without any financial loss.“Sometimes people come to return sold medicines, including antibiotics. I have no option other than taking these back and I have to return the money to maintain good relationship with the customers.” One informant narrated, “Based on the good terms I have with a customer, I sometimes take back the sold Ciprofloxacin because the customer pretends to me that they have been fully cured from the disease.” Another informant stated, “I have to take back sold medicines from customers, otherwise they will be disappointed.” A third said, Informants stated that often they start their pharmaceutical business with little knowledge about medicines. Their source of information is primarily from the representatives of the pharmaceutical industry. They usually acquire their knowledge about the medicines from drug information leaflets, brochures, treatment guidelines and medicine directories provided by different pharmaceuticals companies. They also receive different types of financial incentives from the industry, such as special discounts to sell a particular product, financial incentives, gift boxes, and invitations to attend commercial medical education sessions. They are also informed about new medicines through pharmaceutical representatives, and through the internet websites of different pharmaceutical companies. The informants also become acquainted with medicines by observing what physicians do for various clinical syndromes. They also learn about medicine from short course training provided by the Ministry of Health & Family Welfare (MoFW), and pharmacist training. Further, they may participate in dissemination meetings or seminars arranged by pharmaceutical companies with the aim to increase their sale.“I gathered knowledge about medicine from my 8 years’ job experience in medicines shop. At that time, I asked other colleagues about application of prescribing drug. I also learned about selling medicines from sales representative.” One informant narrated, “It is difficult to get an idea about all medicines nevertheless I have followed working experience with this business and observe how doctors prescribe.” Another respondent stated, The informants stated the influence of pharmaceutical representatives and advertisers contribute to inappropriate sales of antibiotics. Pharmaceutical companies regularly arrange dissemination meetings, conferences, and seminars of their products for local drug dispensers in order to increase their sales in the rural markets. Area managers and medical representatives conduct these sessions. Retail drug outlets are visited on a daily basis to get drug orders by company representatives. They influence dispensers to make larger orders of their medicines with antibiotics: for example, if they purchase five boxes of a certain drug then the additional one box of medicine and antibiotic is given free; or they may receive monthly credit in order to achieve their selling target.Informants stated that antibiotics are effective for different diseases and it helps people to recover quickly from their illness. They often use antibiotics to cure high fever, cold, cough, acute watery diarrhea, skin disease, accidental injuries to cure wound and infections, and any kind of post-surgical infections that are not cured by common drugs. Drug sellers are repeatedly recommended by the pharmaceutical companies antibiotics like Azithromycin for high fever, respiratory tract infections (RTI), skin and soft tissue infections. Antibiotics help in ‘preventing ability’ inside the body to protect it from germs. Informants also explained that beside benefit from antibiotics also causes side effects for instance dizziness, feeling weak, diarrhea, vertigo, and allergy.“I observed antibiotics are recommended by other colleagues of our drug store e.g. Ciprofloxacin, when high fever is not managed by paracetamol.” One respondent pointed out, I have no profound insights about antibiotic use and antibiotic resistance, so what I do is to copy how doctors prescribe antibiotics.” Another informant said, ““In my consideration, paracetamol works for fever, and antibiotics work to add to the strength of the body to kill the germ.” One drug seller mentioned, “Antibiotics are the medicines which are used for 6-7 types of diseases.” Another drug seller narrated his knowledge on antibiotic use as When asked about the meaning of the term “antibiotic resistance”, the dispensers had varied responses. They mentioned that if the wrong doses of antibiotics are prescribed for different illnesses, these antibiotics may not work in the human body if used for a long time. According to another informant’s observation, if antibiotics are not completed according to the prescribed durations, it will not able to kill the germ, the germ will persist in patient’s body and they will be affected by the same disease again, then the antibiotics need to be changed, and stronger antibiotics are needed that are expensive, and the treatment time will become longer.Informants also narrated that when first and second line antibiotics are not effective then they recommend third or fourth line such as Cephalosporins or Quinolones. Due to the availability of these drugs and for higher profit margin the drug sellers are also inclined for marketing these antibiotics.“Many customers do not consume all dispensed antibiotic pills when they feel better from illness, and when they are affected again after a few days, then they consume the remaining antibiotic for a second time but now they don’t feel cured. This time that antibiotic is not working in their body and this situation is called antibiotic resistance.” One informant stated, My customer alleges that antibiotics have a strong role to recover illness quickly and on the other hand it shows some toxicities i.e., weakness, loss of appetite, affects liver & kidney. But I have no clear idea if it has any association with antibiotic resistance or not.” Another informant said, “Informants cited that they regularly sell incomplete courses of antibiotics to their customers, as customers have limited financial capacity. They stated that first people want to start with minimal dosage and if they see good response are hesitant to go for remaining dosage of antibiotics. In that case they do not need to spend a large amount of money at one time. Informants also sell incomplete doses of regimens for maintaining good relation with familiar customers, and thereby helping to ensure that the customer comes back again in future to buy antibiotics and other medicines.I have to sell incomplete doses of antibiotics to maintain good relationship with the customers. If I don’t sell incomplete doses then customers will purchase from another drug store, as a result I will lose the customers and income, so for surviving with the business I sell incomplete doses of antibiotics.” One informant stated, ““It is a village area, people are not financially solvent. I suggest and sell them antibiotics for 3 or 4 days. Meanwhile, if they feel better, they do not want to continue even for these 3 to 4 days, and they stop sooner.” Another informant said, “I saw many people do not follow doctors’ recommendation regarding antibiotics. Suppose someone needs to take a seven days course, but s/he buy it for three days, and after three days if s/he recovered then s/he does not buy the rest.” Another respondent stated, Informants said that they usually recommend antibiotic doses based on age, body weight and severity of illness, as well as their own experience. They provide antibiotics for upper respiratory tract infections which are often viral, and for which they should not be providing antibiotics. They stated that they do not have good knowledge of the correct dose. Further, their selection of the recommended duration of treatment varies and is inconsistent: some of them prefer to recommend a 7-days course, and others suggesting a 3 to 5-day course for the same illness.“If a patient suffers from fever, I ask the patient about how long they suffer from fever. If customers suffer for 3-4 days, then I give an antibiotic; if shorter duration of fever I give them paracetamol only. I suggest customer to consume antibiotics 7 days or at least 5 days.” One informant explained, Dispensers mentioned that certain customers want to purchase a specific brand or type of antibiotics for their illness without a doctor’s prescription. Sometimes they are unable to recall the exact brand name, but customers express their requirements based on certain colors (capsules red and tablets white), size & shape.As a result, Amoxicillin, Ciprofloxacin, and Azithromycin are sold on customers’ specific request. Because customers sometimes show a preference about their choices regarding certain brand of antibiotics, dispensers respect customer desire to ensure that they come again in future so that they do not lose the customer.If a customer requests a specific antibiotic I provide the same. However, if it is not available, I advise to take an alternative antibiotic but sometimes they do not express their interest to buy that medicine.” One respondent mentioned, “All informants reported they provide instructions to their customers about the dosage of the drugs they sell and the duration of its use. Informants mentioned they usually provide oral and written instruction to the customers. Sometimes they cut corners of the strip with numbers that coincide with the number of dosage they need to take per day as a way to remember, in particular for customers who are unable to read or write Fig. .Fig. 3TThe drug seller cuts the medicine strip edges for the uneducated customer to remember daily dose schedule. Medicines sold by the dispensers to the customer are instructed on the number of pills to be taken daily, as well as the duration, verbally and/or written on a piece of paper. Some of the dispensers mention they also write instructions on the package.Dispensers suggest people to complete the antibiotic course according to the recommendations; otherwise the drugs will not work next time. But customers reportedly do not always take this advice. It was observed that the dispensers do not inform the customers about possible adverse effects of antibiotics as some drugs can cause nausea, rash, vomiting, diarrhea, and poor appetite. These side effects result in customers to stop the course prematurely. Additionally, customers do not always share information with the dispensers about previous allergic reactions to, for example, penicillins, and thus they may purchase them again.