Escherichia coli.In Bangladesh, treatment for urinary tract infection has become increasingly difficult due to antibiotic resistance. In addition, the prescription of age and gender-specific drugs is still far from being practiced in Bangladesh. We are examining trends of antibiotic resistance per age and gender in patients with urinary tract infection (UTI) caused by the most frequent agent, E. coli isolates obtained from urine cultures. A sensitivity study using the Kirby-Bauer method was carried out to identify the antibiotic resistance trends.We determined the resistance of 1663 Imipenem with 1.9% resistance of all isolates found to be the lowest percentage of resistance. Meropenem (2.8%), amikacin (2.8%), colistin (2.9%), and nitrofurantoin (15.8%) showed low resistance percentages. The sensitivity analysis suggests that age and gender (area under curve = 0.67) should be taken into consideration to prescribe amikacin. The increasing odds ratios (OR) by age groups suggest that amikacin is a less effective agent for older patients with UTIs. Moreover, nitrofurantoin  = 1.07–1.95) and colistin  were less effective against isolates obtained from males compared to isolates obtained from females. Meropenem was effective against bacteria obtained from all age groups and genders. On the other hand, efficacy of imipenem was lower in isolates obtained from adults older than 40 years .In Bangladesh, meropenem, imipenem, amikacin, colistin, and nitrofurantoin are suitable therapeutic alternatives against urinary tract pathogens. Among the oral agents, amikacin, colistin, and nitrofurantoin should be prescribed, taking consideration of age and gender. These results will assist physicians in prescribing effective primary care antibiotics for UTI patients and encouraging the implementation of health policies for a safe prescription of antibiotics. Escherichia coli; Urinary tract infection; Primary health care; Bangladesh.Clinical research; Diagnostics; Infectious disease; Public health; Urology; Antibiotic resistance;  LiteratE. Coli [There are many bacteria that can cause UTIs, but the most common pathogen is E. Coli . One of E. Coli . From thE. Coli . Around E. Coli .The prime step in the treatment of bacterial UTI is treating patients with an effective antibiotic. However, the selection of the appropriate antibiotic is a big concern when treatment is to be given in primary health care (PHC) before isolating the causative agent and performing the test of sensitivity. Wide-spectrum antibiotics are commonly indicated to treat UTIs when, instead, a narrow-spectrum antibiotic could have been sufficient for an effective treatment . In thisResistance to antibiotics is a critical and extremely important problem in Bangladesh, where the infectious disease burden is high. A recent study in the UK has shown that antibiotic prescribing in primary care is declining, but this is not the case in Bangladesh . Many meE. Coli caused UTIs amongst the population of Bangladesh.Due to age and gender, UTIs may exhibit different epidemiological and etiological properties . Isolate2E. coli isolates obtained from various outlets of Medinova Medical Services Limited (MMSL) in Bangladesh. The research included both the male and female UTI patients above six years of age. The doctor suggested urinary infection based on a clinical examination of the visited patients' signs and symptoms, and then they were referred to the MMSL for urine culture and sensitivity testing. A positive culture of urine confirmed UTI cases. Reports on Urine Culture and Sensitivity were obtained from the MMSL database. We've taken on the reported E. coli samples for the analysis. So, we did not take the patients' informed consent.From November 2017 until August 2018, we performed a cross-sectional study following the STROBE guideline. The sensitivity to amikacin, cefixime, ciprofloxacin, cotrimoxazole, meropenem, ceftriaxone, cephalexin, imipenem, nalidixic acid, and nitrofurantoin was determined for 1663 5 cfu/ml were omitted. Quality assurance has been strictly controlled by the Clinical and Laboratory Standards Institute (CLSI) according to the “Performance Standards for Antimicrobial Susceptibility Testing”  [According to the National Committee for Clinical Laboratory Standards, the antibiotic susceptibility examination was formulated using the disc diffusion method (Kirby-Bauer Method) . The ant manual) .Before the research started, we took the ethical clearance from the North South University review committee. The privacy and confidentiality of data concerning the personal identity of UTI patients was strictly protected.2.1Data was analyzed using R 3.3.1. All categorical variables (presented as frequencies and percentages) were determined using descriptive statistics. For the microbial-resistance to each antibiotic, we designed logistic regression models. The findings are reported by the odds ratios (ORs) and confidence intervals and significance was assumed when p < 0.05. ROC curves were used to predict antibiotic resistance based on age and gender.3E. coli isolates were meropenem (2.8%), imipenem (1.9%), amikacin (2.8%), colistin (2.9%), and nitrofurantoin (15.8%).In The distribution of microbial-resistance to antibiotics by the patient age groups is given in It appears from 3.1E. coli, and the medication may be administered to UTI patients irrespective of their age and gender.The highest area under the ROC curve (AUC) given in 4E. coli isolates obtained from UTI cases in Bangladesh, considering age and gender of the patients. Meropenem and imipenem were the selection of antibiotic therapy showed the highest sensitivity percentages among the isolates. But, as they are administrated intravenously, a closer control is required. In addition, as they are more expensive, they are not commonly indicated for routine UTI treatment. Other studies show similar findings regarding meropenem and imipenem [Our study shows the distribution of antibiotic resistance among imipenem , 19, 20.E. coli isolates have high rates of resistance to nitrofurantoin [E. coli isolates to cephalexin compared with the 2015 study in Sudan [E. coli to cephalexin (33%), cefixime (23%), ciprofloxacin (21%), ceftriaxone (17%), nitrofurantoin (3%) and nalidixic acid (37%) [Cotrimoxazole has historically been the drug's first line, but currently, it has become immune to UTI patients . A few aurantoin , 25, 26.in Sudan . Relatedid (37%) . Anotherid (37%) .E. coli from UTI are mostly susceptible to amikacin, meropenem, colistin, and imipenem. Akram et al. found much higher bacterial-resistance to amikacin in 2007 (51%) but lower resistance to imipenem in India [Our results indicated that in India . Imipenein India . Colistiin India .et al. in 2013: 10% (1987), 18.5% (1990), 43.6% (2002), 3.2% (2009) and 73.7% (2013) [Nitrofurantoin was the first highly effective and safe antibiotic prescribed for UTI, decades ago, several bacterial pathogens are becoming resistant to it over time . The pre% (2013) . However% (2013) , 34. Nit% (2013) .We observed that amikacin and nitrofurantoin were more effective against isolates obtained from people of 60 years of age than their younger counterparts when testing the antibiotics in four different age groups. In the elderly population, however, it is best to stop amikacin and nitrofurantoin while empirically treating urinary tract infection. A research carried out in London in 2008 reflected a similar scenario for nitrofurantoin, but we were unable to compare such values because they used 16 years to be their demarcation age . Both thE. coli isolates from UTI cases, considering age and gender differences. These results are important to physicians as basis for prescription of effective antibiotics for UTI patients, preventing the emergence of new antibiotic-resistant strains by incorrect indications. Limitations include the study's use of the antimicrobial susceptibility testing approach for disc-diffusion, which does not always have a reliable antimicrobial resistance profile. In addition, regional variations in the pattern of resistance were not noted in this paper. A multi-level analysis with several diagnostic centers from urban and rural areas will help assess the overall generalizability of our findings for the UTI patients as a whole in a future objective.This research has many strengths. Our study is the first to highlight the antibiotic susceptibility/resistance patterns of 5E. coli obtained from UTI cases in Bangladesh. In contrast, it was found that oral drugs like amikacin, colistin, and nitrofurantoin would be effective. The most effective drugs were meropenem and imipenem, but caution must be practiced because of potential side effects. E. coli isolates obtained from elderly patients were more resistant to amikacin and nitrofurantoin. In addition, isolates obtained from males exhibited greater resistance to amikacin, colistin, and nitrofurantoin. This study will help health care practitioners in proper antibiotic selection.High resistance to nalidixic acid, ciprofloxacin, cephalexin, cefixime, and ceftriaxone was detected among A. Hossain: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.S.A. Hossain: Conceived and designed the experiments; Analyzed and interpreted the data.A.N. Fatema: Conceived and designed the experiments; Wrote the paper.A. Wahab, M.M. Alam, M.N. Islam, M.Z. Hossain, G.U.Ahsan: Performed the experiments; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.http://individual.utoronto.ca/ahmed_3/index_files/data/data.html.Data associated with this study has been deposited at


Introduction Despite excellent reviews in the past several years, the use of antibiotics as prophylaxis for implant placement remains controversial.Aim  To assess the literature on the efficacy of prophylactic antibiotics prescribed prior to and immediately following implant surgery (PIFS).Outcomes Whether administration of antibiotics reduced implant failure and post-operative complications.Design  Databases searched were PubMed and Medline via Ovid (1946 to February 2018), Cochrane Library (Wiley) and Google Scholar.Materials and methods Quality assessment, meta-analysis with a forest plot and incorporated assessment of heterogeneity. A two-tailed paired t-test was performed, analysing differences in mean failure rates between groups.Results Fourteen publications were collected; 5,334 implants were placed with pre-operative antibiotics, 82 implants with antibiotics PIFS and 3,862 placed with no antibiotics. The overall risk ratio (RR) was 0.47 (95% CI 0.39-0.58), with the implant failure rates significantly affected by pre-operative intervention . The number needed to treat (NNT) was 35 (95% CI 26.3-48.2). The difference between mean failure rates was statistically significant (P = 0.0335).Conclusion Administering prophylactic antibiotics reduced the risk of implant failures. Further investigations are recommended to establish a standardised protocol for the proper use of antibiotic regimen. Importance of antibiotic prophylaxis in preventing implant failure.Different types of antibiotics used for prophylaxis.Future considerations in antibiotic use for preventing early implant failure.1 Since most patients undergoing dental implant placement are relatively healthy and do not have significant medical risk factors, the use of antibiotic prophylaxis for healthy patients has not become standard practice. Although current literature outlines long-term studies showing the efficacy of a single-dose antibiotic in reducing early implant failure, the use of antibiotic prophylaxis and choice of agent remain controversial. The primary concern involves conflicting viewpoints that antibiotic prophylaxis may or may not be as effective as traditionally believed. Xu et al. expressed concerns about the difficulty of limited information available to practitioners for stringent control of post-operative complications following implant procedures.2 Furthermore, current literature demonstrates significant variability among practitioners' antibiotic prescribing patterns, prompting the need to assess how varying interventions affect the overall success of implant operations.3Antibiotic prophylaxis is standard practice for numerous surgical procedures in immunocompromised patients and in patients with significant co-morbidities.3 Implant failures during the early wound-healing period involve inflammatory breakdown at the surgical site, causing tissue scarring and poor osseointegration.4 One of the major disadvantages of these sequelae is the loss of hard and soft tissue at the implant site, which renders significant challenges to the surgeon for future implant placements. A retrospective analysis by Pyysalo et al., in 2014, demonstrated fewer numbers of early implant failures among those that were placed under antibiotic treatments.5Dental implants generally exhibit high initial success rates, but failures occur occasionally.2Despite these reviews, implant placement protocols presently still lack guidelines that would ensure consistent and successful post-implant wound healing.6 The conflicting viewpoints are related to concerns that over-prescribing antibiotics can promote the development of antibiotic-resistant bacteria.1,3,7 Moreover, different regimens outlined in the literature regarding antibiotic type, dosage and the time of administration have all contributed to heterogeneity in antibiotic prescribing patterns.8 For instance, Ata-Ali et al. described antibiotic prophylaxis as necessary to provide pre-operative