Met proto-oncogene (MET) amplification and tyrosine-protein kinase Met (c-Met) overexpression confer gefitinib resistance in non-small cell lung cancer (NSCLC). The natural product Licochalcone A (Lico A) exhibits a broad range of inhibitory effects against various tumors. However, the effects of Lico A on c-Met signaling and gefitinib resistance in NSCLC remain unclear. In the present study, Lico A efficiently overcame gefitinib-acquired resistance in NSCLC cells by suppressing c-Met signaling. Lico A decreased cell viability and colony formation dose-dependently and impaired in vivo tumorigenesis of gefitinib-resistant HCC827 and PC-9 cells. Furthermore, Lico A induced intrinsic apoptosis and upregulated the protein expression levels of cleaved poly (ADP-ribose) polymerase and cleaved caspase 3. Lico A promoted the interaction between c-Met and E3 ligase c-Casitas B-lineage lymphoma (Cbl), which enhanced c-Cbl-mediated c-Met ubiquitination and degradation. Depletion of c-Cbl compromised Lico A-induced c-Met ubiquitination and its inhibitory efficacy in gefitinib-resistant NSCLC cells. Taken together, the results suggest that Lico A is a promising antitumor agent that might be used to overcome c-Met overexpression-mediated gefitinib resistance in NSCLC cells. Non-small cell lung cancer (NSCLC) is a profoundly devastating disease that is the leading cause of cancer-associated deaths worldwide . NSCLC aLicochalcone A (Lico A) is a flavonoid extracted from licorice root, demonstrating a wide range of pharmacological effects, including the anti-inflammatory and antitumor activities , 17. TheIn the present study, the ability of Lico A to attenuate c-Met signaling and suppress c-Met expression in a ubiquitination-dependent manner was examined. Moreover, the potential of this compound to overcome gefitinib resistance was assessed in the NSCLC cells.β-actin (#3700), ERK1/2 (#9102), α-tubulin (#2144), cleaved-caspase 3 (#9664), cytochrome c (#4280), and ubiquitin (#3936) were obtained from Cell Signaling Technology, Inc. . The natural compound Licochalcone A (>97%) was from Selleck Chemicals . Lipofectamine 2000 transfection reagent for transient transfection was purchased from Thermo Fisher Scientific .Human NSCLC cell HCC827 and the immortalized lung epithelial cells HBE and NL20 were obtained from American Type Culture Collection . PC-9 cell line was a product of Sigma-Aldrich . Both PC9 and HCC827 cells carry a Glu746-Ala750 deletion mutation in exon 19 of the EGFR , 27. The3 cells/well) and treated with various concentrations of Lico A. Following 24 h of incubation, cell viability was analyzed with MTS using the CellTiter 96® Aqueous One Solution kit (Promega Corporation) as determined by the manufacturer's protocol.The cultured cells were seeded into 96-well plates .The cells were treated with Lico A or gefitinib, and the whole-cell extract (WCE) was prepared with RIPA buffer and concentrated using the BCA protein assay . The Western blot analysis was performed as described previously . A totalThe Mitochondria Isolation kit  was used for cytosolic and mitochondrial fraction extraction following the manufacturers' instructions.μg/ml puromycin for three weeks. Single colonies were chosen for further study. For transient transfection, the siRNAs, including si-c-Cbl (sc-29242) and siCtrl (sc-37001), were purchased from Santa Cruz Biotechnology . The c-Met cDNA (RC217003) was purchased from Origene . The transient transfection was performed using the Lipofectamine 2000  following the manufacturer's protocol. The whole-cell extract was prepared at 2 days later after transfection.Generation of stable c-Met knock out cell lines. CRISPR-Cas9-mediated gene knockout was performed as described previously (PMID: 32945473). In brief, single-guide (sg) RNAs  targeting c-Met were used for the construction of stable cell lines. In brief, the NSCLC cells were transfected with c-Met sgRNA and selected by 1 β-actin. c-Met primer sequences used were forward, TGCACAGTTGGTCCTGCCATGA; reverse, CAGCCATAGGACCGTATTTCGG.The NSCLC cells were treated with Xanth for 24 h, and total RNA was extracted using the Absolutely RNA Purification Kits (Agilent). SYBR-Green Quantitative RT-qPCR Kit was used in RT-qPCR. Amplification cycles were performed as follows: Stage 1: activation, 50°C for 2 min. Stage 2: presoak, 95°C for 10 min. Stage 3: denaturation, 95°C for 15 sec; annealing: 60°C for 1 min. Stage 4: melting curve, 95°C for 15 sec, 60°C for 15 sec, and 95°C for 15 sec. The RT-qPCR results were normalized to 6 cells in 100 μl RPMI-1640) cells were injected into the right flank of 6-week-old female athymic nude mice. Tumor volume and mouse body weight were recorded every 2 days. The