“Customers are not giving importance to my words. Sometimes I don’t want to give the instruction to customers because they consulted with a doctor or relatives who are more experienced compare to drug sellers like us.” One informant explained, “I usually give prescription and instructions to the customer in relation to consuming medicines. I explain to the rural women that they should complete full course of antibiotics, but they don’t understand.” Another seller narrated, “I don’t bother asking to the customer about diseases as it is not my business. My responsibility is only selling medicines, not to provide instructions to the customers. I give verbal instructions to the customers because I am not a doctor, so I can’t give written instructions.” .Informant stated, There are specific guidelines on how to prescribe or dispense antibiotics in Bangladesh. However, in rural areas where there is often no access to expertise and diagnostic tools it can be challenging to follow these guidelines. Dispensers stated that the “Drug Inspector” is empowered by the Government to act as the Licensing Authority of drugs for the purpose of issuing licenses to manufacture, store, sell, import and export drugs and medicines. Due to shortage of manpower within the Directorate General of Drug Administration, it is not possible for them to inspect all pharmaceutical dispensers at regular intervals. Informants also cited that they do not ever see any Government authority taking the initiative to enforce the existing rules and regulations, or concerning any prohibition or penalty for selling antibiotics without prescription. According to the conditions of receiving a drug license, medicines should be dispensed by registered pharmacists, but in reality no registered pharmacist is found in most of the drug stores. Informants added that they do not ever face any legal difficulty to run their pharmacy business, selling medicines and antibiotics without prescription. If the government would strictly enforce the regulations that require antibiotics to be sold only with a registered doctor’s prescription, dispensers would not sell antibiotics without prescription and thus there would be less chance of developing antibiotic resistance. However, this would also create access challenges for people who really need the medicines.“Recently, the High Court of Bangladesh ordered that prescriptions should be either printed or handwritten in capital letters. However, this is not followed in most instances. Yesterday, I saw one prescription which I didn’t understand. If we obey the rules and regulations and follow in all cases, then the inappropriate selling of antibiotics in Bangladesh will be changed.” One respondent mentioned, “I think some pharmacy store-keepers know the rules of selling drugs and some don’t, however, none fall the regulations strictly. They just sell and want to make profit by selling medicine to the customers” Another narrated, I sell a variety of different grocer products  including some drugs along with antibiotics from here. I am now doing this business to support my son’s education expenses; actually, I have no idea about rules and regulations of our country about selling medicines. Nobody informs me about this. I just sell different medicines with a view to help the local people, because they ask medicines from me as well.” Informants stated, “Provide training to pharmacy drug storekeepers and village doctors on correct procedures for ensuring appropriate antibiotic sales.Conduct awareness campaigns for community members in order to raise knowledge on the proper use of medicines and antibiotics within the community. Print and electronic media can play an important role in disseminating information among the population.Engage local leaders and key institutions - for example, members of parliament, concerned ministries, and regulatory authorities - in awareness campaigns to make them effective. Such awareness programs need to be conducted on an ongoing basis in order to ensure that people will learn and then change their current practice.TULSI LEAF.’ Herbal medicines can be an acceptable and also effective alternative to treat cough and cold.Instead of consuming antibiotics, use traditional medicine for upper respiratory tract infections, like ‘Informants suggested several steps that should be taken to improve the appropriate sales of antibiotics:This study investigated antibiotic dispensers’ perspectives on antibiotic access and use in a rural setting of Bangladesh, both licensed and unauthorized private drug sellers as well as public health facilities participated in the research. Although the National Drug Policy (2005 and 2016) prohibits antibiotic sales without a prescription from a qualified doctor, this is ignored by the dispensers due to lack of monitoring by the drug regulatory authorities, financial incentives, and limited awareness and knowledge of the risks of unnecessary antibiotic use.This study reveals a high degree of inappropriate antibiotic dispensing in the study area, in terms of both selling antibiotics without a proper diagnosis or a prescription, and the provision of incomplete antibiotic regimes in terms of dose or duration. Financial constraints are the major impediment to health-seeking behavior from formal health care facilities. Patients prefer to go to drug outlets where they can get the medicines just by explaining their health problems, without any laboratory diagnosis or a prescription. This results in antibiotics being sold without proper instructions, and with an increased likelihood that they will have an incorrect dose and treatment duration. Interestingly we also found that unused drugs are taken back by the drug sellers and resold, which may be an important health risk as the condition of the drug is uncertain. This is not considered good pharmacy practice.Inappropriate antibiotic use and widespread selling without physician prescription is common in many LMICs –29. But In our study area, 155 out of 278 (51%) unlicensed retail drug shops are running where antibiotics are sold by untrained or poorly trained drug store owners. We did not identify any graduate pharmacists in any of the 301 antibiotic dispensing outlets that we located. Most of the drug shops are run by individual owners with limited expertise, which leads, for example, to improper storage of drugs. This is a violation of Bangladesh’s drug policies 2005 and 2016. There is no one to monitor or enforce this because of the limited work force of the DGDA . SellingThe sellers are influenced by promotional offers of the pharmaceutical companies to sell more medicines, including antibiotics , 23, 25.The reported high percentage of non-prescribed sales of antibiotics is worrisome, given its association with unnecessary use, too short courses and inappropriate drug and dose choice, and subsequently, the development of antibiotic resistance . We founRegulation is crucial to safeguard access to antibiotics, but a transition towards such regulation needs governmental commitment and improvements in health systems that are not possible in many countries . RoutineThis study is part of a multi- centered observational study. As the study was conducted in rural setup of Bangladesh, we expect that the findings will be relevant to antibiotics sales in other socially and culturally similar areas of Bangladesh. Although there are number of antibiotics widely available in this study area, we considered five essential antibiotics in order to compare data with other sites, and thus we may have missed some locally relevant antibiotics. We selected informants for in-depth interview proportionately from three hundred and one located private and public drug outlets, this has likely imbalanced the ratio of interview between them. Since interviews of the drug dispensers were only possible when the shops were open at the same time customers also visit this might have interrupt the flow of discussion.Escherichia coli [The findings from this study indicate that sellers are heavily influenced by the drug industry to sell antibiotics despite the customers are not always in need. However, the drug authority need to enforce on drug sellers that drugs like fluroquinolone not to recommend as first line therapy for uncomplicated urinary tract infections due to the development of resistance specially hia coli , 38.The dispensers are not sufficiently trained for proper dispensing of antibiotics, which results in poor instructions provided to customers and also the potentially risky practice of taking back leftover sold drugs for future sell. All these factors result in widespread misuse of antibiotic in the study area, and consequently the emergence of antibiotic resistance. Improving this situation would require; strengthening the regulatory process by the Directorate General of Drug Administration Bangladesh, close supervision by the relevant authority, provision of reliable information to drug sellers that is independent of the pharmaceutical industry, and promoting awareness programs both for the community and for drug dispensers to increase understanding of antibiotic resistance through effective communication, education and training.Additional file 1. Appendix A. eCRF registry form drug dispenser characteristics (for mapping exercise). Appendix B. eCRF_Dispensers Inventory. Appendix C. Preparatory_Antibiotic Dispensers_In-Depth Interview Guide.