protection against infections and reduce the frequency of implant failures.9 Escalante et al. also supported the benefits of prophylactic antibiotic practices in implant surgery, reducing the possibility of developing post-operative infections at the surgical site.4 Ahmad and Saad, however, suggested that practitioners must administer prophylactic antibiotics with caution and only when determined to be appropriate, not merely as a general measure.1 Over-prescription of antibiotics may not only promote the emergence of antibiotic-resistant microorganisms; it could also induce toxic effects and hypersensitivity reactions.10The Canadian Dental Association (CDA) has emphasised the importance of prophylactic antibiotics in managing the risk of infections associated with surgical procedures.A considerable number of studies have previously reported ambivalent results, causing many to question the benefit of antibiotic prophylaxis. Limited evaluations on the importance of prophylactic antibiotics warrant further research, as the practice directly influences patient outcomes and care, associated costs, suggested lines of management and subsequent treatment planning in implant procedures. This systematic review thus aims to assess the efficacy of antibiotic use, chiefly involving administration before and immediately following dental implant surgery. We also aim to examine how current viewpoints on antibiotic practices in implant dentistry compare to the observed success rates of implants with or without the use of antibiotics.A literature search was conducted in February 2018, across the four electronic databases: PubMed and Medline via Ovid (1946 to February 2018), Cochrane Library  and Google Scholar. No time or language restrictions were applied to attain the maximum number of results regarding implant dentistry. Manual record searching across dental journals and other relevant databases generated additional literature more specific to the review focus. Several keywords were used during the search process, generating records pertaining to dental implants and implant failures. A detailed summary of search method and keywords is shown in Appendix 1.The screening standard was duplicated independently by two review authors. Primary screening involved examining the title and abstract of generated records. Full-text studies that seemed to meet the criteria were included and further assessed. The two review authors independently carried out secondary screening of the remaining records, involving assessment of study methods, results and discussions. Final selection of studies was made by discussion between authors under population, intervention, comparison and outcome (PICO)-based inclusion/exclusion criteria.The population of interest included all patients in need of implant placement and patients referred/scheduled for implant surgery. General inclusion criteria were medically and orally healthy adults (>19 years of age) who were non-smokers or light smokers (<5 cigarettes a day), had no existing periodontal disease or oral infections, and had no procedures that required antibiotic dosage before implant treatments.Interventions were standard oral implant procedures in conjunction with a single-dose antibiotic administered pre-operatively or immediately following surgery (PIFS).Comparison involved the effects of pre-operative antibiotics and PIFS versus the effects of no antibiotics/placebo on implant failure rates. Experimental studies directly pertaining to implant failures in the presence/absence of antibiotic prophylaxis were examined for data extraction. Included study types were randomised controlled trials (RCTs), clinically controlled trials (CCTs) and prospective/retrospective clinical studies (PCS/RCS). For meta-analysis, included study designs were RCTs and CCTs.Outcomes of interest were implant failure rates with or without different types of prophylactic antibiotics.Subsequently, patients were excluded if they possessed present medical conditions requiring antibiotic administration, were immunodeficient, had allergies to certain antibiotics, were non-adults (<19 years of age), were pregnant, or have had prosthetic procedures or endocarditis treatment previously (P). Treatment groups that involved long-term administration of post-operative antibiotics (duration of 2-7 days following surgery) were excluded (I). Studies without treatment interventions such as reviews, case reports and commentaries were excluded (C). Finally, records were excluded if they did not reflect the assessment of implant failures, if they had different populations of interest and if studies had different outcomes of interest (O).11,12 The criteria used in the assessment consisted of seven domains: random sequence generation, allocation concealment, blinding of assessors, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias. A study was determined to be at low risk of bias if it satisfied all of the above criteria, at moderate risk of bias if a study did not satisfy one of the above criteria, and at high risk of bias if two or more domains were not satisfied.The risk of bias assessment for included RCTs was performed following the guidelines of the Cochrane Handbook for Systematic Reviews of Interventions.The data extraction process was duplicated independently by two authors and then double checked between two authors to validate the gathered information. Any disagreements were resolved by discussion or consulting with other review authors.2 statistic to analyse variability due to heterogeneity among the gathered studies. Relative weights of included studies were expressed in percentages and the risk ratio (RR) with 95% confidence interval (CI) was computed per study or subgroup. Lastly, a two-tailed paired t-test was performed to further confer statistically significant differences between the two groups of interest. The overall effect of pre-operative antibiotic prophylaxis on implant failure rates was displayed using a bar chart.The methodology was reviewed by an independent statistician from the Strategy for Patient-Oriented Research (SPOR) networks. The meta-analysis was performed using RevMan 5.3, constructing a forest plot with IA summary of the search process is shown in 13,14,15,16,17,18,19 three CCTs,20,21,22 two pilot randomised controlled trials,3,23 one PCS24 and one RCS.25 All studies stated post-operative follow-up durations, as well as the outcomes of investigation. Four studies13,18,20,25 identified implant failures through poor osseointegration, five studies3,19,22,23,24 investigated pain and swelling, and the remaining five studies21,14,15,16,17 assessed for implant failures associated with post-operative complications and other adverse effects.A detailed summary of the included studies is shown in 19,25 administered antibiotics PIFS and nine studies20,21,13,15,16,17,18,19 used either no antibiotics or placebo tablets as alternatives. Six studies3,18,19,20,23,24 introduced chlorhexidine mouthwash as secondary interventions and the remaining seven did not specify other interventions allowed.All studies contained at least one primary test group with a pre-operative antibiotic intervention . Two stu3,22,23,14,15,16,17,18,19 used oral amoxicillin, two studies24,25 administered penicillin derivatives or clindamycin, two studies13,20 did not specify the antibiotic types used during implant placements and one study21 used several types, with penicillin derivatives being the most common. Of the nine studies that administered amoxicillin, seven studies14,15,16,17,18,19,22,23 used an identical regimen (2 g single-dose amoxicillin) one hour before surgery, and the other two studies3,18 gave 1 g and 3 g single-dose one hour before surgery, respectively. Thirteen studies3,13,14,15,16,17,18,19,20,21,23,24,25, provided precise numerical results of failed implants, while one study22 did not specify how many implants succeeded or failed (reported success rate as a proportion of patients in the test group). Lastly, only three studies13,18,20 reported a statistically significant difference between pre-operative groups and non-antibiotic groups. Four studies3,14,24,25 found no significant difference between single-dose pre-operative interventions and long-term post-operative interventions, and seven studies14,15,17,19,21,22,23 reported a trend favouring antibiotic prophylaxis, but no statistically significant differences.Nine studies13,23 were determined to be at high risk of bias, one study14 at moderate risk and the remaining six studies3,15,16,17,18,19 at low risk of bias , while the CCT group exhibited medium-high heterogeneity (I2 = 64%). Implant failure rates were significantly affected by the difference in prophylactic measures (prophylaxis vs no antibiotic) as shown through the test for overall effect  and the number needed to treat was 35 (95% CI 26.3-48.2).The forest plot analysis of included RCTs and CCTs is exhibited in Lastly, the two-tailed paired t-test showed significant difference in mean implant failure rates between two treatment groups, with a mean failure rate of 1.8% among the antibiotic prophylaxis patients and 6.0% in the non-prophylaxis patients  use of a wide variety of prophylactic regimens; 2) lack of consistent effects of antibiotic prophylaxis; and 3) lack of standardised follow-up durations.Additionally, Romandini 7 The study also revealed the types of pre-operative antibiotics given to patients allergic to penicillin: 48.1% administered clindamycin, 19.2% metronidazole and 4.8% erythromycin.7 Other records further portrayed the predominance of amoxicillin uses,33 shown through the nine studies3,14,15,16,17,18,19,22,23 included in this review as well. Some clinicians, however, chose other alternatives such as clindamycin, Keflex, cefazolin and other penicillin derivatives.34 Pyysalo et al. reported phenoxymethylpenicillin (72.2%), followed by amoxicillin, cephalexin, roxithromycin and clindamycin.5 Ahmad and Saad also mentioned clindamycin as an alternative to amoxicillin and penicillin derivatives.1 In contrast, Escalante et al. proposed azithromycin as an effective alternative prophylactic antibiotic for patients allergic to penicillin.4Myriad antibiotic choices were available to practitioners, often involving three or more types and dosages applicable to each intervention. Lack of consensus between practitioners concerning distinct antibiotic prescribing patterns warrants the need to facilitate calibration to enhance treatment homogeneity. Current literature leans towards the use of oral amoxicillin, where roughly 80% of the respondents used amoxicillin for patients not allergic to penicillin, according to a study conducted in the UK.4,10 In the vast majority of cases, evidence suggests that providing antibiotics pre-operatively rather than post-operatively following routine dental implant placement is the protocol of choice.7 Only in a small number of cases, the presence of a disease may increase the risk of bacteraemia associated with these routine activities accordingly; in this condition, the benefits of post-operative antibiotics are emphasised despite the recommended guidelines.32Although 2 g of amoxicillin is the most commonly administered pre-operative dose among clinicians, the literature is unclear on which antibiotic exhibits the most effective result. As mentioned earlier, the American Heart Association recommends only a single-dose pre-operative antibiotic prophylaxis in most of the cases, while some patients with high-risk heart diseases may require additional post-operative doses to prevent secondary bacteraemia following dental procedures.35 Incorrect use of antibiotics with little consideration of repercussions increases the risk of developing resistance and other adverse side effects, and ultimately devalues the efficacy of antibiotic prophylaxis.36 One of the important factors that should be considered is evaluating the cost of the antibiotics to the desired outcomes. Due to the increase of healthcare costs and limited resources, policymakers and healthcare payers are also concerned about the cost-effectiveness of the excessive prescription of antibiotics. Hence, more studies of economic evaluation to assess the cost-effectiveness of antibiotics by exploring whether antibiotic treatment makes a sufficient contribution to health to justify its costs should be explored more in the future.37 These factors still suggest that antibiotics should be prescribed with caution and for the shortest duration possible to achieve the desired outcome.In addition, most antibiotic interventions varied by their duration, sometimes exceeding the duration needed to prevent post-operative complications. Prolonged administration of antibiotics may contribute to the emergence of resistant bacteria.Our study shows the benefits of pre-operative antibiotics in reducing dental implant failure. However, there is no standardised guide to the use of antibiotics in dental implant surgery, accentuating the need for further investigations to identify the most effective antibiotic regimen for reducing implant failures.Based on our meta-analysis and statistical results, there is adequate evidence to suggest that a single-dose antibiotic prescribed pre-operatively may reduce the occurrence of implant failures. Administering prophylactic antibiotics before implant surgery can provide significant benefits to patients receiving the treatment. The observed overall reduction in risk provides support for use of prophylactic antibiotics in implant dentistry.