tumor-bearing mice had initiated Lico A treatment when the tumor reached a maximum of 100 mm3. The mice were divided into two groups randomly. Lico A treatment  was initiated every 2 days via intraperitoneal injection. The control group (n = 5) was treated with vehicle control. Animal health and behavior were monitored every 2 days. The following formula calculated tumor volume: A × B2 × 0.5, where A was the longest diameter of the tumor and B was the shortest diameter. B2 was B squared. The tumor-bearing mice were euthanized by CO2 when the tumor volume reached 700 mm3 (24 days). The fill rate of CO2 is 30% of the chamber volume per minute (3 liter/min), and the duration time is 5 min. Death was further confirmed by cervical dislocation.All in vivo animal experiments were performed in 2021, which the Institutional Animal Ethics Committee approved at The Third Xiangya Hospital, Central South University. Mice were kept in the colony cages with free access to food and tap water and standardized housing conditions . The proper care and use of experimental animals, including efforts to minimize suffering and distress using analgesics or anesthetics, was based on the Guide for the Care and Use of Laboratory Animals . For tumor transplantation, HCC827-GR .The IHC staining was performed as described previously . Brieflyt-test or one-way ANOVA. A probability value of P < 0.05 was considered to indicate a statistically significant difference.SPSS software  was used for statistical analysis. The quantitative data are presented as the mean ± SD, and the difference was analyzed using the two-tailed Student's To further discover novel antitumor agents that can overcome gefitinib resistance, two gefitinib-resistant cell lines were generated, namely HCC827-GR and PC-9-GR. The MTS data indicated that gefitinib significantly decreased the viability of HCC827 and PC-9 cells but not that of HCC827-GR and PC-9-GR cells . The tryμM Lico A for 48 h. Exposure to higher concentrations (10 or 20 μM) of Lico A exhibited a more potent inhibitory effect  to Lico A inhibited hepatocyte growth factor- (HGF-) induced c-Met phosphorylation. Moreover, the activation of the c-Met downstream kinases, ERK1/2 and AKT, was substantially reduced. However, the total protein levels of c-Met were unaffected . The datectively . This suectively . Moreoveectively . To furtectively . These rPrevious studies have shown that the E3 ligase c-Cbl promotes the degradation of c-Met ubiquitination. Therefore, it was hypothesized that c-Cbl was involved in Lico A-induced c-Met ubiquitination. The interaction between c-Cbl and c-Met was initially determined. The results indicated that Lico A strengthened the interaction between c-Cbl and c-Met in HCC827-GR cells . MoreoveTo verify the in vivo inhibitory effect of Lico A, a xenograft mouse model was established. HCC827-GR-derived xenograft tumors were treated with Lico A and vehicle control. The data indicated that Lico A significantly delayed the tumor growth of HCC827-GR xenografts compared with that of the vehicle control . The aveNSCLC is the leading cause of cancer-associated deaths worldwide and accounts for ~85% of lung cancer cases. Lung squamous carcinomas and lung adenocarcinomas are the main subtypes of NSCLC , 2. EGFRμg/ml [μM or higher Lico A produced cytotoxicity in Chinese hamster ovary (CHO) fibroblasts. Lower concentrations (1.85 to 7.39 μM) exhibited protective activity against chromosomal damage induced by doxorubicin (DXR) or methyl methanesulfonate (MMS) in CHO cells [Natural products are widely studied as potential therapeutic antitumor agents due to their limited toxicity . Lico A μg/ml . ConcentHO cells . While aHO cells , 47. ThuLicochalcone B and Licochalcone D, which are structurally similar derivatives to Lico A, induce apoptosis by dual inhibition of EGFR and MET expression. Licochalcone B and Licochalcone D inhibited both EGFR and MET kinase activity by competing with their ATP-binding pockets , 49. MorIn addition to the secondary mutation in EGFR, multiple EGFR-independent resistance mechanisms have been identified, including the bypass receptor tyrosine kinases (RTKs) activation , hyperactivation of downstream signaling (e.g. PI3K and BRAF mutations), and histological transformations  . MoreoveIn summary, the present study demonstrated that the natural product Lico A inhibited c-Met signaling and disrupted c-Met ubiquitination in a c-Cbl-dependent manner. Targeting c-Met degradation is a promising strategy to abrogate gefitinib resistance in NSCLC cells. The current study provided new insights on the role of Lico A in NSCLC treatment and suggested that this compound may be a promising therapeutic agent for gefitinib-resistant NSCLC.