In terms of shrinkage deformation control, materials with high toughness and low shrinkage are required to repair the existing concrete structures. The implication of this research may provide a theoretical basis for the preparation and application of SHCC with high toughness and low shrinkage.Traditional cement-based repair materials are brittle and prone to cracking. The failure of more than half of repaired concrete structure is due to the re-cracking of the repair material itself or delamination and peeling from the concrete matrix. Thus, a second repair is required in a short period, increasing the maintenance cost. To reduce cracking, Strain Hardening Cement-based Composite (SHCC), with strain hardening and multiple cracking property, is prepared to study the influence of interface roughness and repair layer thickness on the shrinkage, cracking and delamination modes of SHCC-repaired concrete beams. The results show that under the shrinkage stress, multiple fine cracks instead of local fractures occur in the SHCC repair layer, and the interfacial delamination is effectively controlled. Interfacial bonding property is the main factor that affects the shrinkage and deformation coordination of SHCC-repaired beams. When the interface roughness is different, the crack width of the SHCC repair layer is similar. However, it has a greater influence on the interfacial delamination length and maximum delamination height of the repaired beam. With the increase of interface roughness, the delamination length and height of the repaired beam are greatly reduced. Therefore, before using SHCC to repair the existing structures or components, the bonding surface should be roughened to improve the bond strength between SHCC and the old concrete. With the increase of the repair layer thickness, the cracking and delamination of the repair layer tend to be alleviated. Although the crack width of the repair layer can be effectively controlled after cracking, the overlarge shrinkage (985.35 × 10 In recent years, the cost for infrastructure repair and maintenance in developed countries such as the United States, Britain, Japan, and Germany have been increasing, and investment in repair and maintenance of old buildings has accounted for more than 50% of the total construction. In 2018, the United States released the “Legislative Outline for Rebuilding Infrastructure in America”, hoping to use $200 billion in federal government funds to leverage $1.3 trillion in local government and private capital to repair and renew old infrastructure [At the early stage, ordinary cement paste, mortar or ordinary concrete were used as repair material of concrete structures. But cracks or delamination often occurred at the interface between the repair layer and old concrete, causing the interface to de-bond. In order to improve the interface performance between repair layer and old concrete, steel fiber, PAN-based carbon fiber, polypropylene fiber, fly ash, silica fume, latex, expansion agent were added to cement-based repair materials ,4,5,6. TSHCC is usually composed of cement, water, fiber, fine aggregate, fly ash, silica fume (microsilica), etc. However, the use of silica fume is often risky in the aspect of its application to SHCC, because this additive of silica fume usually increases the brittleness of the cement matrix ,18. SHCCThe repaired beam is composed of old concrete and repair materials. The mix proportion of concrete with compressive strength of is shown in SHCC with compressive strength (100 mm × 100 mm × 100 mm) of 48.49 MPa at the age of 28 days curing in an indoor environment  was used as the repair material. The mix proportion of SHCC, which is the result of optimization with local materials, is shown in The specimens for uniaxial tensile tests of SHCC are dumbbell specimens, as shown in The dimensions of SHCC specimen and concrete specimen for free shrinkage tests are 40 mm × 40 mm × 160 mm and 100 mm × 100 mm × 515 mm. After hardening for 24 h under wet burlap, the forms were removed and the specimens were placed in an indoor environment  for the shrinkage test as shown in The dimension of old concrete beam is 515 mm (length) ×100 mm (width) × 100 mm (height). The dimension of SHCC repair layer is 515 mm (length) × 100 mm (width) with a thickness of 20–50 mm, as shown in This test focuses on the influence of interface roughness and repair layer thickness on the performance of SHCC-repaired concrete beam as shown in The casting process of SHCC-repaired concrete beam is as follows:(1) Preparation of old concrete beams: the old concrete beams were casted first, and the mold was removed after 24 h. Then, the old concrete beams were covered with a plastic film and regularly sprinkled for curing. After 28 days of sprinkling curing, they were kept indoors  for natural curing until 6 months. The compressive strength of the cubic concrete specimen (100 mm × 100 mm × 100 mm) curing in the same environment for 6 months is 41.04 MPa.(2) Treatment of the old concrete bonding surface: the old concrete bonding surface during casting was treated as shown in (3) The sand filling method was used to evaluate the roughness of the old concrete bonding surface , as showh is the average depth of sand filling (mm); V is the sand volume (mm3); A is the area of the cross-section (mm2).Where (4) The repair layer was poured following the design requirements in (5) Then, the time of the first crack, the crack width (determined by crack width measuring instrument), the length of the cracks (determined by ruler), the number of the cracks were recorded. At the same time, interfacial delamination of SHCC-repaired beams, including delamination position, delamination time, delamination length and delamination height was also recorded.The stress–strain curves of SHCC under uniaxial tension are shown in tF, first cracking strain tε, elastic modulus tE, ultimate tensile strength tuf and ultimate tensile strain tuε of SHCC under tensile load as shown in tF is the stress value corresponding to the endpoint of the elastic stage of the tensile stress–strain curve. The first cracking strain tε is the strain value corresponding to the initial cracking strength. The tensile modulus tE is the slope of the elastic stage of the stress–strain curve. The ultimate tensile strength tuf is the stress value corresponding to the highest point on the stress–strain curve. The ultimate tensile strain tuε is the strain value corresponding to the softening point on the stress–strain curve. It can be seen from tF and tuf increase with the increase of curing age, with rapid growth in the early stages and slow growth in the later stages. The ultimate tensile strength at different ages is about 1.2~1.4 times the first cracking strength. (2) Before 14 days, tε and tuε increase with the increase of curing age; while after 14 days, tε and tuε decrease as the curing age increases. (3) tE increases with the increase of curing age, with rapid growth in the early stages and gradually stable growth in the later stages. (4) The reason for the above results is as follows: with the hydration of cement, both the strength of matrix and the bond strength between PVA fiber and the matrix increase. At the early curing age, the strength of matrix is much smaller compared with the bond strength. If the tensile stress of SHCC is bigger than the strength of matrix, crack occurs. At the same the PVA fiber here spans between this crack. Because the bond strength between PVA fiber and the matrix is bigger than the tensile stress, the PVA fibers can transfer the load to the matrix near this crack. As a result, the tensile strain is increasing. In addition, with the increasing of curing age, the strength of matrix is almost the same with, but still less than the bond strength between PVA fiber and the matrix. In this situation, some of the PVA fibers cannot transfer the load to the matrix near the crack, resulting in the decrease of strain capacity.From Crack patterns of SHCC under uniaxial tension at different ages are shown in −6 and 687.78 × 10−6, and the drying time when they reach half of the final shrinkage value are 9.45 days and 21.73 days, respectively, which agrees with the test results.t is the drying time, day; a is the final shrinkage value of the specimen; b is the drying time when the shrinkage of the specimen reaches half of the final shrinkage value, day.Studies show that the shrinkage curve of cement-based materials can be fitted with Hyperbola function, exponential function and logarithmic function ,29,30,31The shrinkage of the old concrete in the repaired beam has been basically completed after 6 months curing. The SHCC-repaired layer shrinks after being poured. The deformations of the old concrete and the repaired layer are inconsistent, making the shrinkage deformation of the repaired layer constrained by the old concrete, which generates tensile stress in the repaired layer. Meanwhile, tensile and shear stresses could be generated at the interface between the old concrete and the repair layer. When the tensile stress exceeds the tensile strength of the repair material or interfacial bond strength, the repaired beam is prone to cracking and interfacial delamination. The restrained shrinkage stress and failure mode analysis of the repaired beam are shown in xxσ in the repair layer would occur in the middle of the repair layer bottom, and the maximum interface tensile stress xyσ and interface shear stress yyσ would occur at the end of the repaired beam [If the shrinkage deformation of the repair layer does not change along its height, under the restraint of the old concrete, the maximum tensile stress red beam . In factred beam . Due to It is found that under the restraint of the old concrete, SHCC-repaired concrete beams show cracking or delamination or a combination of the two to varying degrees, as shown in The influence of interface roughness on cracking and delamination modes of SHCC-repaired concrete beam is shown in For SHCC-repaired beams with different interface roughness, compared with the crack damage degree (crack width ≤ 0.05 mm), the delamination damage degree (delamination height ≥ 0.07 mm) is more serious. The effect of roughness on the delamination of SHCC-repaired beams is more significant, and it has less influence on the surface cracking. Under the action of shrinkage tensile stress, the SHCC repair layer is in the stage of tensile strain hardening, and the crack width is controlled due to the bridging of the fiber without local fracture. The SHCC-repaired beam with a naturally smooth surface has serious delamination, indicating that the mechanical bond and friction resistance of the smoother old concrete bonding surface are small. The SHCC repair layer has a larger early shrinkage after pouring, and smaller bond strength between the repair layer and the old concrete leads to delamination.The influence of repair layer thickness on the cracking and delamination mode of SHCC-repaired concrete beam is shown in SHCC-repaired concrete beams of different thicknesses have a slightly increased degree of delamination damage (delamination height of 0.05~0.08 mm) compared with the crack degree damage (crack width ≤ 0.05 mm). When the repair layer has a relatively thin thickness, the moisture transmission pathway between the interior and the outside of the repair layer is shorter, the moisture exchange rate is faster, and the water loss degree is larger, causing the SHCC to shrink, and the repaired concrete beam is more prone to cracking and delamination. When the repair layer thickness (within 50 mm) increases gradually, the degree of cracking and delamination of the repair layer tends to decrease.(1) When SHCC is used as the repair material, under the action of shrinkage stress, there are many fine cracks instead of local fractures in the repair layer, and the interfacial delamination is effectively controlled.(2) The interfacial bonding property is the main factor that affects the shrinkage and deformation coordination of SHCC-repaired concrete beams. When the interface roughness is different, the maximum shrinkage crack width of the SHCC repair layer is 0.05 mm, and the average crack width is less than 0.04 mm. The crack widths are not much different. However, the interface roughness has a greater influence on the interfacial delamination length and maximum delamination height of the repaired concrete beam. With the increase of interface roughness, the delamination length and height of the repaired concrete beam are greatly reduced. Therefore, before using SHCC to repair the existing concrete structures, the bonding surface should be roughened to improve the bond strength between SHCC and the old concrete. However, besides the interface roughness, the interfacial bonding and shrinkage properties of repaired beams are also affected by the properties of the old concrete and SHCC, requiring an in-depth study.(3) When the SHCC repair layer is relatively thin, the cracking and delamination degree is greater. As the repair layer thickness increases, the cracking and delamination degree of the repair layer tends to decrease. When it is greater than or equal to 30 mm, the downward trend is insignificant. In view of the overall performance and economic benefits of the repaired concrete beams, without affecting the bearing capacity and durability of the repaired beam, a thinner SHCC repair layer is preferred to repair and strengthen the existing concrete structure or component.-6. Although the crack width of the repair layer can be effectively controlled after cracking, the overlarge shrinkage of the SHCC will result in the cracking of the repair layer and the delamination of the repair interface under the restraint of concrete; thus, SHCC fails to efficiently repair the concrete. In terms of shrinkage deformation control, materials with high toughness and low shrinkage are required to repair the existing concrete structures. By combining the advantages of different shrinkage-reducing measures, multiple regulation methods can be adopted to solve the coordinated development of strength, toughness, durability, shrinkage and deformation of SHCC. (4) The final shrinkage value of SHCC prepared in this paper is about 985.35 × 10(5) Further studies about the bond property between SHCC and concrete, the mechanical property of SHCC-repaired concrete beams are needed for the design and application of SHCC to repair reinforced concrete structures in sever environment, for example marine environment.