Concrete made with sea sand and seawater is rich in chlorine ions which are the main factors that induce corrosion of the reinforcement. In this study, an innovative method to rehabilitate reinforcement is presented; the concentrations of chloride ions and the corrosion inhibitor in concrete were measured. Electrochemical chloride extraction (ECE) was applied as a control experiment via using saturated Ca(OH)2 solution as an external electrolyte. Bidirectional electromigration (BIEM)technology combined with the corrosion inhibitor could not only remove the chloride ions but also protect the steel bar in concrete, and animidazoline inhibitor mixed in concrete is more effective than thetriethylenetetramine inhibitor due to the specific molecular structure. It was found that the optimum ratio of N/Cl reached the maximum value 3.3, when the concentration of inhibitor was 1. Meanwhile, the experimental results also revealed that the corrosion inhibitor and chloride ion concentrations reached necessary levels on the surface of the steel, and the corrosion inhibitor migrated effectively. Overall, the contents of imidazoline and triethylenetetramine inhibitor in seawater concrete are0.75% and 1%, respectively. The results demonstrate that the addition of the corrosion inhibitor and the application of bidirectional electromigration would effectively improve the durability of reinforced concrete containing sea sand and seawater. The last decade has witnessed an increasing demand forsand in the construction sector. However, there exists a short fall of river sand supplies, which has triggered a search for alternative sand resources. One possible alternative is desalinated sea sand, which, compared to river sand, offers several advantages: it has a lower mud content, harder particles and better gradation. Sea sand was first used in Great Britain in concrete structures ,2. Besid2 current density, when the concentration of chlorides could reach 50%–90% on the surface of the reinforcement embedded in the concrete. Ihekwaba [Many studies have been carried out on the durability of sea sand and seawater concrete. Some of them focused on developing different types of inhibitors. Tatematsua  synthesiIhekwaba  researchIhekwaba  technoloIhekwaba  confirmeIhekwaba  researchIhekwaba ,21 was tIhekwaba . All of At present, the durability of new concrete engineering is guaranteed by adding a corrosion inhibitor into the concrete. However, a large dose of inhibitor is required, and there are some common problems, such as slow penetration, lack of penetration depth and overall difficulty in getting the corrosion inhibitor and dispersant to reach the surface of the steel . Organic3), ether , ethylenediamine , triethylamine  and 1,3-dibromopropane  were provided by chemical reagent factory , and were AR analytical reagent grade.Sulfoxide chloride , lauric acid (≥98%), NaHCO3 . Two light round steel bars (HPB235) with 10 mm diameters were embedded in the reinforced concrete specimens. The compressive strength of the concrete was C35, and the thickness of the protective layer was 40 mm, which according to the Chinese standard “Code for design of concrete structures (GB50010-2010),” and the environmental category of this study, is 3a. Ordinary Portland cement (OPC) was used with water-cement ratio of 0.48, and sea sand was used as fine aggregate. Sea sand and seawater were collected from Ningbo, China. The coordination of the reinforced concrete specimens is shown in The basic principle underlying bidirectional electromigration technology was shown in our previous research ,22. DuriDifferent amounts of imidazoline (IMDZ) and triethylenetetramine (TETA) were incorporated into concrete. The previous research found that the optimized amount of incorporated triethylenetetramine inhibitor was more than 2% of cementitious materials . The corAfter the power was turned on, the reinforced concrete specimens were taken out from device of electrochemical migration, and the concrete surface was cleaned. The specimens were then placed in a ventilated area for 72 h; after 72 h of standing, most of the residual electricity in the test specimen will dissipate, which will not affect the corrosion potential test results of the steel bars, and the performance of the concrete will also recover to more than 90%; then depolarization treatment was performed and the influence of residual electricity on the steel bars in the concrete after completion of the power supply was assessed for influence on the test results. The electrochemical refractometer 600  was used to obtain the potentiometric polarization curve to investigate the effect of triethylenetetramine inhibitors on the corrosion resistance of concrete ,26.The electrochemical workstation was used for tests. A three-electrode system was adapted during tests. The electrochemical workstation started scanning at −70 mV with respect to the open circuit potential, with a scan interval of −70 to +70 mV, which is a weak polarized zone with scan rate being 0.15 mV/s. The data were analyzed with GamryEchem Analyst , an analysis software built into the electrochemical workstation Reference 600.After the corrosion potential of the steel was tested, the concentration of residual chloride ions in the concrete after bidirectional electromigration was measured. The powder pick-up area was divided into areas I and II directly below the reinforcement. Three holes were created in each powder extraction area, as shown in We used a drill with a diameter of 12 mm to take the powder which was extracted layer-by-layer every 5.0 mm until the surface of the steel bar was reached. After the powder was collected, 2.0 g of powder was accurately weighed and mixed with 20.0 g of deionized water. After soaking for 24 h, the Chloride-Meter DY-2501 precision ion meter was used to measure the chloride ion concentrations of the aqueous solutions ,28.An additional hole was drilled in the reinforced concrete test piece. An impact drill with a diameter of 12 mm was used to collect powder at a depth of 10 mm along the direction of the protective layer. The concrete powder was oriented from the drill and passed through a sieve with a mesh size of 200. The sieved concrete powder was stored in a small sealed bag until the content of the corrosion inhibitor in the concrete being measured.Since the corrosion-inhibiting group of the corrosion inhibitor contains nitrogen, the nitrogen element content, which indicates the corrosion inhibitor content in the concrete, was detected by an organic element analyzer. The concrete powder was stored in a small sealed bag wrapped with a tin capsule or silver sac and placed in an organic element analyzer . A small amount of pure oxygen was added to help the sample fully burn. After combustion, the sample afurther catalytic oxidation-reduction, in which nitrogen was converted into detectable nitrogen. Finally, a thermal conductivity detector  was used to complete detection. The signal output by the TCD detector was directly converted into elemental content using integral calculations in a computer program.The corrosion potentials of the steel bars in the reinforced concrete specimens after bidirectional electromigration (BIEM) are shown in To analyze changes in the corrosion potentials of the steel bars, the corrosion potentials of the steel bars with different corrosion inhibitor contents are listed in As shown in As shown in The distributions of residual chloride ions in the concrete specimens after ECE and BIEM treatment are shown in As shown in The content of nitrogen in concrete powder was determined using an organic element analyzer; thus, the content of corrosion inhibitor in concrete was obtained. The content of corrosion inhibitor in concrete specimens is shown in −2, 0.012×10−2 or 0.0145×10−2 mol/g, respectively. In −2, 0.0098×10−2 or 0.0145×10−2 mol/g, respectively. The content of triethylenetetramine inhibitor in the middle of concrete specimen is higher than that of imidazoline inhibitor, which can be learned from the distribution of residual chloride ion in concrete. The reason is that the dissociation degree of triethylenetetramine is limited in high alkali environment, and amounts of non-dissociated triethylenetetramine inhibitor accumulate in the middle of concrete specimens, which also affects chloride ion migration [As shown in igration . The conIt can be seen that the content of triethylenetetramine inhibitor decreases with the depth of concrete in the outer 20 mm, while it slightly increases with the concrete depth in the 30–40 mm. As seen in In order to investigate the influence of the amount of inhibitor on the corrosion potentials of steel bars, the relationship between the corrosion potential and the amount of inhibitor is presented in It can be seen from The chloride ion concentrations on the steel surface of the concrete specimens containing different concentrations of triethylenetetramine inhibitor were analyzed and the effect of the corrosion inhibitor concentration on the chloride extraction was studied, as shown in It can be seen from − on the steel surface was studied, as shown in The respective amounts of corrosion inhibitor and chloride ions on the surfaces of the steel bars in concrete specimens were synthetically analyzed, and the effect of the corrosion inhibitor concentration on the amount of N/ClPrevious study has shown that when the concentration of corrosion inhibitor near the steel bar is low, the corrosion inhibitor is not effective for protection of concrete . For theThe presence of Cl- impedes the function of corrosion inhibitor even if the content of corrosion inhibitor is quite high. The high content of Cl-in the concrete is possibly attributable to the inhibition of the corrosion inhibitor from absorption of Cl-into concrete . Thus, t(1)The corrosion potential showed that the imidazoline and triethylenetetramine inhibitor migrated to the steel surface under the application of BIEM, and improved the corrosion potential of the steel bar, and thus, had a protective effect on the steel bar. When the concentration of imidazoline reached 0.5%, the corrosion potential of the steel bars was lower than −250 mV.(2)After BIEM treatment, the residual chloride ion concentrations in the reinforced concrete specimens decreased along the thickness of the protective layer. When the corrosion inhibitor content was high, the chloride ion concentrations in the samples containing the corrosion inhibitor were higher than those of electrochemical sample. With the addition of corrosion inhibitor, the chloride concentrations in the concrete specimen were obviously higher than those of samples with ECE treatment.(3)When the inhibitor content was low, the content of corrosion inhibitor in the reinforced concrete specimens decreased along the concrete protective layer initially and then increased. When the inhibitor content was large, the corrosion inhibitor content in the reinforced concrete specimens was protected along the concrete. The tendency of the layer direction increased first, before decreasing due to the limited dissociation of triethylenetetramine in the high-alkali environment, where some undissociated triethylenetetramine accumulated in the center of the concrete specimen.(4)This study showed the corrosion inhibitor can enhance the durability of the concrete containing sea sand and seawater. If the durability index of concrete becomes seriously degraded, it can continue to be energized to improve the durability of the concrete.In the above study, an amount of imidazoline and triethylenetetramine inhibitor were mixed into the concrete specimens which contained sea sand and seawater, and bidirectional electromigration (BIEM) technology was used. The effect of corrosion inhibitor on the durability of concrete was as follows:


Hyperhomocysteinemia (hHcy) represents a strong risk factor for atherosclerosis-associated diseases, like stroke, dementia or Alzheimer’s disease. A methionine (Met)-rich diet leads to an elevated level of homocysteine in plasma and might cause pathological alterations across the brain. The hippocampus is being constantly studied for its selective vulnerability linked with neurodegeneration. This study explores metabolic and histo-morphological changes in the rat hippocampus after global ischemia in the hHcy conditions using a combination of proton magnetic resonance spectroscopy and magnetic resonance-volumetry as well as immunohistochemical analysis. After 4 weeks of a Met-enriched diet at a dose of 2 g/kg of animal weight/day, adult male Wistar rats underwent 4-vessel occlusion lasting for 15 min, followed by a reperfusion period varying from 3 to 7 days. Histo-morphological analyses showed that the subsequent ischemia-reperfusion insult (IRI) aggravates the extent of the sole hHcy-induced degeneration of the hippocampal neurons. Decreased volume in the grey matter, extensive changes in the metabolic ratio, deeper alterations in the number and morphology of neurons, astrocytes and their processes were demonstrated in the hippocampus 7 days post-ischemia in the hHcy animals. Our results suggest that the combination of the two risk factors (hHcy and IRI) endorses and exacerbates the rat hippocampal neurodegenerative processes. Methionine (Met) is an essential amino acid present in food, which is regularly consumed within the Western diet ,2,3,4,5.Furthermore, protein intake rich in Met content or dysregulation of Met metabolism within the “Met-homocysteine” cycle can concomitantly lead to the elevation of homocysteine (Hcy) in the circulating plasma. Hcy is a sulphur-containing amino acid produced by metabolic conversion of Met to cysteine. Its elevated level in plasma, hyperhomocysteinemia (hHcy), is one of the known risk factors for cardio- and cerebrovascular disorders. Clinical relevance of elevated total Hcy level in plasma has been proven by many studies on different tissues ,15. ReceIn our previous work on the rat hHcy model induced by a Met-enriched diet , we haveOur experiment was accomplished according to Directive 2010/63/EU of the European Parliament and the Council on the protection of animals used for scientific purposes. Animals used in this experiment were handled in accordance with the directive for Animal Care and Health of the State Veterinary and Food Department of the Slovak Republic  for animal experiments. n = 38) were used in this experiment. Animals were kept under the standard conditions (air-conditioned rooms with a temperature of 22 ± 2°) and 12 h day/night cycle. Water, as well as food, were available ad libitum. Global IRI was evoked by the 4-vessel occlusion (4-VO) model in accordance with Pulsinelli et al.  were obtained in one acquisition using the chemical shift imaging (CSI) method ; 22 × 22 mm2 FOV; TR/TE = 1500/20 ms; 36 averages and 6 kHz acquisition bandwidth. Eddy currents and B0 drift compensations, as well as OVS (outer volume suppression) and VAPOR (variable pulse power and optimized relaxation delays) suppression, were initiated. Linear and second order shims were automatically adjusted with the cuboid shim volume. The average linewidth of water peak was 14.8 ± 1.9 Hz.For identification of magnetic field (B) method . The 2D 2-weighted MRI slices  software on 12 consecutive coronal TI slices . The sel1H MRS data curve fittings were performed by LCModel software . Subsequently, the metabolite levels of total N-acetyl aspartate , myo-Inositol (mIns), choline  and creatine  containing compounds were expressed as following ratios: tNAA/tCr, tCho/tCr, mIns/tCr and mIns/tNAA.During the spectroscopic data assessment, one representative voxel from hippocampus  was sele2-infinity leaving morphology unspecified. A total of 534 fields of view were analysed. The total number of Fluoro-Jade C+ (green fluorescent cytoplasm), NeuN (red fluorescent cytoplasm and processes), GFAP (green fluorescent cytoplasm and processes) was directly counted in the sections of the hippocampal CA1 area of each staining, respectively . The sampling grid size was set up to: 6 × 6 mm for each area. All calculations are expressed as the total number of labelled cells per mm2.Images of the coronal brain sections from the first experimental group were exported in tiff format. Images were evaluated using ImageJ software . Firstly, red, green and blue (RGB) channels were converted to 8 bit grey-scale images. Threshold levels were set from 13 pixels (min) to 255 pixels (max). Particle analysis was finished based upon size restrictions of 0 mmt-test.Data acquired from image analysis of brain sections were analysed using GraphPad Prism software, version 6.01 for Windows . The data were evaluated using one-way and two-way analysis of variance (ANOVA). Statistical analysis for spectroscopic and MRI volumetric data was accomplished using the SPSS software package . The differences in hippocampal volumes and metabolite ratios between animal groups  were analysed by independent-samples 2-tailed n = 5), and reached 11.22 ± 3.86 μmol/l (n = 5). Analysis of plasma level of Hcy in animals has shown that total plasma Hcy level in animals with 28 days of Met diet (Met-C) was significantly higher when compared to the naïve male control Wistar rats , in comparison to both controls (C and Met-C). Interestingly, in the same group with the Met diet (Met-IR-3d), the differences reached 58.33-fold increase , when compared to the naïve ischemized group. To show the extent of neurodegeneration, FJC was used for detection of disintegrated neurons in the tissue slices of the CA1 area of the rat hippocampus. As expected in both control groups, we could not detect any FJC positive neurons . Unsurprp < 0.001), resp. 21-fold (p < 0.001) compared to both control groups (naïve and Met treated) and differences between naïve ischemized and Met ischemized groups represented 36.4% (p < 0.01). Remarkably, longer reperfusion time led to an apparent reduction of the cellular FJC positivity representing 32.3% (p < 0.001), resp. 44.6% (p < 0.001), when compared to the naïve IR-7d and IR-3d groups and Met-IR-3d group, probably as a result of an extensive tissue breakdown. This can be seen by the morphologically changed neurons (arrowhead), as well as scarred, unstained neuronal tissue which substitutes neuronal loss (asterisk), likely as a result of prolonged ischemia and neuronal tissue remodelling (In the prolonged reperfusion period (7 days) in both groups of animals (naïve and Met diet treated), we documented persisted increase of FJC+ neurons , NeuN antibody labelled nuclei as well as the cytoplasm of the majority of neuronal cell types of all regions in the adult brain . No immunoreactivity was observed in the astrocytes, neither in the nuclei nor the cytoplasm. The preponderance of the cytoplasmic immunopositivity was concentrated in the soma, extending to a short distance into the processes  decrease in the number of NeuN+ neurons in comparison to the naïve control. In the same group with the Met diet, we found a statistically significant decrease of NeuN+ neurons in comparison to the naive controls to 44.9% (p < 0.001) as well as to 51.4% (p < 0.001) compared to the Met controls (In the naïve ischemized group with 3 reperfusion days (IR-3d), we detected 36.7% (controls . Additiop < 0.001) in the naïve ischemized group, as well as in the ischemized Met group up to 49.2% (p < 0.001), when compared to the naïve controls and 43.1% versus Met controls (p < 0.001). In addition, Met diet induced diminishing in the number of maturate neurons between naïve and Met group up to 31.7% , in comparison to the naïve control (Immunolabelling of GFAP in the naïve controls (C) presented astrocytes with radially arranged thin processes. Astrocytes were dispersed in all layers of the hippocampus —arrow. I control . p < 0.01) when compared to the naïve controls. However, in the same Met ischemized group, surprisingly, we found a diminished number of astrocytes (p < 0.01) versus naïve control, and 34.4% (p < 0.001) versus Met-C . In addition, an extensive elevation was detected when compared to the 3 days reperfusion group, up to 28.9%  showed a significant decrease in tNAA/tCr ratio, and a significant increase in mIns/Cr when compared to the non-ischemized (C and Met-C) groups (Quantification of ) groups . However) groups .p = 0.031). The hippocampal volume was significantly increased in Met-IR-3d and significantly decreased in Met-IR-7d group, compared to the naïve control (C) as well as to the Met-C groups (p < 0.001) when compared to the 3rd post-ischemic day. With respect to the naïve control volume threshold level, we found an increased level in Met-C (10 ± 2%) and in the Met-IR-3d (23 ± 6%) animal groups, however, in the Met-IR-7d group (−6 ± 2%) was confirmed hippocampal volume decrement  or dysregulation in neurogenesis, as a reaction to the mild hHcy conditions . Based o1H MRS and MRI-volumetry analysis were applied as a non-invasive approach to determine the metabolic ratio and alternations in the hippocampal volume in ischemized animals with the Met diet. In vivo As generally accepted, tCr emerges as a constituent of all brain cells. tNAA is considered to be the characteristic neuroaxonal marker, tCho and mIns are regarded as the glial components, e.i. tCho (mainly for oligodendrocytes) and mIns for astrocytes ,52. 1H MRS revealed changes in all selected metabolites in the hippocampal area  can impair cardiac contractility, alter substrate metabolism, and cause cardiac remodelling and dysfunction . AnotherBased on these results, we suggest that Met-enriched diet inducing a mild form of hHcy in combination with IRI in rats represents an experimental model, which can evolve a toxic body environment with potentially detrimental impact to the neuronal tissue, seen on the morphological and metabolic levels. This is presented by the changed metabolic ratio, volume disturbances, attenuated neurites and activation of astrocytes in the rat hippocampus. In conclusion, our study utilizes a novel approach which combines histological analyses with the non-invasive in vivo 1H MRS, MRI methodology, with the aim to reveal the potential histo-morphological as well as the metabolic hippocampal dysregulation in rats with Met diet subjected to IRI. Both methods showed that hippocampi of ischemized animals with Met diet manifested exaggerated neurodegeneration, mainly in the selectively vulnerable CA 1 hippocampal region. Evidence of a possible causal link between the ischemic insult with hHcy induced by the Met diet and incipient neurodegenerative processes would contribute to better understanding of the potentially deleterious impact of both modifiable risk factors (hHcy and IRI) in clinically relevant neurological disorders. 1H MRI and histology Furthermore, there are different areas in the hippocampus with different vulnerability to acute or chronic insults that could influence the overall metabolic ratio. Our results support these findings and the need for a multiparametric MRI approach, in combination with histologic analysis to characterize the complex evolution of neurodegenerative diseases [The limitation of this study was in the measurement of metabolites because their level could fluctuate in chronic conditions due to the occurrence of cytotoxic oedema (tissue water), which could result in a lack of correlation between diseases .