However, there was a statistically significant increase (p < 0.01) in the use of penicillins. This study, carried out in daily practice conditions, confirms that the educational strategy was associated with an overall reduction in the use of antibiotics and an improvement in the antibiotic prescription profile in acute respiratory tract infections.The objective of this study was to assess the impact of an education intervention for primary health care physicians, based on the knowledge of clinical practice guidelines and availability of rapid antigen detection test for group A streptococci (GAS), on the improvement of antibiotic prescription for patients with acute respiratory tract infections. Before and after the intervention, physicians collected data from ten consecutive patients who attended during a 3-week period. This process was performed twice a year for 6 consecutive years (2012–2017). A total of 18,001 patients were visited by 391 primary care physicians during the study period, 55.6% before intervention and 44.4% after intervention. After intervention, the antibiotic prescription decreased significantly, from 33.0 to 23.4% ( There is a large variability in the prescription of antibiotics between health care professionals and countries for the management of patients with acute respiratory tract infections. Inappropriate and excessive antibiotic consumption is the most important factor for the development of antibiotic resistance3. In countries with the highest antibiotic use, it is where more multiresistant pathogens are isolated4. Also, consumption of one antibiotic agent is associated with a greater propensity to resistance6. In addition, there has been an alarming decrease in the research of new antibiotic drugs8, with a clinical shortage of new molecules being approved in the past decades10. A few antibiotic classes in the pipeline are innovative treatments that will add value to the current antibiotic treatment arsenal.Infections are one of the most common reasons for consultations in primary care, accounting for approximately one-third of visits to the general practitioner, with half of these visits due to acute respiratory tract infections11. A high percentage of antibiotics are prescribed in the primary care setting, with acute lower respiratory tract illness as one of the most common condition12. The debate continues about the effect of antibiotics on the course of the disease, concerns regarding complications if antibiotics are not prescribed, or the definition of the clinical characteristics that identify patients at risk for which antibiotics are necessary. Single quality improvement antibiotic prescribing strategies  have shown to be effective for the rational use of antibiotics14. Also, active clinician education may be more effective than passive education, particularly for addressing the antibiotic treatment decision15.Unnecessary prescribing of antibiotics is a major contributor to the problem of antimicrobial resistance, and quality improvement efforts appear generally effective at reducing both inappropriate use of antibiotics and choice of antibiotic drugsTherefore, the aim of this study was to assess changes in antibiotic prescription following an education intervention for primary health care physicians, based on the knowledge of clinical practice guidelines and availability of rapid antigen detection test for GAS, and on the improvement of antibiotic use for patients with acute respiratory tract infection.A total of 18,001 patients were visited by 391 primary care physicians during the study period, of whom 10,002 (55.6%) were attended before implementation of the educational intervention and the remaining 7999 (44.4%) in the post-intervention phase of the study. The number of physicians and patients included in each edition of the study is shown in Table As shown in Table p < 0.01 (χ2)). In relation to the different pathological conditions (Table p < 0.05 (χ2)) and chronic bronchitis ). The use of antibiotics also showed an important decrease in cases of acute pharyngotonsillitis (from 44.4 to 32.7%) and exacerbation episodes of chronic obstructive pulmonary disease (COPD) (from 85.2 to 75.6%), although pre- and post-intervention differences were not statistically significant (p = 0.08 (χ2)).After the educational intervention, there was a statistically significant decrease in the prescription of antibiotics, from 33.0% (3221/9760) to 23.4% (1828/7812) (p < 0.01 (χ2)), acute otitis media ), acute pharyngotonsillitis ), acute bronchitis ), acute exacerbation of COPD or chronic bronchitis ), and influenza ). Also, the use of penicillins increased significantly in the post-intervention phase for the indications of acute otitis media, acute sinusitis, acute pharyngotonsillitis, acute bronchitis, and pneumonia, whereas the use of amoxicillin/clavulanate decreases significantly for the same indications. In acute exacerbation episodes of COPD or chronic bronchitis, there was a significant decrease in the use of macrolides.Table p < 0.01 (χ2)) in the mean (SD) percentage of use of penicillins (19.7 [6.1] vs. 41.7% [9.4]) and significant reductions in the use of amoxicillin/clavulanate (32.7 [10.1] vs. 27.0% [6.1]), macrolides (19.0 [5.9] vs. 11.1% [2.5]), and cephalosporins (8.2 [2.5] vs. 4.2% [0.9])  fulfilled the criteria for performing the test and underwent GAS testing. The test was positive in 520 cases (positivity rate 38.5%) and negative in the remaining 830. Antibiotics were prescribed in 98.1% of positive cases (510/520) and in 11.7% (97/839) of negative cases. The rate of antibiotic prescription for acute pharyngotonsillitis after the intervention was 32.7% as compared to 44.4% before the intervention (Table The main finding of this study is a significant reduction in prescribing antibiotic treatment (from 33.3 to 23.4%) by primary care physicians for patients with acute respiratory tract infections after implementation of an educational strategy, based on a better knowledge of clinical practice guidelines. Also, the profile of antibiotics prescribed was more in line with guidelines recommendations, with an overall increase of penicillins and a decrease of amoxicillin/clavulanate and macrolides.n = 382) received two visits by peer academic detailers, the first presenting the national clinical guidelines for antibiotic use and recent research evidence on acute respiratory tract infections, and the second based on feedback reports on each general practitioner’s antibiotic prescribing profile from the preceding year16. There was a reduction in prescribing of antibiotics for acute respiratory tract infections  and for prescribing non-penicillin antibiotics when an antibiotic was issued16. In a study