There are many atrial fibrillation (AF) screening devices available. Validation studies have mainly been performed in optimal settings in the young population.We aim to compare the yield of AF detection, compliance, and patient‐based experience in an ambulatory elderly population by using intermittent electrocardiogram (ECG) recordings and continuous event recording simultaneously.The study participants were part of the STROKESTOP II study, a Swedish screening study for AF. All participants were 75/76 years of age, were clinically free of AF, and had N‐terminal pro b‐type natriuretic peptides levels ≥125 ng/L. AF screening was performed in parallel during a 2‐week period, using a continuous event recording device  and 30‐second intermittent recordings using a handheld ECG device (Zenicor II) four times daily. Participants were asked to fill out a questionnaire with regard to compliance and ease of use of the devices.P = .002). No new cases of AF were detected using intermittent ECG monitoring only, but some episodes were detected in parallel for patients. On a graded ordinal scale of 1 to 5, with 1 reflecting “very easy to use”, continuous monitoring was graded 2 (interquartile range [IQR]: 1‐3) compared to intermittent 1 (IQR: 1‐1) (P < .001).During continuous event recording, 6% (n = 15/269) were diagnosed with AF and intermittent ECG detected AF in 2% (n = 5/269) of the participants (Continuous event recording detected three times more AF compared to intermittent ECG in an elderly ambulatory population. Compliance and user‐friendliness were rated higher for the intermittent ECG device. The prevalence of clinically diagnosed AF is approximately 3% in the adult population,AF can be asymptomatic, and individuals with asymptomatic AF have been suggested to have a higher risk of stroke than those with symptomatic AF.With the advent of new technology, many new devices for AF detection have been developed. The validation of these devices is commonly performed in an optimal in‐office setting, in the young population.We aim to compare the yield of AF detection, compliance, and patient‐based experience in population screening in elderly individuals, by using intermittent ECG recordings vs continuous event recording.22.1This is a substudy of STROKESTOP II, a Swedish mass‐screening study for AF in individuals aged 75 and 76 years. The study protocol has been published previously.2.2Consecutive participants were included during the last 8 months of the STROKESTOP II study. All participants were free of AF at baseline and had NT‐proBNP ≥125 ng/L. All participants received oral and written information about the substudy and provided informed consent.2.3During inclusion in the STROKESTOP II study, all participants filled out a health questionnaire from which baseline medical data were gathered. In addition to the one‐lead ambulatory handheld Zenicor II device used in STROKESTOP II, participants were equipped with a one‐lead continuous event recorder, R‐test 4 evolution , and were instructed to use the recording devices in parallel for 2 weeks. Both devices had buttons for activation if symptomatic arrhythmia occurred. The participants were also asked to fill out a questionnaire with regards to their experience of the two different AF screening devices including information on completion of the 2‐week registration (yes/no), problems leading to discontinuation (free text), ease of use , and effect on daily life. They were also asked to fill out a symptom diary during the 2 weeks.2.4To identify ECGs with suspected AF, all intermittent recordings were inspected manually in addition to the validated computerized algorithm used by Zenicor.2.5The R‐test 4 evolution device was programmed to store not only AF suspicious activity, but also other significant arrhythmias Table . The R‐t2.6p‐waves, and duration of at least 30 seconds.The diagnostic criteria for AF used in the study are according to ESC guidelines: absolute irregular rate‐to‐rate intervals, no discernable, distinct 2.7Participants with other significant bradyarrhythmias such as second‐degree atrioventricular block Mobitz type II, sinoatrial block or sinoatrial arrest for >2 seconds during daytime or >3 seconds at night‐time, or sinus bradycardia with a frequency of less than 30 beats/min were offered cardiologist follow‐up. Similar follow‐up was offered to participants with multifocal or broad complex tachycardia consisting of eight or more consecutive beats.2.8U test. Chi‐square test was used for proportions. Comparisons of the two screening methods were performed using McNemar's test for dichotomous variables and paired sample t‐test for continuous variables. All tests were two‐sided, and a value of P < .05 was regarded as significant. All analyses were performed using IBM SPSS Statistics, version 24 software .All continuous variables were analyzed according to non‐normal distribution, as most variables were non‐normally distributed. Together with all ordinal data, they were reported as median with interquartile range (IQR), and analyzed using Mann‐Whitney 2.9The study complies with the Declaration of Helsinki. The protocol was approved by the regional ethics committee . All participants provided informed consent.3Of the 3763 participants in STROKESTOP II, 269 (7%) were included in this comparison study between June 2017 and January 2018.3.1P = .002). Using parallel monitoring, no new cases of AF were detected using intermittent ECG monitoring only. Using continuous event recording, AF was detected on average day 4 (IQR: 1‐8) compared to day 8 (IQR: 4‐14) using intermittent ECG (P = .135). There was a significant difference in AF detection between the two devices already after 3 days of monitoring  of the participants and intermittent ECG detected AF in 2% (n = 5)  reported symptoms in their diaries.Individuals diagnosed with AF had lower systolic blood pressure compared to those free of AF. AF was more common in patients reporting diabetes, but was less commonly associated with hypertension, vascular disease, and previous stroke/transient ischemic attack Table .3.2In total, other significant arrhythmias were overall more commonly detected using continuous monitoring compared to intermittent recordings Table .3.3The detection algorithm for the intermittent ECG device detected all episodes manually interpreted as AF. For the continuous recorder, 73% (n = 11) of episodes manually interpreted as AF were categorized as AF by the algorithm of the device, and 27% (n = 4) were categorized as other arrhythmias. Hence, for intermittent ECGs, only episodes marked as AF needed manual interpretation, whereas for continuous ECG all categories marked as arrhythmia needed to be interpreted to detect all AF cases.The number of ECG strips that needed manual interpretation per participant was 3 (IQR: 1‐8) for intermittent ECG compared to 55 (IQR: 40‐70) for continuous event recorder. The average time spent on analysis of ECG recordings per participant was 4.5 minutes for continuous event recording and 0.75 minutes for intermittent ECG. For continuous event recording, 4 (IQR: 0‐10)% of the ECGs were non‐interpretable compared to 0 (IQR: 0‐2)% of intermittent ECGs.3.4P = .559 for intermittent ECG and P = .804 for continuous event recording).Intermittent ECG was graded as easier to tolerate compared to continuous ECG by the participants Figure . Median 4In this ambulatory elderly population, continuous event recording detected three times more new cases of AF compared to intermittent ECG. Both methods were well tolerated and, even in this ambulatory setting, ECG quality was good. The device‐based algorithms differed in categorization of AF events, leading to a more time‐consuming interpretation for the continuous event recording device.Continuous event recording detected more AF cases during the first 3 days than intermittent ECG did during 2 weeks. Overall, the detection of 6% new AF is significantly higher than the outcomes of previous screening studies using intermittent ECGs (3‐4%).One might speculate that in order to increase the ease of use and reduce the interpretation burden for the investigator, the duration of continuous event recording could be reduced.An important aspect of AF screening is to diagnose AF in asymptomatic patients. It might be possible that patients who experienced symptoms of palpitations were more keen to participate in our study, which might have had an impact on generalization and compliance. In a real‐world setting, compliance might be lower and the perceived differences in ease of use between the devices might lead to lower compliance for continuous monitoring.To our knowledge, there are no prior studies comparing continuous event recording and intermittent ECG, except our smaller pilot study, where the two devices were used in parallel for 2 weeks; according to our previous results, continuous event recording detected >2.5 times more participants with AF compared to the intermittent ECG.p‐waves.In prior studies, both devices have shown an increased detection of AF compared to 24‐ to 48‐hour Holter monitoring. When intermittent ECG recordings, using the Zenicor device, for 10 seconds twice daily during 30 days were compared to 24‐hour continuous ECG recording, AF episodes were detected in 82% using intermittent recordings compared to 32% using continuous recording.In our study, intermittent ECG was graded as more user‐friendly than continuous event recording, and previous studies have shown similar results.There is an ongoing discussion regarding the stroke risk in screening‐detected AF, as patients with screening‐detected AF may have short and rare AF episodes. In our study, screening‐detected AF was treated equally to clinically detected AF as we hypothesized that patients with AF detected during such short monitoring period are likely to have a high AF burden. In a large matched cohort study of stroke risk in incidentally detected ambulatory AF, individuals with incidentally diagnosed AF were found to have twice as high incidence of stroke compared to individuals with no AF. Anticoagulant treatment reduced stroke risk by >60% and mortality by >40% in these individuals with incidentally detected AF.4.1The participants were all part of the STROKESTOP II study. It is possible that they were healthier than the general population, as participation in screening studies is known to be higher in healthier individuals.Participants with high NT‐proBNP levels are more likely to have AF, and this could lead to an increased detection, with a detection bias. As the participants are their own control, this will not affect the results of this study.p‐wave analysis difficult. This could introduce a misclassification bias by underestimation of true cases. Neither of the screening devices used have 100% sensitivity for AF detection; hence, we may have underestimated the true AF prevalence.Both AF screening methods used in the study are one‐lead ECGs, making 5Continuous event recording detected three times more new cases of AF compared to intermittent ECG when performed simultaneously in an ambulatory setting for 2 weeks. In our elderly population, both methods were well tolerated, although intermittent ECG was graded as more user‐friendly. The ECG quality was good for both methods, but device‐based algorithms differed in categorization of AF events, leading to a more time‐consuming interpretation for the continuous event recording device.Tove Fredriksson has received unrestricted research grants from Boehringer‐Ingelheim and Stiftelsen Hjärtat. Katrin Kemp Gudmundsdottir has received a research grant from Stiftelsen Hjärtat. Viveka Frykman has received lecture fees from MSD, Boehringer‐Ingelheim, Bayer, and Medtronic. Leif Friberg has received consultancy fees from Bayer, Boehringer‐Ingelheim, BMS/Pfizer, and Sanofi. Faris Al‐Khalili has received lecture fees from Bayer, Boehringer‐Ingelheim, and BMS/Pfizer. Johan Engdahl has received consultancy fees from Sanofi and Pfizer; lecture fees from Bayer, Boehringer‐Ingelheim, Astra Zeneca, and Medtronic; and unrestricted research grants from Pfizer and Boehringer‐Ingelheim. Emma Svennberg has received lecture fees from Bayer, Bristol‐Myers Squibb‐Pfizer, Boehringer‐ Ingelheim, and Sanofi.Table S1. Settings for R‐test 4 evolution.Click here for additional data file.