However, information on the effects of harvest aids on seed nutrition (composition)  in soybean is very limited. The objective of this research was to investigate the influence of harvest aids on seed protein, oil, fatty acids, sugars, and amino acids in soybean. Our hypothesis was that harvest aid may influence seed nutrition, especially at R6 as at R6 the seeds may still undergo biochemical changes. Field experiments were conducted in 2012 and 2013 under Midsouth USA environmental conditions in which harvest aids were applied at R6 (seed-fill) and R7 (yellow pods) growth stages. Harvest aids applied included an untreated control, 0.28 kg ai ha−1 of paraquat, 0.28 kg ai ha−1 of paraquat, and 1.015 kg ai ha−1 of carfentrazone-ethyl (AIM); 6.72 kg ai ha−1 sodium chlorate, 1.015 kg ai ha−1 carfentrazone-ethyl; and 2.0 kg ae ha−1 glyphosate. Results showed that the application of harvest aids at either R6 or R7 resulted in the alteration of some seed composition such as protein, oil, oleic acid, fructose, and little effects on amino acids. In addition, harvest aids affected seed composition constituents differently depending on year and growth stage. This research demonstrated the possible alteration of some nutrients by harvest aids. This research helps growers and scientists to advance the understanding and management of harvest aids and investigate possible effects of harvest aids on seed nutrition.Interest in using harvest aids (defoliants or crop desiccants) such as paraquat, carfentrazone-ethyl, glyphosate, and sodium chlorate (NaClO The use of the early-maturing soybean cultivars in the Early Soybean Production System (ESPS) has become popular throughout the Midsouth USA ,2. Growi-1 rate to soybean near seed maturity had significant effects on germination and/or growth of the resulting F1 generation [−1, glyphosate at 1.7 and 3.4 kg ha−1, and ametryn at 1.1 and 2.2 kg ha−1, 3 to 4 weeks before harvest, reduced yield compared to when applications were made 2 weeks before harvest [Previous research on harvest aids reported that the application of glyphosate at five rates, from 0.56 to 3.36 kg, 3 weeks before soybean harvest, resulted in decreased soybean yield , and appneration . Other hneration . It was  harvest ,18,19,20 harvest  found thAlthough information on the effect of harvest aids on seed composition is very limited, available research showed that harvest aids, for example, paraquat, glyphosate, and ametryn, resulted in a decrease of seed oil content when harvest aids were applied 4 and 3 weeks before harvest date . HoweverANOVA of seed composition constituents showed that, generally, Year, Growth Stage (Stage), and treatments (Treat) were the main factors influencing some seed composition constituents, including seed protein, oil, and some fatty acids, sugars, and amino acids .F values, these three factors are more important than their interactions (Year × Stage × Treat). Therefore, since the F value for Year × Stage × Treat interactions was smaller  compared with that of the effect of Year or Stage or Treat alone, the data were combined across the two years. The most affected compounds were protein, oil, oleic acid, and fructose. Amino acids were affected by Year, Stage, and their interactions: for example, the significant effects of Year on asparagine (ASP), glycine (GLY), valine (VAL), and tryptophan (TRY); the effects of Stage on isoleucine (ISO-LEU) and lycine (LYS); the effects of Year, Year × Stage on threonine (THR), proline (PRO), and serine (SER); and the effects of Stage, Year × Treat × Stage on leucine (LEU)  . ANOVA fne (LEU) . For exa3 was applied, but all other harvest aids were similar to the non-treated  at 2.15 kg ai ha−1, followed by a cultivation prior to the growth stage R1 that also cleared the furrows to facilitate irrigation. Furrow irrigation was applied at a rate of 25.0 mm ha−1 on 22 May, 25 June, and 30 July 2012. In 2013, the same rate of irrigation was applied 19 June, 25 June, 1 July, 8 July, 18 July, and 7 August. Irrigation was applied as needed based on the recommendation of the research team.An experiment was conducted in 2012 and 2013 on Dundee silty clay  located 2 km north of Elizabeth, MS, on property leased by the USDA-ARS Crop Production Systems Research Unit at Stoneville, MS. Field preparation began for each growing season in the previous autumn by disking the field level and then forming 40 cm high ridges spaced 102 cm apart. Just prior to planting, the ridges were harrowed to form a 40 cm seedbed. The cultivars used in this study were Asgrow AG4303  in 2012 and Pioneer P94Y23  in 2013. Soybean cultivars were planted in twin rows spaced 25 cm apart and centered 102 cm between rows. Each plot was eight rows in width with each row being 12 m long. Planting dates for the experiment were 23 April 2012 and 18 April 2013, with a planting rate of 30 seed m3) , and glyphosate (N-(phosphonomethyl) glycine) . Though glyphosate would not be a harvest aid on glyphosate-resistant soybean cultivars as were used in this experiment, it may be used by some growers to kill late-emerging weeds that would interfere with harvest. Treatments applied to sub-plots were: an untreated control, 0.28 kg ai (active ingredient) ha−1 of paraquat, 0.28 kg ai ha−1 of paraquat + 1.015 kg ai ha−1 of carfentrazone-ethyl, 6.72 kg ai ha−1 sodium chlorate, 0.015 kg ai ha−1 carfentrazone-ethyl, and 2.0 kg ae  ha−1 glyphosate. The application rate of harvest aids was used based on the label recommendations. These treatments were randomized within each whole plot and applied only to the center four rows. Plots were machine-harvested with a Kincaid 8X-P combine equipped with a Juniper HarvestMaster weight system for yield determination approximately 21 days after the final treatment application. Mature seed samples from each sub-plot were collected and used for seed composition analysis.The harvest aids used were: paraquat  , carfentrazone-ethyl , sodium chlorate  contents in mature seeds from each plot were analyzed with a Diode Array Feed Analyzer AD 7200 . Briefly, seeds were ground by a Laboratory Mill 3600  and approximately 25 g of seed were analyzed for protein, oil, and fatty acid contents according to ,37,38,39Harvested mature seeds from each plot were analyzed for the amino acids alanine (ALN), cysteine (CYS), valine (VAL), methionine (MET), isoleucine (ISO-LEU), leucine (LEU), tyrosine (TYR), phenylalanine (PHE), lysine (LYS), histidine (HIS), arginine (ARG), tryptophan (TRY), asparagine (ASP), threonine (THR), serine (SER), glutamine (GLU), proline (PRO), and glycine (GLY). The analysis was conducted by a near-infrared (NIR) reflectance diode array feed analyzer  as described by ,43,44,45p ≤ 0.05 was used in SAS  and analyzed according to [The experimental design was a split-plot replicated four times with two harvest aid applications at Growth Stages R6 and R7. The main plot was Growth Stage, and harvest aid treatment was sub-plot. Statistical analyses to evaluate the effect of Year, Growth Stage, and Treatment, and their interactions, were conducted using PROC MIXED  . Replicarding to ,51.The use of harvest aids has become an agricultural practice to improve seed quality and harvest efficiency and allows the harvest of soybean 7 to 10 days earlier when compared to non-treated soybean. However, producers must be careful to not make harvest aid applications too soon, which can result in a reduction in yield  and seed


Immunoglobulin G4 (IgG4)-related disease is an increasingly recognized immune-mediated entity that can affect virtually every organ system. Depending on the location of the disease, it can present a wide range of clinical manifestations and even mimic malignancies. Appendiceal involvement in patients with IgG4-related disease is particularly rare and very few cases are reported in the literature.We report a case of IgG4-related appendiceal disease in a 42-year-old woman who presents with a subacute onset of right lower quadrant abdominal pain.Abdominal computed tomography showed a markedly enlarged appendix, raising the concern of malignancy. The diagnosis of IgG4 appendiceal disease was confirmed by postoperative histopathologic and immunohistochemical examination.The patient underwent right hemicolectomy.After the surgery, the patient had an uneventful recovery and reported a resolution of her symptoms. The serum IgG4 was revaluated 5 days after surgery and returned to its normal values. At the 3-year follow up, the patient had no recurrence of symptoms and her imaging exams remain unremarkable.This study reports the fifth case of IgG4-related appendiceal disease. Increasing awareness of this condition may influence the management of these patients, once patients with IgG4-related disease should be monitored after treatment, due to the risk of recurrence or involvement of other organs. Histologically, these lesions consist of infiltration of lymphocytes and IgG4-positive plasma cells and fibrosis. IgG4-related appendiceal disease is extremely unusual. To the best of our knowledge, there are only 4 cases that describe appendiceal disease associated with increased IgG4 plasma cells–8 and there is only one that fulfills all pathological diagnostic criteria.Immunoglobulin G4 (IgG4)-related disease is an increasingly recognized immune-mediated entity that includes a group of conditions with specific clinical, serologic, and pathologic features, which is characterized by a significant steroid responsiveness.The aim of this study is to describe the fifth case of IgG4-related appendiceal disease, but the second one that fully satisfied all the pathological criteria, and review the literature. The patient has provided informed consent for publication of the case.2A previously healthy 42-year-old woman was admitted in the emergency department with a 1-week history of right lower quadrant abdominal pain. Physical examination revealed a painful mass in the right lower quadrant with no rebound or guarding, normal bowel sounds, and no hepatosplenomegaly. Physical examination was otherwise normal, and the initial laboratory tests were normal.Abdominal computed tomography (CT) scan with intravenous iodinated contrast media was requested to evaluate his symptoms. CT scan showed a marked enlargement, thickening, and mucosal hyperenhancement of the appendix Fig. . There wThe patient underwent an open right hemicolectomy Fig. A. A frozImmunostaining was performed according to standard protocols using avidin–biotin complex labeled with peroxidase. Appropriate positive and negative controls were run concurrently for all the markers tested. The inflammatory cells were composed of mixed reactive population of T and B cells with plasmacytosis. There were mostly CD3-positive lymphocytes, whereas CD8 and CD20-positive lymphocytes were less numerous. Immunostaining for IgG and IgG4  is a newly recognized immune-mediated disorder that can affect various organs and usually presents as tumefactive lesions that mimics malignant conditions.–14 Pancreas was the first and is the most frequent digestive organ to be involved in IgG4-RD followed by bile ducts. IgG4-related lesions of the small bowel and colon are extremely rare. Only 2 cases of solitary IgG4-RD at the cecum and sigmoid colon have been reported, and only 1 case at the ileocecal region mimicking malignancy has been reported. The involvement of the appendix is also rare.