that assessed views of experts from five countries who develop guidelines and interventions for respiratory tract infections, a consistent set of recommendations included to address general practitioner concerns about recommendations and explain the need for guidelines, to provide interventions that engage physicians, and to provide consistent educational messages about antibiotic use17. In a systematic review of 39 studies assessing the effectiveness of different educational interventions , all interventions reduced the use of antibiotics prescribed by health care professionals in the outpatient setting, although effectiveness depends to a large extent on the particular prescribing behavior and the barriers to change in the particular community18.The present results are consistent with previous studies in which educational interventions led to reduced antibiotic prescribing for respiratory tract infections. In a multi-faceted educational intervention in Norwegian general practice, general practitioners 20 for acute pharyngotonsillitis, antibiotic overuse is a widespread practice and a key driver of antibiotic resistance. In the present study, before implementation of the educational strategy, 85.2% of patients with acute exacerbation of COPD received antibiotics, which is >81.9% reported in a study of 332 Spanish general practitioners who received guidelines and participated in discussion sessions21. In patients with pharyngotonsillitis, the percentage of antibiotic prescription of 44.4% was <49.1% found in an observational study with the participation of 126 general practitioners from eight autonomous communities in Spain22. The availability of the rapid antigen testing for GAS at the time of consultation also promoted a reduction in antibiotic prescribing as shown by others23. In other pathological conditions, such as acute bronchitis and acute otitis media, there was a significant reduction in prescribing antibiotics after the physician’s educational intervention. The percentage of antibiotic use in the pre-intervention phase in acute bronchitis was 72%, which is higher than 62.3% found in a similar study21, and decreasing to 56% after the intervention.There are difficulties in clinical practice to distinguish bacterial from viral respiratory tract infections. Although the criteria for the use of antibiotics in COPD exacerbations have been established by Anthonisen et al.24. The ability of professionals to introduce changes in their clinical practice after an educational intervention has been documented in other studies18. The correlation of behavioral changes with antibiotic prescription practices may potentially result in a reduction of antibiotic resistance as antibiotic overuse is the principal cause of emergence of resistance3. The reduction in the use of broad-spectrum antibiotics may also contribute to reducing bacterial resistance of pathogens usually involved in acute respiratory tract infections25. In a retrospective observational study including all episodes of respiratory tract infections registered during a 1-year period in a north-eastern Spanish region, a high prescription of broad-spectrum agents and antibiotics not recommended as the first choice was observed, with a high risk of increasing antimicrobial resistance26.Another effect observed after the educational intervention has been a change in the prescribing antibiotic profile, with a clear decrease in the use of broad-spectrum macrolides and amoxicillin/clavulanate and an increase in the prescription of narrow-spectrum antibiotics (penicillin). These data are consistent with the WHO recommendations in its classification of antibioticsNevertheless, this study also has certain limitations. As this was a retrospective study with voluntary participation of physicians, the potential for selection bias and confounding cannot be excluded. Moreover, this study was conducted only in Spanish patients, and it is, therefore, uncertain whether these findings can be extrapolated to a more diverse group of patients and to other health care systems. Although the large study population and the repeated educational interventions along the years consistently showing a benefit of the intervention to a more rational antibiotic prescription in acute respiratory tract infections, the lack of long-term follow-up, and the possibility for a waning off of the benefit of the intervention should be also taken into account as a limitation of the study. The study was designed as 10-day short-term periods before and after the intervention, and was repeated twice a year over various consecutive years. The consistency of findings in the reduction of antibiotic prescription in the different editions from 2012 to 2017 following this study design reinforces the impact of an educational intervention combined with GAS testing to sensitize primary care physicians on the better prescription of antibiotics for acute respiratory tract infections. However, other factors that might have influenced antibiotics prescribing over this time cannot be excluded.In conclusion, this study carried out in daily practice conditions based on an educational strategy, with advice on the implementation of recommendations of clinical practice guidelines and the use of rapid antigen testing for GAS at the time of consultation in acute pharyngotonsillitis, shows an overall reduction of the use of antibiotics and an improvement in the antibiotic prescribing profile of primary care physicians for patients with acute respiratory tract infections. However, the amount of antibiotics prescribed in primary care is an important issue that needs exploration in further studies.This was a retrospective observational study using data collected from an anonymized database that belonged to subjects who were previously involved in the HAPPY AUDIT project . In such a project, the primary objective was to assess the impact of an education strategy, based on the therapeutic adequacy of the use of antibiotics in respiratory infections combined with the use of rapid antigen testing for GAS, on changes in antibiotic prescription for acute respiratory tract infection. The study was conducted in the primary health care setting in the Region of Murcia, Spain  between 2012 and 2017. The study protocol was approved by the Ethics Committee for Clinical Research of Hospital Virgen de la Arrixaca, Murcia, Spain. Written consent was waived because data used in this study were extracted from an anonymous database.The study population consisted of male and female patients aged >14 years who had received a physician’s diagnosis of acute respiratory tract infection (ICD-10) in the last month and who had not received antibiotics in the previous month prior