However, theoretical advantages of neoadjuvant treatment, including the increased R0 resection rate, early delivery of systemic therapy to all patients, directly addressing occult metastatic disease, and improved patient selection for resection, may also apply to these patients. A systematic review by intention-to-treat showed a superior median overall survival (OS) for any neoadjuvant approach (19 months) compared to upfront surgery (15 months) in (B)RPC patients. A neoadjuvant approach was recently supported by three randomized controlled trials (RCTs). For resectable PDAC, neoadjuvant treatment was superior in a Japanese RCT of neoadjuvant gemcitabine with S-1 vs. upfront surgery, with adjuvant S-1 in both arms . A Korean trial of neoadjuvant gemcitabine-based chemoradiotherapy vs. upfront resection in BRPC patients was terminated early due to superiority of the neoadjuvant group . The PREOPANC-1 trial for (B)RPC patients also showed favorable outcome for neoadjuvant gemcitabine-based chemoradiotherapy vs. upfront surgery . FOLFIRINOX is likely a better neoadjuvant regimen, because of superiority compared to gemcitabine in both the metastatic and adjuvant setting. Currently, five RCTs evaluating neoadjuvant modified or fulldose FOLFIRINOX are accruing patients.Approximately 20% of pancreatic ductal adenocarcinoma (PDAC) patients have (borderline) resectable pancreatic cancer [(B)RPC] at diagnosis. Upfront resection with adjuvant chemotherapy has long been the standard of care for these patients. However, although surgical quality has improved, still about 50% of patients never receive adjuvant treatment. Therefore, recent developments have focused on a neoadjuvant approach. Directly comparing results from neoadjuvant and adjuvant regimens is challenging due to differences in patient populations that influence outcomes. Neoadjuvant trials include all patients who have (B)RPC on imaging, while adjuvant-only trials include patients who underwent a complete resection and recovered to a good performance status without any evidence of residual disease. Guidelines recommend neoadjuvant treatment for BRPC patients mainly to improve negative resection margin (R0) rates. For  In contrast to the decreasing cancer-related death rates for many other solid organ malignancies, PDAC survival has not shown much improvement over the last decades . As a cos (RCTs) . These teatments . FollowiResection remains the only curative-intent treatment. However, even curative-intent surgery typically does not overcome the aggressive biology, resulting in recurrent disease within 2 years after resection in the vast majority of patients . StudiesThe objective of this paper is two-fold. First, we aim to give a general overview of the current treatment strategies for (B)RPC patients, to discuss the rationale for neoadjuvant and adjuvant therapy, and to consider the challenges when comparing these treatment approaches. Second, we aim to summarize the currently available evidence for neoadjuvant treatment with a special focus on neoadjuvant FOLFIRINOX, including published and ongoing phase II-III trials for neoadjuvant treatment.To identify relevant studies for neoadjuvant treatment, a comprehensive search of Clinicaltrials, Embase, and MEDLINE was performed. Search terms included “neoadjuvant,” “FOLFIRINOX,” “folinic acid,” “fluorouracil,” “irinotecan,” “oxaliplatin,” “pancreas cancer,” “drug combination,” and relevant variants thereof. Only articles written in English were assessed. Articles were selected based on relevance for our objectives, considering methodological quality, study type, number of included patients, and additional value to current knowledge. A selection was made for prospective studies with restriction to phase II and III trials and publication dates from 2006 to 2019. Furthermore, references of included articles were assessed for additional relevant literature.resectable PDAC, BRPC, and LAPC. Historically however, BRPC was not recognized as a unique disease stage. In 2001, a first definition of marginally resectable tumors was proposed  is rarely known before pathological examination of the resected specimen. However, patients with extensive apparent contact on imaging often undergo a surgically incomplete (R1) resection, suggesting imaging is indeed a good predictor of the presence of tumor cells surrounding and/or invading the vessel wall. Lack of international agreement on the definition of an R0 resection  and standardized protocols for pathological examination  may explain variation in published R0 resection rates , 18. At ble PDAC , 21. Simble PDAC , 12.resectable PDAC. Initial adjuvant treatment strategies included both chemotherapy and radiotherapy. In 2004, the long-term results from the ESPAC-1 (European Study Group for Pancreatic Cancer) trial were published (n = 73), adjuvant 5-FU based chemoradiotherapy followed by 5-FU , adjuvant 5-FU alone , and observation alone . The trial was not powered for a direct comparison of the four groups, yet survival was longer in patients who received chemotherapy compared to patients who did not . Furthermore, comparison of patients with or without chemoradiotherapy showed inferior median OS for patients who received chemoradiotherapy . The CONKO-001 (Charité Onkologie 001) trial found that adjuvant gemcitabine was superior to observation alone with a 5-year survival rate of 21 vs. 10% (p = 0.01) (n = 366) to gemcitabine plus capecitabine (n = 364) (p = 0.032). In 2018, the results of the PRODIGE 24/CCTG PA.6 trial comparing adjuvant gemcitabine to modified FOLFIRINOX (mFOLFIRINOX) exceeded expectations (p = 0.003). mFOLFIRINOX is currently the best adjuvant treatment for patients with a good performance score.Upfront surgery followed by adjuvant chemotherapy has long been the standard of care for patients with potentially ublished . This mun = 364) . Median The strategy of chemotherapy following surgery has several drawbacks. First, approximately 20% of patients with (B)RPC on imaging will never undergo a resection because of occult metastatic or locally irresectable disease . More adConversely, the neoadjuvant approach has some potential drawbacks. First, patients might have progressive disease during neoadjuvant treatment, precluding curative-intent resection. However, it is unlikely that patients with progressive disease during chemotherapy would have been cured with upfront resection, since cure is exceedingly rare with a 10-year OS of only 4% after surgery . Furtherresectable PDAC treated with upfront resection and adjuvant mFOLFIRINOX: a survival estimate far superior than previously reported for other treatments RPC on imaging are eligible for neoadjuvant trials after adequate biliary drainage. Thus, direct comparison of outcomes of neoadjuvant and adjuvant trials is biased, because neoadjuvant trials can include almost all patients whilst for adjuvant trials only the 25% of patients with the best tumor biology and performance status can be included.resectable PDAC . A second meta-analysis by Dhir et al. provided an update of the literature published since 2009, which marks the endorsement of the AHPBA/SSAT/SSO consensus criteria, as well as the introduction of newer preoperative regimens (resectable PDAC and BRPC was similar (18 vs. 19 months). Furthermore, the R0 resection rate of 85% was much higher than previously reported in the setting of upfront resection. The third meta-analysis by Versteijne et al. included only studies that did not exclude patients who didn't undergo resection after neoadjuvant treatment or patients who didn't undergo adjuvant chemotherapy after resection (p < 0.001), but the R0 resection rate was better after neoadjuvant treatment .One of the first studies describing neoadjuvant treatment for pancreatic cancer was published in 1980 . Over tiregimens . In thisesection . These cesection . This reesection . This meFollowing the ACCORD-11/PRODIGE-4 trial for metastatic PDAC by Conroy et al. in 2011, FOLFIRINOX emerged as a potential preoperative treatment for non-metastatic PDAC . No RCT n = 2005) to patients who underwent upfront resection (n = 6015). The neoadjuvant patients had a longer median OS compared to patients who underwent upfront resection . Moreover, compared with a subgroup of patients who received adjuvant therapy after upfront resection, the neoadjuvant group still had better survival . Second, a large observational cohort study from Verona Hospital included all consecutive BRPC (n = 267) and LAPC (n = 413) patients (nab-paclitaxel (22%) were the most commonly used regimens, and additional radiotherapy was applied in 23% of patients. Resection rate was 24% for BRPC patients, with an R0 resection rate of 58% for all patients combined. No differences were found in R0 resection rates between BRPC and LAPC patients and chemotherapy regimens used.In addition to these meta-analyses, two large retrospective studies investigated the neoadjuvant approach , 46. Theresectable PDAC  phase II studies on neoadjuvant FOLFIRINOX for (B)RPC have been published to date  47–49).