–8 Patients with IgG4-RD are at risk to have recurrent disease or involvement of other organs over time; furthermore, lymphomas can occur.,4,8 Considering that these patients should be followed up at regular visits.Gastrointestinal IgG4-related diseases are difficult to be diagnosed by clinical and radiological findings alone, owing to its nonspecific presentation. Most of reported cases were diagnosed only after surgical resection. The cutoff threshold appears to vary by organ due to the nature of fibrosis and limited published data. The International Committee has proposed organ-specific cutoff, which ranges from >10 per high—power field to >200 per high-power field. However, it is important to highlight that appendiceal involvement has not been included in the Consensus since there were no published case reports at that time. Therefore, specific cutoff value was not described for this organ. The Committee also considered IgG4/IgG plasma cell ratio a more powerful tool than the IgG4+ plasma cell counts in establishing the diagnosis of IgG4-related disease. An IgG4/IgG ratio >40% is established as a comprehensive cutoff value in any organ. Other minor features such as eosinophilia, phlebitis without obliteration, increased serum IgG4, and response to steroids are also described. Prominent neutrophilic infiltration, granuloma, and necrosis are features that would exclude the diagnosis of IgG4-RD and increase the suspicion for infections, sarcoidosis, or granulomatous diseases.According to the Consensus created by the International Committee on IgG4-related disease held in 2011, the 3 major histopathological features associated with this condition are dense lymphoplasmacytic infiltrate, storiform fibrosis, which is characterized by an irregularly cartwheel-like fibrotic pattern, and obliterative phlebitis, in combination with increased IgG4+ plasma cells infiltrating the tissue.–8 The average age of patients was 43 years, ranging from 20 to 57 years, and the majority was woman . All patients presented with right lower quadrant abdominal pain, 4 of them (80%) with an acute or subacute onset and 1 (20%) with a chronic presentation (4–5 months). The last patient had also weight loss complaint. Regarding imaging findings, CT showed appendiceal mass-forming lesions in 2 cases,,8 and appendiceal enlargement in one of the previous reported cases. One study did not describe if preoperative radiological study was performed. All studies that described the imaging features on CT demonstrated no extra-appendiceal disease. Finally, only 1 case demonstrated slight periappendiceal inflammatory changes and also only 1 patient showed distension of the appendiceal lumen.Table  reported the patient follow-up, who was also free of disease and had good recovery from symptoms 3 months after surgery.All patients underwent surgical treatment, most of them with right hemicolectomy , due to the possibility of malignancy, and one of them with appendectomy, since the presumptive diagnosis was acute appendicitis. The prognosis of this entity is not described in the literature due to its rarity. Apart from our study, only Veerankutty et al reported a patient who satisfies the full spectrum of histological criteria recommended by the International Committee. We also report the first case with pre- and postoperative serum IgG4 level, demonstrating the reduction of its level after surgery. It‘s important to highlight that the pretreatment elevation of this serum immunoglobulin is not a sensitive criterion for the diagnosis of IgG4-RD, which can be normal in up to 40% of patients. Moreover, there is no evidence that the decrease of this serological marker in the postsurgical setting is a reliable criterion for the diagnosis of this disease. Therefore, the serum IgG4 level is not considered a major criterion for this condition . However, according to the Consensus, elevated serum IgG4 level (> 135 mg/dL) can be considered an additional criterion to suggest this diagnosis in patients with “Probable Histological Features of IgG4-Related Disease” . Our case fulfilled all 3 histopathological features as well as the immunohistochemical criteria (IgG4/IgG ratio > 40% in the tissue), thus the serum IgG4 concentration was not completely necessary in this case, but it helps to reinforce this diagnosis.Besides our study, only Kwon et alThe main differential diagnosis of IgG4-related appendiceal disease on imaging is acute appendicitis and malignancies, including adenocarcinoma and neuroendocrine tumors. Although rare, IgG4-related appendiceal disease should be suspected when appendiceal involvement is disproportionately more intense than the periappendiceal inflammatory changes, which can be slight or even absent. Furthermore, when other findings of IgG4-RD are present, such as involvement of the pancreas, biliary tree, kidney, and/or retroperitoneum, the diagnosis appendiceal involvement should be considered. In these situations, a therapeutic test with steroids may be considered. However, the mass-like form of the disease is challenging to be diagnosed based solely on imaging features and a definitive diagnosis may require histopathology.Recognition of this entity has an important clinical implication, considering that a steroid test can be performed before surgery in order to avoid an unnecessary surgical procedure. Increasing awareness of this condition may also influence the management of these patients, once patients with IgG4-related disease should be monitored after treatment, due to the risk of recurrence or involvement of other organs.Adriano Basso Dias: Manuscript writing.Natally Horvat: Study concept and manuscript writing.Maria Dirlei Begnami: Major role in the acquisition of data.Emerson Shigueaki Abe: Critical revision of the manuscript for important intellectual content.Publio Cesar Cavalcante Viana: Critical revision of the manuscript for important intellectual content.Marcel Cerqueira Cesar Machado: Study concept, critical revision of the manuscript for important intellectual content.


The use of mineral additives from the power and metallurgy industries in the production of building materials still raises questions about the ecological safety of such materials. These questions are particularly associated with the release of heavy metals. The article presents research related to the leaching of chromium from concretes made of Portland cement CEM I and slag cement CEM III/B (containing 75% of granulated blast furnace slag). Concrete was evaluated for leaching mechanisms that may appear during tank test over the long term (64 days). It has been presented that the dominating process associated with the leaching of chromium from both types of concrete is surface wash-off. Between the 9th and 64th day of the test, leaching of Portland cement concrete can be diffusion controlled. It has been proven that the participation of slag in the composition of concrete does not affect the level of leaching of chromium into the environment from concrete. In the European cement industry, waste materials are used in the production of Portland clinker and cement. At present, alternative fuels  are used on a large scale as partial substitutes for natural fuels, and waste from other industries as an alternative to raw materials naturally extracted from the environment. In addition, cement plants significantly reduce the production and use of Portland clinker (the most expensive component of cement) by introducing so-called mineral additives into the composition of cement as main components. Among the most commonly used are wastes from the power industry and metallurgy, such as silica fly ash, granulated blast furnace slag and silica dust ,2,3. They enable the adjustment of concrete properties so that this composite can meet increasingly difficult requirements in applications such as communication and underground infrastructure; engineering and mining works; and environmental protection facilities ,4.The use of waste in the cement industry and construction has therefore become an irreversible process, consistent with the idea of sustainable development. Over time, this solution has raised questions not only about the quality of the end products, but also about their impact on the environment. In many countries, various organizations have begun establishing a system for the environmental assessment of materials used in construction, which is to take particular account of the degree of release of heavy metals into the environment. The process of performing an environmental assessment on a material (concrete) is extremely difficult and complex. It requires consideration of factors related to the internal structure of the composite and external factors affecting concrete objects and structures in their working environment , which may affect the release of hazardous substances. Attention should be paid to the level of release of heavy metals from concrete (concrete objects and structures), depending on the application conditions, considering the “life cycle” of the composite ,6.An important aspect, from the scientific point of view, is to know the processes that accompanies the release of heavy metals from building materials, especially during exposure to external factors . The materials may then release additional portions of heavy metals or lose their functional properties  .From a point of view of the author, it is wise to control the content of heavy metals at every “stage of life” of the mineral material, from production to application in the environment. Such a modern approach will protect the environment against the uncontrolled spread of metals. Only knowledge of the processes affecting the level of leaching can strengthen this control and predict the development of the situation under certain concrete application scenarios. A modern approach is needed to show the mechanisms that accompany release from these structures. The comprehensive leaching assessment system will give information on how concrete will behave over a long period of time in a given application environment and whether it will affect both its structure and the leaching of heavy metal. Only such a modern approach will give full control and environmental protection in the future. There are not many publications worldwide that would present this approach. Such publications most often concern only Portland cement.2 impact), structural, physical and chemical changes may occur in the concrete matrix, which contributes to the release of larger portions of heavy metals. Available literature data show very few sources that would describe the relationship of leaching and the mechanisms associated with it. The author has therefore attempted to determine the mechanisms that accompany the long-term leaching from concrete. The author’s research can be used to predict long-term heavy metal leaching. Such research also would allow one to avoid the destruction of concrete during the leaching process. The author of paper would like to emphasize the importance of research of heavy metal leaching from mineral matrices using diffusion tests; e.g., a tank test. Such tests allow the evaluation of the release in aqueous conditions of the long residence time of the sample. Construction materials are considered neutral to the environment. However, this external environment  is not without influence on concrete. During long-term exposure of concrete in water or an environment with reduced pH or under the influence of carbonation . According to Glasser [3A·CaCrO4·12H2O). Replacement of SO42− by CrO42− is possible, but for a high degree of chromium oxidation. Therefore, immobilization of chromium is difficult and usually does not exceed 80%. Glasser [It is also known that hexavalent chromium is permanently bonded by substituting the sulphate group in ettringite structures , on the other hand, is ingrained into the structures of phase C-S-H , where substitution takes place ,12,182CrO4 in amounts ranging from 0.5% to 5% in relation to the weight of Portland cement in binders and observed the delay of setting time of cement paste hydration along with the increase of potassium chromate additive. Tamás et al. [The effect of this reaction is to stabilize the disturbed structure of the C-S-H phase and to inhibit its transition into crystalline phases . The ress et al.  demonstrs et al. . Namely,On the basis of the works ,24,25, iExternal factors include, among others, application scenario, liquid to solid ratio (L/S), time of contact with the leaching medium, ambient pH, temperature and mechanical influences .The internal factors characterizing the tested structural material include: the porosity, thermal conductivity, shape, specific surface area, size and reactivity of the material , and its age .Much attention is paid in literature to the influence of pH on the release of heavy metals ,24,25,26As a result of the decreasing pH of cement mortar , the solubilities of heavy metals change. They usually form slightly soluble compounds in a strongly alkaline environment, whereas at a lower pH they show an increased solubility. Amphoteric metals  have the lowest solubility at pH between 8 and 10 ,28. StudThe results of van der Sloot’s works  also indThe author conducted research on the leaching of chromium from concrete on Portland cement CEM I 32,5R (abbreviation: CEM I) and on slag cement CEM III/B 32,5N-LH-HSR/NA (abbreviation: CEM III/B). The content of granulated blast furnace slag in slag cement was 75%. 