to their inclusion in the study. Patients with severe or terminal illness were excluded.Primary care physicians were invited to participate in the study, which included a systematic registration of patients with acute respiratory tract infection attended in their daily practices during 10 consecutive days before and after the intervention. Registration of patients was carried out by the attending physicians who participated in the study. This first phase of the study was followed by the intervention, which was an accredited education session. This included a 4-h face-to-face course divided into two parts. In the first part, participants were instructed regarding recommendations of current clinical practice guidelines for the management of respiratory tract infections and the use of antibiotics. In the second part, they were instructed regarding the use of rapid antigen testing for GAS in patients with acute pharyngotonsillitis, with information on technical aspects of the test, indications , and interpretation. In addition to theoretical training, participants performed an exercise for the acquisition of practical skills. The key characteristics of the GAS test include a time needed for results of ~10 min, sensitivity 95%, specificity 93%, positive predictive value 79.2%, and negative predictive value 98.5%.After the education sessions, participants performed a second 10-day registration of consecutive patients with acute respiratory tract infection attended in daily practice. Also, they have available the rapid antigen testing for GAS in their consultation. This three-step process  was performed twice a year (March–April–May and November–October–December) over 6 consecutive years (2012–2017). Clinicians and patients involved at different time points were not the same persons, that is, both clinicians and patients participated only once.27 using the Audit Project Odense method29.Data were recorded according to the methodology of HAPPY AUDIT projectDuring the pre-intervention and post-intervention phases of the study, the following data were collected: demographics ; duration of symptoms (number of days); symptoms ; rapid antigen testing for GAS ; chest radiography ; etiology of infection ; and diagnosis according to ICD-10 codes in primary care, including common cold (code J00), acute otitis media (code H66.9), acute sinusitis (code J01), acute pharyngitis (code J02.9), unspecified acute tonsillitis (code J03.90), acute bronchitis (code J20), pneumonia , exacerbation of COPD or chronic bronchitis , influenza (code J11.1), and other infections of the respiratory tract . Use or no use of antibiotics and the class of antibiotics administered  were recorded. Other variables were the presence of penicillin allergy, patient’s demand for antibiotic treatment, and patient’s referral to the hospital or to a specialist.χ2 test was used for the comparison of categorical variables, and the Student’s t test was used for continuous data. Statistical significance was set at p < 0.05. The SPSS statistical program version 18.0 was used for statistical analysis.Categorical variables are expressed as frequencies and percentages, and continuous variables as mean and standard deviation. The Further information on research design is available in the Reporting Summary


Impressed current cathodic protection (ICCP) is a widely used method to protect steel reinforcements against corrosion. This research aimed to study the effect of cathodic protection on reinforced concrete with fly ash using electrochemical noise (EN). Two types of reinforced concrete mixtures were manufactured; 100% Ordinary Portland Cement (OCP) and replacing 15% of cement using fly ash (OCPFA). The specimens were under-designed protected conditions (−1000 ≤ E ≤ −850 mV vs. Ag/AgCl) and cathodic overprotection (E < −1000 mV vs. Ag/AgCl) by impressed current, and specimens concrete were immersed in a 3.5 wt.% sodium chloride (NaCl) Solution. The analysis of electrochemical noise-time series showed that the mixtures microstructure influenced the corrosion process. Transients of uniform corrosion were observed in the specimens elaborated with (OPC), unlike those elaborated with (OPCFA). This phenomenon marked the difference in the concrete matrix’s hydration products, preventing Cl In the construction industry, corrosion of steel reinforcement is one of the main issues. It is considered the leading cause of the premature deterioration of reinforced concrete structures ,2,3. The2), generating between 5–8% CO2 total emissions to the environment. In the future, the CO2 emissions are predicted around 10–15% [2 emissions and replace, in a portion, Portland cement [The cement industry is one of the two largest producers of carbon dioxide CO, generatd cement ,13,14,15New alkali-activated materials have been employed in place of OPC with a different type of ashes. The employ of different ashes showed good results increasing the durability of concrete. OPC has been replaced by ashes as slags furnace ash (SFA), metakaolin (MK), and fly ash (FA), among others ,17,18. SAnother critical factor of these SCM is that they cure faster than OPC, making them even more suitable for precast components.− ions are considered the most critical factor influencing the corrosion intensity of reinforced concrete [− ion causes the breakdown of the steel’s normal passive condition in concrete and corrosion development. An efficient method to control steel reinforcement corrosion in concrete structures is the impressed current cathodic protection (ICCP). ICCP is an efficient method to stop or control the steel reinforcement’s corrosion in concrete structures. ICCP is known for active or passive steel conditions; it is necessary to have specific conditions for both pH and potential in the electrochemical system [Corrosion of reinforcing steel is the leading cause of deterioration in concrete. When steel corrodes, the resulting rust occupies a greater volume than the steel. The action of Clconcrete ,28,29. Tl system ,31,32,33Electrochemical noise (EN) technique for corrosion applications has allowed many advances in recent years interesting for corrosion science. A particular advantage of EN measurements is detecting and analyzing the early stages of localized corrosion.Electrochemical noise describes the spontaneous low-level potential and current fluctuations occurring during a corrosion process. During the corrosion process, predominantly electrochemical cathodic and anodic reactions can cause small transients in electrical charges on the electrode. These transients manifest in potential and current noise exploited in a corrosion map ,35. The Transients are linked to anodic and cathodic reactions because of stochastic