–49.47–49ble PDAC . Twenty-Although, the three studies slightly differ in the treatment regimen and sequence, neoadjuvant (m)FOLFIRINOX treatment with or without chemoradiotherapy is feasible with high R0 resection rates. The survival estimates are promising, but need confirmation in larger RCT's.p = 0.96). In the Korean randomized phase II-III trial, BRPC patients were randomly assigned to receive gemcitabine-based chemoradiotherapy (45 Gy in 25 fractions and 9 Gy in 5 fractions) (arm A) or upfront surgery followed by chemoradiotherapy following the same protocol as the neoadjuvant group (arm B) (p = 0.028), better 2-year survival rate (41 vs. 26%), and a superior R0 resection rate  in the neoadjuvant group compared to upfront surgery. Consequently, the study was discontinued due to superiority and lack of equipoise (NCT01458717). At ASCO 2018, the Dutch phase III PREOPANC-1 trial presented preliminary results, after inclusion of 246 (B)RPC patients who were randomly allocated to neoadjuvant gemcitabine-based chemoradiotherapy followed by a resection and adjuvant 4 cycles of gemcitabine (arm A), or upfront surgery followed by 6 cycles of gemcitabine (arm B) (p < 0.001) and superior DFS  in the neoadjuvant group, but a difference in OS could not be demonstrated . To allow for comparison with adjuvant trials, a subgroup analysis was performed of patients who received at least one cycle of adjuvant chemotherapy, showing a median OS of 42 months in the neoadjuvant group and 19 months in the upfront surgery group (p = 0.006). Final results are awaited soon. The PACT-15 trial was an Italian multicenter phase II trial, in which 93 resectable PDAC patients were randomly assigned (1:1:1) to receive adjuvant gemcitabine (arm A), adjuvant PEXG  (arm B), or 3 cycles of PEXG pre- and postoperative (arm C) . Three non-randomized studies on regimens other than FOLFIRINOX have been published (resectable PDAC. (resectable PDAC were randomized to either neoadjuvant gemcitabine and S-1 chemotherapy (2 cycles) or upfront surgery, both followed by 6 months of adjuvant S-1 (p = 0.015). No differences were found regarding the resection rate, R0 resection rate, and postoperative morbidity. Although S-1 is only used as standard-of-care in East Asia, the study does provide additional proof of the superiority of neoadjuvant therapy over upfront resection for patients with resectable PDAC.A number of phase II–III trials have been conducted using other neoadjuvant regimens, yet several of these RCTs were terminated early due to slow accrual. This emphasizes the difficulties in conducting large neoadjuvant RCTs in pancreatic cancer.  (arm B) . Both gr (arm B) . After 8 (arm C) . Median ublished –57. The ublished . Eighty le PDAC.  This stuvant S-1 . This stresectable PDAC patients. The results of the R0 resection rates were notable, with a two-fold increase in two out of the three evaluable RCTs. However, it remains unclear whether superior R0 resection rate is an appropriate intermediate outcome for OS in the neoadjuvant setting. The results of ongoing larger RCTs may further clarify the survival benefit of neoadjuvant treatment as opposed to upfront resection for (B)RPC patients.In summary, although based on only three RCTs, a neoadjuvant approach seems to be consistently superior to upfront resection for R0 resection rates, at least equal or superior for DFS, and at least equal or superior for OS in both BRPC and The NCCN guideline, ASCO Clinical Practice Guideline, and European Society for Medical Oncology (ESMO) Clinical Practice Guideline are commonly used guidelines for pancreatic cancer treatment , 58, 59.resectable PDAC, but advise to consider neoadjuvant treatment in patients with high-risk features, preferably in the setting of a clinical trial. High-risk features include imaging findings suspicious of advanced or metastatic disease, significantly elevated Carcinogen Antigen (CA) 19-9, large primary tumors or regional lymph nodes, excessive weight loss, and notable pain. The adjuvant treatment of first choice is mFOLFIRINOX. For BRPC patients, neoadjuvant treatment is recommended, with therapeutic options including FOLFIRINOX or gemcitabine/nab-paclitaxel, both with or without subsequent chemoradiotherapy. The 2019 ASCO Clinical Practice Guideline  can be offered. Following neoadjuvant therapy, patients may be candidates for additional chemotherapy following surgery, depending on their performance status and initial response to the neoadjuvant treatment. The ASCO guideline recommends a total of 6 months of chemotherapy, considering both neoadjuvant and adjuvant treatment. Adjuvant chemoradiotherapy may be offered to patients who underwent primary resection with microscopically positive margins (R1) and/or node-positive disease after completion of systemic adjuvant chemotherapy. The 2019 ESMO guideline radiotherapy, and the timing of surgery after neoadjuvant treatment still need to be further investigated. Several ongoing phase II and III trials are investigating these aspects of neoadjuvant treatment regimens in patients with (B)RPC. resectable PDAC. In the PANDAS-PRODIGE44 trial, 90 BRPC patients will receive neoadjuvant mFOLFIRINOX with (arm A) or without capecitabine-based chemoradiotherapy (arm B), both followed by surgery and adjuvant gemcitabine or modified LV5FU (NCT02676349). This study uses R0 resection rate as primary endpoint. The PANACHE01-PRODIGE48 is a three-arm trial with 2:2:1 allocation to 4 cycles of neoadjuvant mFOLFIRINOX (arm A) or FOLFOX (arm B), both followed by 8 cycles of adjuvant chemotherapy, or upfront surgery followed by 12 cycles of adjuvant chemotherapy (arm C) (NCT02959879) . This study has completed accrual and randomized 147 patients to either 3 cycles of perioperative mFOLFIRINOX (arm A) or perioperative gemcitabine with nab-paclitaxel (arm B). The primary outcome is 2-year OS, and results are anticipated in 2020. The ALLIANCE A021501 was initially designed to evaluate the additional value of hypofractionated radiation therapy to systemic therapy as neoadjuvant treatment for BRPC of the pancreatic head, with 18-month OS rate as primary outcome(NCT02839343). , or upfront surgery followed by 6 cycles of adjuvant gemcitabine-capecitabine (arm B) (NCT02919787) ].Of the nine RCTs, two originate from France: the PANDAS-PRODIGE44 trial for BRPC patients, and the PANACHE01-PRODIGE48 trial for 2959879) . The choal NCT025716. ThisU NCT026749. This 2919787) . In thisresectable PDAC (NCT00727441). This study is awaiting final results. In the randomized NEONAX trial, 166 patients with resectable PDAC are randomized to receive 6 cycles of gemcitabine with nab-paclitaxel perioperative  (arm A), or all cycles adjuvant (arm B)  and four ongoing single-arm phase II trials are investigating neoadjuvant regimens other than FOLFIRINOX  . The thr (arm B) . In the Most ongoing studies of both neoadjuvant FOLFIRINOX and other neoadjuvant regimens are underpowered to detect a clinically relevant difference  in OS. Some studies are hypothesis-generating in their selection of intermediate outcome, such as R0 resection or treatment completion rates. Other studies do have survival as primary outcome, but have a sample size that is too small to detect a clinically relevant survival difference of 3 or 6 months. Assuming an alpha error of 0.05 and a power of 80%, a sample size exceeding 300 patients is needed to detect a difference in median OS of 6 months. An explanation for inadequate sample size is often a concern for feasibility. The PREOPANC-2 trial appears to be the only RCT that may be adequately powered to assess whether neoadjuvant FOLFIRINOX is superior to other regimens. Furthermore, the ALLIANCE A021806 is the only adequately powered RCT comparing perioperative (8 + 4 cycles) mFOLFIRINOX with adjuvant mFOLFIRINOX (12 cycles).Selection bias hampers comparing survival outcomes between neoadjuvant and adjuvant trials. Patients in neoadjuvant trials may have occult metastatic disease at surgery or may not fully recover from surgery; patients in adjuvant trials were selected after overcoming these hurdles. Only a direct comparison in an RCT will avoid this inevitable selection bias. Despite the limited number of published RCTs comparing a neoadjuvant approach to upfront surgery, patients with resectabel PDAC and BRPC seem to consistently benefit from a neoadjuvant approach with regards to the R0 resection rate. Furthermore, the DFS and OS were at least equal or superior with a neoadjuvant approach compared to upfront surgery. The currently published RCTs supporting neoadjuvant treatment over upfront resection included mostly single-agent based regimens. The multi-agent regimen FOLFIRINOX has considerable toxicity requiring a good performance status. FOLFIRINOX has already proven superior to gemcitabine in the metastatic and adjuvant setting. Ongoing RCTs will investigate whether FOLFIRINOX is indeed the superior regimen in the neoadjuvant setting. Likely, neoadjuvant FOLFIRINOX may further improve the outcomes of this vulnerable patient group. In addition, future RCTs should study the optimal number of neoadjuvant cycles, the value of additional neoadjuvant chemoradiotherapy, the optimal patient selection for surgical resection, and the need for subsequent adjuvant chemotherapy. For patients with a good performance status, we advocate patient participation in one of the large ongoing RCTs evaluating the potential benefit of neoadjuvant FOLFIRINOX for (B)RPC patients.