3; sand—685.2 kg/m3; gravel 2–8 mm—600.4 kg/m3; gravel 8–16 mm—628.6 kg/m3; water—180.0 kg/m3; water/cement ratio (w/c)—0.6. The gravels we used were natural aggregates with a density 2.63 kg/dm3.In the research, concrete was designed and made using both cements. The composition of the concrete mix was as follows: cement—300.0 kg/mCubes measuring 10 cm × 10 cm × 10 cm were formed from the obtained concrete mixes. After 24 h the concrete cubes were subjected to leaching tests taking into account the form of the material , time (up to 64 days of the test) and the impact of water.The assessment of inorganic component leaching from mineral materials in the monolithic form involves tank tests, often referred to as diffusion tests. The Netherlands Standardization Institute (NEN), European Committee for Standardization (CEN) and Environmental Protection Agency (EPA) have created a leaching test for different types of materials . The most common test is based on the EA NEN 7375 standard . The testotal in eluates were determined with an inductively-coupled plasma-mass spectrometer (ICP MS) by Perkin Elmer. Each measurement was carried out with three repetitions holding relative standard deviation (RSD) <5%. The test was performed at the temperature range 19–21 °C. The eluates used in the test were extracted according to the following time schedule: after 0.25, 1, 2.25, 4, 9, 16, 36 and 64 days. After each extraction of the eluate, the liquid was completely replaced and the samples were immersed in the liquid again. The eluates were filtered through the membrane filters with the pore size of 0.45 μm. The concentrations of CrIn each of the eight eluates, the concentration of chromium was analyzed.In the course of the study, the level of leaching per eluate fraction, total leachability per unit of surface area and the emerging leaching processes were determined according to . i (mg/m2);i (μg/dm3);V—volume of eluate (dm3);2); A—sample surface area (mf—factor: 1000 (μg/mg).In the course of calculations for the analyzed heavy metal, the leachability in particular fractions was determined using the Equation :(1)Ei*=cn comprising fraction i = 1 to n, in mg/m2 of sample surface area; i, in mg/m2; and N is the total number of leachant replenishment periods.lculated  accordinn comprising fraction i = 1 to n, in mg/m2 of sample surface area; i, in mg/m2; it is the replenishment time of fraction i, in s; and ti−1 is the replenishment time of fraction i−1, in s.Derived cumulative leaching  formula :(3)εn=Ei—Surface wash-off (SWO);—Depletion (DEP);—Dissolution (DIS);—Delayed diffusion or dissolution (DDD).Based on the above calculations, it can be determined which mechanisms accompany heavy metal leaching; i.e., whether each is dominated by diffusion (DIF) or other mechanisms, such as:it for n = 1 to N should be plotted in order to visually assess the measurements obtained. In the same graph, the logarithm of the calculated total leaching For such an analysis, it is recommended  that theThe eluate fractions obtained and tested during periods 1–8 should be divided into increments long enough to recognize the mechanisms involved in release of heavy metals. Eluate fractions collected in periods 1–8 should be grouped into the ranges like shown in a−bCF concentration factor (Equation (4)), slope (rc) of linear regression line of log ε versus log t and the standard deviation (sdrc) shall be determined for a given heavy metal under assessment and for each of the ranges identified.The On the basis of the slope of the regression function in individual increments , it can Graphical representations of the results obtained for chromium are shown in On the basis of the above analyses, it can be concluded that the leachability of chromium from concrete with Portland cement and with slag cement is not controlled during the entire test cycle by one leaching mechanism. However, it can be claimed that the release of chromium in both the longest (increments 2–7) and the shortest and earliest test period (increment 1–4) occurs by surface wash-off (SWO). The tests also showed that the matrix with CEM I does not dissolve, which means that the matrix did not deteriorate during long (64 days) chromium leaching tests. In later test periods (between the 9th and 64th day of the test) the leaching of chromium from concrete may be accompanied by diffusion processes.Dissolution occurs in the case of concrete with CEM III/B (increment 5−8), which may indicate the destruction of the concrete during long-term exposure to water. However, during this time there are no increased doses of released chromium per area unit, compared to concrete with Portland cement . StudiesThe use of alternative fuels and by-products from the power and metallurgy industries as valuable components of clinkers, cements and mineral composites has become in the last twenty years a fully intentional activity of the cement and construction industry, consistent with the idea of sustainable development. This has led to the development of a wide range of industrial by-products that are able to meet the challenges of modern construction. On the other hand, however, the system of environmental assessments has become extremely topical, which should cover the difficult subject of releasing heavy metals from mineral composites into the environment through research procedures. Concrete is commonly present in the environments surrounding people. Therefore, it must not affect the quality of the environment with which it is in contact, and particularly, it must not impair human health.The paper presents considerations on the mechanisms accompanying the leaching of heavy metals from building materials, focusing on the release of chromium from concrete produced with Portland cement and slag cement. The results of chromium leaching from concrete seasoned up to 64 days were obtained. Processes accompanying the release of chromium from concrete matrices were evaluated. It was found that the dominant process which controls the leaching of chromium from the analyzed concretes is surface wash-off. It has also been proven that the use of large amounts of granulated blast furnace slag in the cement composition does not release additional portions of chromium from concretes in long contact with water. Concrete with slag cement can be influenced by dissolution. In practice, therefore, more tight concrete of this type should be used , which would allow obtaining a compact structure with a lower capillary content. This can protect slag concrete from dissolving during long term contact with water. As a result, this will contribute to not releasing further portions of chromium into the environment.There is not much research in the literature on the mechanisms controlling the leaching process; hence, the author took up this subject. However, this issue should be further developed considering other concrete exposure conditions and other kinds of concrete. If such complex tests are carried out, it will be possible to plot the matrices of the use of individual concretes in various application scenarios, so additional heavy metal concentrations are not released.


Diabetes mellitus (DM) has become a major health problem in most countries of the world. DM causes many complications, including hyperglycemia, diabetic ketoacidosis, and death. In Asia, mulberry has been used widely in the treatment of DM. Combination of drugs with herbal medicine may reduce the unwanted side effects caused by drugs. In this study, the influence of extended mulberry leaves extract (MLE) intake on metformin (Met) was evaluated in terms of pharmacokinetics and pharmacodynamics in DM-induced rats. Three week-treatment of MLE alone produced the anti-hyperglycemic effect (around 24%) if compared to the control. Interestingly, Met administration after MLE treatment for 3 weeks enhanced about 49% of the anti-hyperglycemic effect of Met. In addition, the extended intake of MLE potentiated the anti-hyperglycemic effect of Met on various concentrations. This potentiated anti-hyperglycemic effect of Met appears to be due to the pharmacokinetic change of Met. In this study, 3 week-treatment of MLE reduced the elimination of Met in DM-induced rats. In addition, MLE reduced the human organic cation transporter 2 (hOCT2) activity in a concentration-dependent manner. Thus, these findings suggest that MLE lowered the elimination of Met via inhibiting the hOCT2. Diabetes mellitus (DM) is a major health problem in most countries of the world. According to the World Health Organization (WHO) , 422 milFive classes of anti-hyperglycemic drugs—including sulfonylurea, biguanides, α-glucosidase inhibitor, meglitinides, and thiazolidinediones—are available for the treatment of DM . MetformMorus alba L., is the common deciduous tree in Asia. Its products, such as fruits, leaves, and roots have been used traditionally as an anti-hyperglycemic folk remedy in the Asian country. Anti-hyperglycemic effects of mulberry have been frequently reported in literatures [Plants have been used as a therapeutic purpose in many Asian countries, and many drugs are derived from plants. Mulberry, eratures ,7,8,9,10eratures ,15,16,17Hypoxis hemerocallidea and l-canavanine inhibited the P-glycoprotein (P-gp) efflux of nevirapine in the Caco-2 cell [Houttuynia cordata ethanol extract reduced renal excretion of Met via decrease of organic cation transporters (OCT) uptake of Met [Morus alba L. extract intake on Met in terms of pharmacokinetics and pharmacodynamics in DM-induced rats.Combinations of drugs with herbal medicine may reduce the unwanted side effects caused by drugs. For instance, cinnamic acid derivatives when combined with Met reduced the expression of fatty acid synthase and β-Hydroxy β-methylglutaryl-CoA (HMG CoA) reductase involved in the secondary complications of DM ,19. Howeo-2 cell . Capsaico-2 cell ,26. Whilo-2 cell ,28. Moree of Met . Thus, bMorus alba L. was added into purified water and then extracted under the pressure at 121 °C for 3 h. These extracts were fermented by adding 1% (w/v) enzyme (Viscozyme®) under pH 5.0 at 45 °C for 15 h. MLE was dried using a freeze-dryer [Streptozotocin  and phenformin  were purchased from Sigma-Aldrich . Met (purity ≥ 98%) was a gift from the Korea United Pharm Inc. . High-performance liquid chromatography (HPLC) grade acetonitrile (ACN) and methanol (MeOH) were purchased from JT baker . All the other agents were of analytical grade and were obtained by commercial sources. The mulberry leaf extracts (MLE) were obtained from the MSC . Briefly, ze-dryer . MLE useMale Sprague–Dawley rats were used in this study. Rats aged 7 weeks were obtained by Samtako , and they were housed at 22 °C with a 12-h light–dark cycle and given free access to food and water. Before the experiments, animals were acclimated for 1 week. All experiments were performed according to the guidelines established by the Chungnam National University Institutional Animal Care and Use Committee. This study was approved by the Local Ethical Committee of Chungnam National University .