processes (rupture and re-passivation of the passive film) and deterministic processes (formation and propagation of pitting) ,37. The LI) Chaos analysis, Recurrence quantification analysis and Fractal analysis. The Fast Fourier Transform includes power spectral density, noise impedance, etc; and time-frequency domains method; have the analysis Hilbert-Huang transform, Discrete Wavelet transform, Stockwell transform and others) [LI, skewness, and kurtosis values have been related to different corrosion types and the asymmetry of the distribution of EN data [Ma, et al. ,39 indic others) ,45,46,47 EN data ,50,51,52This research aimed to study the Effect of cathodic protection on reinforced concrete with fly ash using electrochemical noise. Two types of reinforced concrete mixtures were elaborated: 100% Ordinary Portland Cement (OCP) and replacing 15% of cement using fly ash (OCPFA). The specimens were under-designed protected conditions (−1000 ≤ E ≤ −850 mV vs. Ag/AgCl) and cathodic overprotection (E < −1000 mV vs. Ag/AgCl) by impressed current, and specimens concrete were immersed in a 3.5 wt.% sodium chloride (NaCl) Solution. This concentration was used to simulate an aggressive environment that contains chlorides where civil works can be built based on reinforced concrete.This research were designed for eigth specimens: OCP and FA concretes mixtures according to NMX-C-414-ONNCCE  of 30 MPThe dosage of concrete mixtures was carried out according to the method of ACI 211.1 . This meThe OPC and FA mixtures were made with a ratio of water/cement of 0.66. The specimens were cylindrical with dimensions of 15 high by 7.5 cm in diameter . In all After the curing step, the specimens of both mixtures were immersed in a 3.5 wt.% NaCl solution and an impressed current cathodic protection system were induced. Anode, ¼ in. graphite bars with 0.635 cm. in diameter and a length of 15 cm. were used. The capacity of the employed rectifier was 20 V/10 A. Four of the specimens were treated to a potential protection criterion (−1000 mV ≤ E ≤ −850 mV vs. Ag/AgCl), and the rest of them with overprotection (E < −1000 mV vs. Ag/AgCl) . The proThe electrochemical noise (EN) technique was used to evaluate 100% Ordinary Portland Cement (OCP) and the other with replacing 15% of cement using fly ash (OCPFA), immersed in 3.5 wt.% in NaCl solutions. The EN measurements were carried out using ACM Instruments —Gill-AC potentiostat/galvanostat/ZRA (Zero Resistance Ammeter).nR) and corrosion rate. To each experiment was employed two nominally identical specimens as the working electrodes (WE1 and WE2) and a saturated calomel electrode as the reference electrode (RE) [The EN experiments were carried out in based to ASTM G199-09 standard to determine the noise resistance (ode (RE) ,57. The ode (RE) ,52.nR, Kurtosis, and skewness) was obtained from the signal without DC. For PSD  analysis, a Hann window was applied before being transformed to the FFT frequency domain (fast Fourier transform). Data analysis was processed with a program made in MATLAB 2018a software .DC trend signal was removed from the original EN signal by the polynomial method, from signal without DC statistical data  was as well utilized for concrete surface morphology analyzed to different magnifications, operating at 20 kV, WD = 10 mm. The chemical composition of these concrete specimens was obtained by energy dispersive X-ray spectroscopy .The specimen 2OPC-OP b shows tFor specimens with three months of exposure c,d, it ix(t) is the EN time series, tm, is the DC component, ts is the random component, and tY is a stationary component. The latter two are the functions that define the corrosion system [nx) and polynomial of “n” grade (op) at n-th term (ia) in “n” time to obtain a signal without trend (ny) [EN signal is composed of random, stationary, and DC variables. It is necessary to separate DC from random and stationary components to analyze EN data because DC creates false frequencies and interferes in visual, statistical, and PSD analysis. In this way, when DC is removed, corrosion data presented at low frequencies are conserved . EN can n system . The polend (yn) ,42,43,44−4 V maximum. Low potentials are associated with high stability produced by oxidation-reduction events. The ECN signal corroborated it with low amplitude transients and fluctuations of ×10−7 (A/cm2) order. The behavior of EPN and ECN signals is related to the uniform corrosion process. In the case of FA samples, the potential transients show individual events of low frequency. On the other hand, OPC-P samples presented a higher instability associated with the passive layer’s break. When concrete contains FA and the passive layer is broken, it will be regenerated easier than samples with only OPC.−10 (A/cm2) amplitude. The low current value indicates passivation in the sample after three months of exposure [The sample of OPC-P exposed three months c,d preseexposure . The decThe three months samples with OPC presented potential and current transients characteristic of unstable uniform corrosion, with low frequency and low amplitude behaviors. However, the decrease of frequency presented at two months of exposure is related to a higher harmful activity. The samples with FA presented lower current demand related to lower corrosion kinetic and suggest a passivity phenomenon.nR) is determined by standard deviation from time series values. These statistical values give corrosion kinetics information. Cottis and Turgoose [x1 is the transient ECN or transient EPN, Electrochemical noise resistance (Turgoose  found a nR) is defined as the ratio of the standard deviation of potential to the standard deviation of current: (Equation (4)).Noise resistance  can be applied as an analog relation to determining corrosion. pR is expressed in Ω·cm2, corri is the corrosion current density, and B in V. is a constant resulting from a combination of the anodic and cathodic Tafel slopes. Recommended values of B are 0.026 and 0.052 V for active and passive systems, respectively [ectively .(5)Rn=Βr.m.sI is obtained by Equation (6), where X is the average of EN data, n the data number, and σ the standard deviation:Some authors related the EN signal to statistical analysis with the metal surface to the corrosion process . The Ir.LI) is defined as the ratio of the standard deviation of ECN, r.m.sI (root mean square of the noise current) (Equation (7)).The localization index  was not considered because Mansfeld and Sun [LI can present limitations and should be used with discretion. In 2001, Reid and Eden [This research employed Kurtosis and skewness to try to define the corrosion type. Localization index  that generates uncertainty in the results. The following Equation can be provided, where N is the number of data studied [Statistical calculations have a standard error , after which spectral density is calculated with Equations (11) and (12) ,67.