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.EO'R received funding for the Memorial Sloan-Kettering Cancer Center for pancreatic cancer research in general from the following companies: Genentech, Roche, BMS, Halozyme, Celgene, ActaBiologica, OncoMed, Parker Institute, AstraZenica, Silenseed. Consulting/Advisory: Cytomx, BioLineRx, Targovax, Halozyme, Celgene, Bayer, Polaris. EO'R receives personal fees/consulting/advisory funds from Cytomx, BioLineRx, Targovax, Halozyme, Celgene, Bayer, Polaris. The remaining 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. The handling editor declared a past co-authorship with one of the authors BK.


L-Methionine (L-Met) is a sulfur-containing amino acid, which is one of the eight essential amino acids to human body. In this work, the fermentative production of L-Met with genetically engineered Escherichia coli W3110-BL in a 5-L fermentor was enhanced through supplement of Ca2+ into the fermentation medium. With the addition of 30 g/L calcium carbonate (CaCO3), the titer of L-Met and yield against glucose reached 1.48 g/L and 0.09 mol/mol glucose, 57.45% higher than those of the control, respectively. The flux balance analysis (FBA) revealed that addition of CaCO3 strengthened the tricarboxylic acid cycle and increased the intracellular ATP concentration by 39.28%. The re-distribution of carbon, ATP, and cofactors flux may collaborate to improve L-Met biosynthesis with E. coli W3110-BL. The regulation of citrate synthase and oxidative phosphorylation pathway was proposed to be important for overproduction of L-Met. These foundations provide helpful reference in the following metabolic modification or fermentation control for further improvement of L-Met biosynthesis. S-adenosyl methionine (SAM), a common methyl-group donor involved in the biosynthesis of nucleic acids, phospholipids, proteins, epinephrine, melatonin, creatine, and other molecules. Along with L-lysine, Met is a dominant amino acid used in animal feed (Methionine (Met) is one of the eight essential amino acids of humans. It is extensively applied in the fields of pharmaceutics, food, and feed. Met can be also used as an important participant in the synthesis of mal feed . In receL-methionine (L-Met) synthesis is well studied in many microorganisms  was constructed via deletion of metI for partial inactivation of the L-Met import system MetD, metJ for elimination of negative transcription regulation by L-Met, and lysA for blocking the biosynthesis of the by-product L-lysine (metH encoding B12-dependenthomocysteine-N5-methyltetrahydrofolate transmethylase (MetH), metF encoding 5, 10-methylenetetrahydrofolate reductase (MetF), cysE encoding serine acetyltransferase (CysE), and metBL enconding cystathionine gamma-synthase (MetB)/bifunctional aspartate kinase/homoserine dehydrogenase II (MetL) were enhanced by replacing their native promoters on the chromosome by a strong promoter trc, respectively  are essential elements in the nutrition and are required for almost all living things. Inorganic salts starvation or limitation in microbial cells will lead to critical stress response  in the fermentation medium exhibited positive effects on various fermentation product, such as vitamins  were reported to play an important role to stimulate L-Met production with Bacillus thuringiensis EC1. After optimization of their concentrations, an improved L-Met yield of 3.18 g/L was achieved (2+) on cell growth, morphology, and accumulation of target metabolites has also been reported by numerous researchers , as one of the metabolic flux analysis (MFA) approaches, is to analyze the distribution of metabolic flux based on the quantitative equilibrium model of the metabolic network  . The inp13C , owing t13C , descriputamicum  and E. c2SO4, 1.5 g/L yeast extract, 2 g/L KH2PO4, 10 g/L Na2S2O3, 1 mg/L Vb12, 1 mL/L salt solution , 50 mg/L L-lysine, 100 μg/mL Amp, and 0.1 mmol/L isopropyl β-d-1-thiogalactopyranoside (IPTG). Different amounts of calcium carbonate (CaCO3) were supplemented when required.The seed medium was Luria-Bertani (LB) medium  supplemented with 100 μg/mL Amp. The fermentation medium contained 20 g/L glucose, 5 g/L  with a working volume of 3 L fermentation medium. The fermentation was initially performed at 37°C with agitation of 300 rpm and aeration of 1.5 vvm. 100 mg/mL Amp was supplemented to maintain plasmid retention as needed. When the optical density of the fermentation broth measured at 600 nm (OD600) reached 1.8-2.0, IPTG was added at the final concentration of 0.1 mmol/L and the inducible expression was conducted at 28°C. During the entire fermentation process, the dissolved oxygen (DO) level was maintained above 20% by controlling the agitation rate at 300–500 rpm  thed strain , the synMetabolic networks are non-linear complex networks regulated by kinetic mechanisms, usually involving hundreds of metabolites and thousands of reactions. Constructing genome-scale metabolic models manually is time-consuming and labor-intensive. Therefore, it is necessary to build mathematical models of these metabolic networks to describe, understand, and eventually predict the system metabolic behavior . GeneralL-Met production by addition of CaCO3, a metrological model FBA, based on mass conservation and pseudo-steady-state (PSS) of metabolic intermediates , were determined based on the extracellular measurement. The positive definite matrix was calculated by Matlab 2016a software   equipped with a RID detector . For metabolites separation, an Aminex HPX-87H column  was used, working at 35°C and a flow rate of 0.6 mL/min with 5 mmol/L sulfuric acid as mobile phase. Biomass, residual glucose, and eviously . The speeviously . The speeviously .E. coli cell pellets were collected, washed and suspended in 100 mM potassium phosphate buffer (pH 7.0). The pellets were disrupted by sonication  in an ice bath using an ultrasonic processor VCX 130PB . Cellular debris was removed by centrifugation at 12,000 g and 4°C for 5 min. The crude extracts were used for assays of enzyme activity and intracellular calcium irons level. Protein concentration was determined by the Bradford method  (DTNB) colorimetric assay method  was used for Citrate synthase (CS) activity detection. According to the instruction of Kit, CS activity was assayed y method .The calcium concentration was assayed using a Calcium Assay Kit  with methylthymol blue (MTB) as a color agent . The abs3 addition were detected by the qRT-PCR. Samples were taken at the logarithmic growth phase (15 h) during the fermentation process with or without addition of CaCO3 and treated by flash-freezing in liquid nitrogen. The total RNA was extracted using RNAiso Plus reagent  according to the manufacturer’s instructions. The RNA quality was determined using 1% agarose gel electrophoresis and the quantity was measured with a NanoDrop 2000c UV–vis Spectrophotometer . Each RNA sample was reverse-transcribed to cDNA by the PrimeScriptTM RT reagent Kit  according to the manufacturer’s manual. The qRT-PCR was carried out on a LightCycler 480 System  using TB Green real-time PCR mix  with the specific primers  were calculated in Microsoft Excel. The statistical differences were determined according to student’s L-Met biosynthesis, the engineered E. coli W3110-BL was grown in batch cultures performed in 5-L fermentor, during which the pH of the fermentation medium was maintained at 6.8 ± 0.2 either with 30% NH3⋅H2O or with CaCO3 in different concentrations . As shown in 3⋅H2O, only 0.94 g/L L-Met was accumulated; while with CaCO3 as pH regulator, L-Met accumulation was enhanced compared with that of the control (with 30% NH3⋅H2O). Furthermore, L-Met titer was increased as the CaCO3 amount raised from 0 to 30 g/L, with the maximum value of 1.48 g/L at 30 g/L CaCO3 (57.45% higher than that of the