® Active, Hoffmann-La Roche, Basel, Switzerland). Glucose level was measured at 0, 1, and 5 days post-injection of STZ. Fasting glucose level above 200 mg/dL was set as the criteria of DM.DM was induced by an injection of STZ. Animals were fasted overnight and then STZ was injected via tail vein at a dose of 45 mg/kg. To overcome the acute hypoglycemia, 5% glucose solution was provided a day. Animals were fasted at least 8 h before collecting whole blood via jugular vein, and then fasting blood glucose level was measured using a blood glucose meter . AUG was calculated by a trapezoidal rule.t-test.To evaluate the influence of repeated administrations of MLE on the anti-hyperglycemic effect of various doses of Met, the MLE was orally administered for 21 days and then the increasing doses of Met were orally administered on day 22. Briefly, animals orally received saline or MLE (600 mg/kg) of the same volume for 21 days. On day 22, animals received the increasing doses of Met . Fasting glucose level was measured at 0, 2, 4, and 12 h post-administration of Met. AUG was calculated as described in g for 10 min and the plasma was stored at −70 °C. The plasma samples were analyzed by HPLC with a UV detector. PK parameters for oral administration were determined as a non-compartmental model using WinNonlin 5.3.1 software .Pharmacokinetic interaction between herb and drug has been reported in many studies. To evaluate the interaction between the MLE and Met, pharmacokinetic analysis of Met was assessed after the repeated administrations of MLE or saline. Briefly, MLE  or saline was orally administered to animals for 21 days, then the animal received the Met per oral at a dose of 50 mg/kg. After the administration of Met, the blood samples were taken via jugular vein at 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h. Blood samples were centrifuged at 1000× To evaluate the interfering effect of MLE presence to Met analysis in rat plasma, the Met-spiked samples with the MLE (10 µg/mL) in rat plasma were used and the validation parameters such as accuracy and precision were evaluated in the presence or absence of MLE in rat plasma.For the calibration curve, the peak area ratios of Met  to IS were plotted against corresponding nominal concentrations of Met. The linearity of the regression curve in the range 0.4–10 µg/mL was assessed on the base of the residual plot. Concentrations of the samples were interpolated from the regression curve. The correlation coefficient of the calibration curve was >0.99.Inter- and intra-day reproducibility was measured to assess accuracy and precision using quality control (QC) samples . The relative error (RE) and relative standard deviation (RSD) were used to evaluate the accuracy and precision of the bioanalytical method. The ±15% fixed range applied as an acceptance of criteria.In addition, to assess the interference in the chromatogram from the MLE at the retention time of Met, the chromatogram of plasma samples obtained from rats received the MLE for 21 days was analyzed.2 atmosphere.Human embryonic kidney cells (HEK-293 cells) over-expressing hOCT2 were used to access the changes in Met uptake by MLE. HEK-293 cells over-expressing hOCT2 was obtained from ThermoFisher Scientific . Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 100 µg/mL of streptomycin, and 100 U/mL of penicillin was used for culture of the cell. The cell was humidified at 37 °C with 5% COd-lysine coated 24-well plates (5.0 × 105 cells/well). DMEM with 10% fetal bovine serum was added into the plates and incubated for 24 h. After the incubation, cells were washed twice using prewarmed Hank’s balanced salt solution (HBSS). Prewarmed HBSS containing Met (2.5 µM) with or without MLE (ranged from 0.1 to 100 µg/mL) was added into the plates. For the positive control, metoprolol  was used as the hOCT2 inhibitor. Plates were incubated for 10 min and cells were washed with iced blank HBSS. Then, the 150 μL of 1 N NaOH was added into the cell to lysis the cell. After 15 min of standing, 150 μL of 1 N HCl was added into the plates and cells was harvested. The lysates were centrifuged at 122,000× g rpm for 10 min. The concentration of Met was measured by LC-MS/MS.Briefly, HEK-293 cells over-expressing hOCT2 were located into the poly-g for 10 min and serum (supernatant) was separated. Serum biochemical parameters  were measured using a VetScan VS2 .To evaluate the hepatotoxicity of the extended intake of MLE, the serum biochemical parameters including alkaline phosphatase (ALP), alanine aminotransferase (ALT) were measured after the administration of MLE  for 21 days. In brief, the rat was received the MLE  or saline for 21 days. On day 0 and 21 days, the whole blood was collected via jugular vein and then stored at room temperature for 10 min. Then, blood samples were centrifuged at 1000× g for 5 min. Five hundred µL of the organic layer was transferred into a tube and dried at 40 °C under nitrogen stream. The residue was reconstituted in 50 µL of mobile phase, vortexed, and an aliquot was injected onto the HPLC system.To extract the Met in plasma samples, the previously described method was partially validated using a rat plasma. In brief, aliquots (0.1 mL) of plasma were added to 10 µL of 0.1 N HCl. Then, plasma was added into 50 µL of IS (5 µg/mL in MeOH). After adding 500 µL of ACN, they were shaken for 10 min and centrifuged at 10,000× v/v%) adjusted to pH 4 with orthophosphoric acid. Flow rate was 2 mL/min. For the UV detection of Met, the wavelength was set at 218 nm.The HPLC system  with ultraviolet detector was used for the determination of Met in plasma. Chromatographic separation assay was performed using a Luna SCX analytical column  maintained at 30 °C. The mobile phase consisted of ACN:ammonium phosphate monobasic (10 mM) solution  and the isocratic mode was used with a flow rate of 0.5 mL/min.For the Met uptake study in HEK-293 cells overexpressing hOCT2, LC-MS/MS system was used. The HPLC system  was used with an Agilent 6495 triple-quad mass spectrometer controlled by MassHunter software. Mass spectrometer was maintained with a gas temperature of 200 °C, gas flow of 14 L/min, nebulizer pressure of 20 psi, capillary voltage of 3000 V, and cell accelerator voltage of 5 V. Positive multiple reaction-monitoring (MRM) was used to analyze Met using phenformin hydrochloride as an IS. For the separation of Met, a HILIC column  was used and maintained at 30 °C. Mobile phases consisted of 10 mM ammonium acetate in DW (pH 6.8):ACN .All data were expressed as mean ± standard error (S.E.) except the pharmacokinetic parameters. Statistical differences among groups were evaluated using a Student’s DM induced rats were used in all period of experiments. After the injection of STZ, hyperglycemia was induced and homogeneously maintained . AnimalsAt 4, 9, 15, 18, and 21 day after administration of MLE, the relative blood glucose levels were significantly lowered as compared to those in saline treatment group  . During Time course of the relative blood glucose level after the oral administration of Met in the range 25–100 mg/kg in the saline or MLE treatment groups are illustrated in max mean values of 1.7 and 1.9 h, respectively. These results were supported by the previous study wherein reported the slow absorption  of Met in DM rats [1/2λz was quite similar between the saline and MLE treatment group in the range of about 3–4 h. This is in line with previous studies in which the T1/2λz was ranged from 3 h to 5 h [0–24 in MLE group was 1.7-fold larger than that in the saline group. However, there was no significant difference between groups. This phenomenon might be relevant to individual variability. The Cl/F in MLE group was significantly decreased by 50% compared to that in the saline group. This result indicates that the extended MLE treatment was reduced the elimination rate of Met.To evaluate the influence of repeated administrations of MLE to the pharmacokinetics of Met, the pharmacokinetic profiles of Met were assessed in rats orally received the MLE or saline.  DM rats . The T1/h to 5 h ,32. MeanBefore the pharmacokinetic analysis of Met, the influence of MLE to Met analysis in rat plasma was evaluated by adding the MLE to the Met spiking plasma. Accuracy (RE) and precision (RSD) values of inter- and intra-day were listed in After the repeated administrations of MLE, the elimination rate of Met was lowered compared to the saline group. It has been reported that Met was predominantly transported to the kidney via hOCT2 for the elimination . Thus, wSerum biochemical parameters including alkaline phosphatase (ALP), alanine aminotransferase (ALT) were evaluated and listed in Evaluation of drug interactions is essential to applications of drugs and herbal medicines in clinical situations. In general, herbal medicine or health supplements are taken long-term. Thus, the extended influence of MLE to Met was evaluated in terms of pharmacokinetics and pharmacodynamics. This is the first study to evaluate the extended influence of MLE to Met.In the present study, the extended intake of MLE (3 weeks) lowered the blood glucose level in DM-induced rats. This result is similar with previous literatures reported that the blood glucose lowering effects of the mulberry, leaf, and fruit extract ,37,38,39The extended intake of MLE (3 weeks) was associated with a 24% increased anti-hyperglycemic effect if compared with the saline treatment group. When the MLE was administered for 3 weeks, the anti-hyperglycemic effect of Met was enhanced in all doses of Met. Met treatment with MLE intake led to an enhanced increase in the anti-hyperglycemic effect. This increase indicates that the extended intake of MLE enhanced the anti-hyperglycemic effect of Met. Enhanced anti-hyperglycemic effect of Met by MLE was shown in all doses of Met, except the high dose of Met (100 mg/kg). This phenomenon might be due to the saturation of Met by the overdose . In humaAloe vera and Andrographis paniculata showed the inhibitory effects on CYP3A4 resulting in the increased efficacy of anti-hyperglycemic drugs such as pioglitazone, repaglinide, and glimepiride [This enhanced effect could not be explained by only the individual anti-hyperglycemic effect of MLE. Interactions between herbal extract and anti-hyperglycemic drug have been reported to affect either efficacy or pharmacokinetics of the drug ,21,22. Fmepiride ,42. In omepiride . Thus, wThe bioanalytical method for Met was partially validated in the presence or absence of MLE in rat plasma. Whether the MLE was added or not in rat plasma, the inter- and intra-batch precision and accuracy of Met were estimated below 15%, which is the acceptance criteria recommended by US Food and Drug Administration . It suggHouttuynia cordata ethanol extract reduced the rat OCT2-mediated renal excretion of Met and increased systemic exposure of Met. Also, there is another hypothesis. Met is metabolized by hepatic CYP2C11, 2D1, and 3A1/2, and eliminated via the kidneys in rats [Herein, the extended intake of MLE lowered the Cl/F of Met. This extended Met elimination phase suggests that the extended MLE intake altered the pharmacokinetics of Met in DM-induced rats. This pharmacokinetic feature could be explained by a hypothesis. It is demonstrated that Met relies on facilitated transport by transporters for delivery to the organs or tissues . In part in rats . In stud in rats ,46. SlowThis study is the first published report of the pharmacokinetic alternation of Met through 3 week-intake of MLE. MLE intake for 3 weeks produced an anti-hyperglycemic effect. Orally administered Met after the MLE intake for 3 weeks enhanced the anti-hyperglycemic effect of Met compared to Met alone. This enhanced effect of Met might be due to the increase of systemic exposure of Met. In addition, extended MLE intake altered the pharmacokinetic properties of Met via the reduction of Met uptake to kidney by inhibiting the hOCT2. Therefore, the extended MLE intake with Met could reduce the unwanted side effects of Met by decreasing the dose of Met.