(11)ψ0). The cut frequency indicates the slope begins. The slope could be helpful to find the corrosion mechanism [xβ and is represented by Equation (13):For PSD interpretation, it is necessary to evaluate the slope and the frequency zero limits (echanism . The sloψ0) give material dissolution information because PSD is related to the total energy present in the system [The frequency zero limits 2 still exists in complex binder pastes at the age of 360 days. With the extension of the curing ages, a large amount of low CaO/SiO2 ratio of C-S-H gel is generated, which causes complex binder pastes to gradually compact.Li and Hou ,73 studyFly ash is a pozzolanic material. It is a finely divided amorphous alumino-silicate with varying amounts of calcium. Calcium mixed with Portland cement and water will react with the calcium hydroxide The hydration of Portland cement releases various calcium-silicate hydrates (C-S-H) and calcium-aluminate hydrates. These pozzolanic reactions are beneficial to the concrete. Pozzolanic reactions increase the quantity of the cementitious binder phase (C-S-H). To a lesser extent, calcium-aluminate hydrates improve the long-term strength and reduce the system’s permeability ,75,76.2O and K2O), carbon, and sulfate (SO3) can also affect fly ash performance.The fly ash is calcium content is perhaps the best indicator of how the fly ash will behave in concrete . HoweverElectrochemical noise transients are related to different types of corrosion processes. The pitting process consists of high insensitivity transients with a high repetition rate. If the behavior is mixed, there are transients and oscillations of short amplitude. The uniform process is related to oscillations of low amplitude from the pattern noise. EPN and ECN signals of specimens subjected to cathodic protection current is related to uniform corrosion process show transients low frequency. OPC-P samples presented a higher instability associated with the break of the passive layer. When concrete contains FA and the passive layer is broken, it will be regenerated easier than OPC samples. The EN signal for overprotected samples at 2 and 3 months has current and potential values with low frequencies and amplitudes. Variation of fluctuations is high, associated with an unstable system. Samples with FA presented lower current demand related to lower corrosion kinetic and suggest a passivity phenomenon.nR is indeed equivalent to the polarization resistance [nR values are proportional to the corrosion rate according to Ohm’s law and the Stern-Geary Equation [nR in OPC samples at 2 and 3 months have low values, which indicates a higher corrosion rate. The overprotection FA samples at three months have high Rn values, representing a better behavior in the corrosion kinetics.It has been determined that sistance . TherefoEquation . The Rn In , a studyStudies of reinforced concrete applying protection methods focus on adding corrosion inhibitors or rehabilitating structures by electrochemical extraction of chlorine ions or realkalination of concrete. The application of cathodic protection current to reinforced concrete has been commonly studied by electrochemical techniques such as OCP, PP, or EIS. However, EN is not used.Chloride-induced localized corrosion of embedded steel has been widely known for many years to be one of the most dangerous corrosion types in reinforced concrete ,80,81. CUsing impressed current cathodic protection is essential to define the protection levels to obtain good results and differentiate the effects of cathodic protection and supplementary materials. In this study, the protected and overprotected OPCFA samples have good corrosion resistance. Lu et al.  investigAn ICCP system can promote hydrogen activity at the concrete-steel interface. The consumption of electrons from the corrosion process, so safe CP limits, must be established to prevent the risk of phenomena such as hydrogen-embrittlement of steel reinforcements or the concrete-steel interface degradation ,87,88,89Moreover, the initiation of fracture can be caused by molecular hydrogen absorbed on the steel surface.Research with similar additions of fly ash and cathodic protection was carried out by Garcia et al. . Garcia Garces  Study ofThese results demonstrate the using supplementary cementitious materials in OPC increases the corrosion resistance when exposed to chlorides. These results would significantly contribute to creating a more sustainable concrete industry.SEM and EDS observations indicated that the microstructures could be observed the hydration products (C-S-H gel) in both mixtures. Hydration influences some results on the Ca/Si ratio obtained, showing that the OPCFA mixtures had better hydration, indicating fewer pores in its microstructure.− ions to the steel surface. Contrary to the mixture made with OPCFA where most of the specimens showed passivity.Results indicated that electrochemical noise tests have marked the difference the two mixtures microstructure, obtaining a uniform corrosion process for the specimens elaborated with the OPC mixture. Microstructure facilitated ClAfter trend removal, EN signals conserved transients and fluctuation behavior and gave practical corrosion information removing DC.Ψ0 parameters should be considered as a counterpart to calculate corrosion resistance of reinforced concrete mixtures.EN results show that Rn and Overprotected samples presented less corrosion resistance than protected samples. This behaviour could be to steel embrittlement by hydrogen adsorption.Samples with FA presented a higher trend to passivation than samples with OPC. Besides, FA samples showed higher corrosion resistance.Statistical and PSD results showed that uniform corrosion predominates the systems. Although current values are shallow, the variation is considerable, and complicated the analysis for current signals, as potential is more stable than current, makes statistical and PSD analysis easier.The current protection and cathodic overprotection did not indicate some tendency in both mixtures behavior, for these exposure times exposure and ratio w/c.The discordance of statistical results could be related to developing a different corrosion process on the surface when LI indicates localized corrosion and skewness uniform corrosion. It suggests that localized and uniform corrosion occurs on the surface, but uniform corrosion is the predominant system.After studying the Effect of cathodic protection on reinforced concrete with fly ash using electrochemical noise.