control). The yield of L-Met against glucose was 0.089 mol/mol, considerably increased compared with that of the control (0.057 mol/mol). However, with CaCO3 further increased to 40 g/L, decreased L-Met production was observed. Therefore, from the view of fermentation optimization, 30 g/L CaCO3 was determined to be most conducive to L-Met biosynthesis.For 3 as pH regulator, the pH of the fermentation medium was not constant. As was seen from 3 addition, the pH was approximately at 6.8  in the initial stage and slowly varied (first decreased and then increased) between 5.5 and 8.2 with different patterns till the end of the fermentation.It should be noted that, when using CaCOL-Met production, other two different modes of batch fermentation were also tried. In Mode I, the pH was maintained at 6.8 ± 0.2 by automatic addition of 30% NH3⋅H2O as well as supplement of 30 g/L CaCO3; in Mode II, CaCl2 (33.3 g/L) was used to displace CaCO3. Herein, the mode with pH controlled at 6.8 ± 0.2 by automatic addition of 30% NH3⋅H2O was named as Control-I, and the one only with 30 g/L CaCO3 as pH regulator was named as Control-II. Except for the pH in Control-II that was varied between 5.5 and 8.2 over time  or CaCl2 (Mode II), the L-Met accumulation was also greatly improved, 59.57 and 58.51% higher than that of the Control-I, respectively.As shown in 3 were compared and analyzed further. Interestingly, although the extracellular pH values of E. coli cells in these two cases were different, there was no significant difference with respect to L-Met biosynthesis. This indicated that calcium ions rather than environmental pH were most responsible for the enhancement of L-Met accumulation.The fermentation patterns in Control-II and Mode I which both contained 30 g/L CaCO3 is much lower than that of CaCl2 in aqueous solution. And it was considerably higher in each mode (1–10 mmol/L) in comparison with that in Control-I (0.5–8.0 μmol/L), implying that the consumption of calcium ions (intracellular free Ca2+ concentration) was highly correlated with the accumulation of L-Met.As shown in 3, the maximum concentrations of main organic acids including α-ketoglutarate, succinic acid, lactic acid, formic acid, and acetic acid were increased to different degrees. As for α-ketoglutarate and succinic acid, the highest accumulation in the case of CaCO3 addition was increased by 50 and 20.3%, respectively, compared with that of the control. It was speculated that the presence of CaCO3 probably increased the carbon flux of the TCA cycle. After the glucose was exhausted, the accumulations of α-ketoglutarate and succinic acid were both decreased, likely due to their conversion to other metabolic products. By contrast, the biosynthesis of formic acid, lactic acid, and acetic acid was not immediately influenced by glucose limitation in the presence of CaCO3. All the main organic acids formed in the fermentation process might be re-utilized for maintenance of cell growth or L-Met biosynthesis in the late fermentation phase.The formed by-products were detected and the results were illustrated in 3 addition on the L-Met biosynthesis pathway in E. coli W3110-BL, the intracellular metabolic flux distribution was estimated using the FBA method. 12 reaction rates including the specific consumption rate of glucose (r1)/oxygen and the specific formation rate of formic acid (r20), lactic acid (r21), acetic acid (r25), α-ketoglutarate, succinic acid (r28), L-Met (r36), biomass (r39), carbon dioxide, pyruvic acid, and other amino acids  were calculated and summarized in 5H11NO2.6.In order to reveal the specific role of CaCOCarbon recoveries as biomass and various metabolites were 97.4–102.1% of the theoretical maximum, suggesting that the major metabolites were properly identified and quantified. The reduction degree calculated based on the equation ε = 4 C + H − 2 O − 3 N  were welL-Met biosynthesis, thus the quality of the outcome computed from the model was valid in estimating the effect of CaCO3 addition on E. coli intracellular flux distributions in batch cultures.The metabolic model included the main biochemical pathways in carbon central metabolism: glycolysis, PP pathway, TCA cycle, amino acid synthesis, transport reactions, oxidative phosphorylation, and biomass formation. The average intracellular metabolic flux distribution in the late logarithmic growth phase (19 h) was calculated and shown in 3 addition. The carbon flux into L-Met biosynthesis was increased by 44.83% in the case of CaCO3 addition, whereas the carbon flux into the TCA cycle was also increased considerably. Under the aerobic condition, the glycolysis and TCA cycle were correlated by the conversion of PEP to oxaloacetate catalyzed by phosphoenolpyruvate carboxylase as well as the formation of citrate from oxaloacetate and acetyl-CoA catalyzed by the citrate synthase. It could be seen from As shown in 3 addition, the relative changes in transcription levels of 15 genes involved in TCA cycle were determined by qRT-PCR. As shown in gltA encoding CS, acnA encoding aconitase (ACO) isoenzyme, icdA encoding isocitrate dehydrogenase (ICDH), sucC encoding succinyl-CoA synthetase (SCS) isoenzyme, sdhC/sdhD encoding succinate dehydrogenase (SDH) isoenzymes, and fumB encoding fumarase (FH) isoenzyme were higher than those of the control (without CaCO3 addition), indicating an up-regulated expression of these genes. By contrast, the expressions of 6 genes including acnB encoding ACO isoenzyme, sucB encoding α-ketoglutarate dehydrogenase (KGDH) isoenzyme, sdhA/sdhB encoding SDH isoenzymes, fumA encoding FH isoenzyme, and mdh encoding malate dehydrogenase (MDH) were down-regulated. While, there were no significant changes in the relative transcriptional quantitation of sucA encoding KGDH isoenzyme and sucD encoding SCS isoenzyme. Although the transcriptional responses of these 15 genes to CaCO3 supplement were different, it could be certain that, the expression of CS, ICDH, and SCS were enhanced due to the presence of CaCO3.With CaCO2+ on intracellular CS activity during L-Met biosynthesis, CS specific activity was monitored in cell-free crude extract by means of DTNB colorimetric assay method. As illustrated in 2+ before 15 h was not observed. However, the specific activity of CS was much higher than that of the control after 15 h, especially during 22–48 h. The increase in CS activity was allowed for perturbation of the acetyl-CoA branch point with enhanced TCA influx in the presence of CaCO3.To evaluate the effect of Ca3 addition could direct more carbon to participate in L-Met biosynthesis, through fluctuation of the carbon balance in some essential metabolic pathways, particularly in the TCA cycle. CS, responsible for the entry reaction of a two-carbon unit into the TCA cycle, was thus considered to be a promising metabolic control node for L-Met overproduction.The results indicated that, CaCO2 in the related biochemical pathways was estimated and presented in 3 was obviously improved compared with the condition of CaCO3 absence. ATP was generated at a relative high level, particularly in the oxidative phosphorylation pathway. Therefore, CaCO3 addition could provide more energy for L-Met biosynthesis.The formation and consumption of ATP, NADH, NADPH, and FADH2 was TCA cycle and it was solely used for oxidative phosphorylation. These results were in good agreement with those of other studies  in the cellular metabolic network. NADH and FADH2 function as electron carriers and participate in almost all oxidation-reduction reactions. They play crucial roles in cellular energy generation in the living cells. Generally, the ATP yielded during direct glucose metabolism is very limited, while both NADH and FADH2 involved in the process of cellular respiration can help in producing more ATP through electron transfer and oxidative phosphorylation reaction. NADPH usually acts as a hydrogen donor to participate in various metabolic reactions  were estimated by FBA. The results indicated that addition of CaCO3 (30 g/L) into the fermentation medium resulted in an increased metabolic flux in TCA cycle and a relatively high level of ATP and cofactors generation, which may collaboratively contribute to the enhancement of L-Met biosynthesis. This study lay important fundament for further modification of the potential metabolic control node to channel more metabolic flux into L-Met over-production.The positive impact of CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.H-YZ, W-JW, KN, Z-QL, and Y-GZ participated in designing of work. The analysis and interpretation of experimental the data was jointly conducted by all authors. H-YZ and W-JW prepared the manuscript with the help of Z-QL. W-JW, Y-YX, and BZ performed the experiments.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.