Methionine (Met) as an essential amino acid has key importance in a variety of metabolic pathways. This study investigated the influence of three dietary Met supplements butanoic acid (DL-HMTBA)) on the metabolome and inflammatory status in the small intestine of pigs. Epithelia from duodenum, proximal jejunum, middle jejunum and ileum were subjected to metabolomics analysis and qRT-PCR of caspase 1, NLR family pyrin domain containing 3 (NLRP3), interleukins IL1β, IL8, IL18, and transforming growth factor TGFβ. Principal component analysis of the intraepithelial metabolome revealed strong clustering of samples by intestinal segment but not by dietary treatment. However, pathway enrichment analysis revealed that after L-Met supplementation polyunsaturated fatty acids (PUFA) and tocopherol metabolites were lower across small intestinal segments, whereas monohydroxy fatty acids were increased in distal small intestine. Pigs supplemented with DL-HMTBA showed a pronounced shift of secondary bile acids (BA) and sphingosine metabolites from middle jejunum to ileum. In the amino acid super pathway, only histidine metabolism tended to be altered in DL-Met-supplemented pigs. Diet did not affect the expression of inflammation-related genes. These findings suggest that dietary supplementation of young pigs with different Met sources selectively alters lipid metabolism without consequences for inflammatory status. Besides the economic relevance, balanced amino acid supplementation is a key element for reducing the crude protein level in feed and thus agricultural nitrogen emissions. Vice versa, low crude protein diets need to be supplemented with essential limiting amino acids, especially Met, to meet the amino acid requirements of pigs2. In Europe, the most common authorized feed additives are DL-Met, L-Met and the hydroxyl analog DL-2-hydroxy-4-methylthiobutyrate (DL-HMTBA) (Reg (EC) No 1831/2003)1. Some studies state an equal bioavailability of these Met sources4, whereas more recent studies show relatively lower bioavailability of DL-HMTBA compared to L- or DL-Met sources6. When supplemented on equimolar levels, several studies did not identify a difference between supplements in performance parameters7 or, as shown in cherry valley ducks, carcass traits8.Methionine (Met) is of great importance in the nutrition of livestock. In pigs, it is the second performance-limiting amino acid9. In the intestinal epithelium, Met is involved in transsulfuration, transmethylation and transamination reactions10. The Met derivate S-adenosylmethionine represents a methyl group donor11, which is particularly important for DNA methylation12, as well as metabolism of neurotransmitters14 and phosphatidylcholines15. Additionally, Met plays a significant role in antioxidant defense through its own antioxidative capacity and as a precursor of cysteine and glutathione17. The amino acid taurine derived from cysteine is essential for bile acid (BA) conjugation14.Across vertebrates, Met is a nutritionally essential amino acid with key functions in several biological processes20. L-Met is the biologically active form, while D-Met and DL-HMTBA have to be converted to L-Met first21. The common intermediate metabolite for L-Met synthesis is 2-keto-4 (methylthio) butanoic acid (KMB), which is synthesized from D-Met by D-amino acid oxidase, from D-HMTBA by D-2-hydroxyacid dehydrogenase and from L-HMTBA by L-2-hydroxy acid oxidase21. Subsequent transamination reactions convert KMB into L-Met21. Central organs for D-Met and DL-HMTBA metabolization are liver and kidney; however, some metabolization also occurs in other organs like stomach (DL-HMTBA) and small intestine (D-Met)22.A significant portion of dietary Met (~ 20–30%) is metabolized in the intestinal epithelium23. Giving the involvement of the small intestine in Met metabolism and additional effects of Met source on absorptive efficiencies, it seemed fair to hypothesize that different dietary Met sources may alter the intracellular metabolism of Met with possible outreach on other metabolic pathways. Therefore, one aim of the current study was to unravel the consequences of different Met supplements on the small intestinal metabolome of pigs.Using the same pigs as in the present study, we had identified earlier that pre-feeding with DL-Met increased L-Met absorption in the porcine small intestine, especially in the middle jejunum25, it was further of interest if the aforementioned Met sources may have different effects on gut health. In growing pigs, gut health is crucial for fattening performance and subsequently for economic efficiency as chronic subclinical inflammation inhibits body weight gain27 and impacts small intestinal function28. Several studies investigated the effect of different Met sources on gut health, linking it to possible oxidative stress and inflammation29. A positive effect of Met supplementation was mostly attributed to improved anti-oxidative status31. However, Met can also have adverse effects if supplemented in excessive amounts. Based on animal experiments, Met was even described as the most toxic amino acid if overdosed32. Therefore, the second aim of the study was to assess the effects of the three different Met supplements on the intestinal inflammatory status as one key readout for gut health.Given the mutual interactions between feed, gastrointestinal mucosa and microbiota23. This resulted in a two-factorial design with the factors intestinal region  and dietary treatment . To include the assessment of Met supplements on porcine intestinal health, we accompanied the metabolomics analyses by mRNA expression analyses of inflammation-related genes.To achieve the two aims, we used the same pigs as in our previous study and performed a detailed metabolomics analysis on epithelia in four small intestinal segments, i.e. duodenum (DUO), proximal jejunum (PJ), middle jejunum (MJ) and ileum (ILE). The pigs had been supplemented with either 0.21% L-Met, 0.21% DL-Met or 0.31% DL-HMTBA to achieve equivalent Met supplementation levels based on their respective equimolar bioavailabilityThe metabolomics dataset covered 749 compounds of known identity , monohydroxy fatty acid metabolism (P = 0.026) and secondary bile acid metabolism (P = 0.035). Pathways that tended to be altered by treatment were sphingosine metabolism (P = 0.064), tocopherol metabolism (P = 0.064) and histidine metabolism (P = 0.067).Metabolites showing main effects of treatment, region × treatment interactions or trends thereof were included in a pathway enrichment analysis to elucidate changes in metabolic pathways that could be related to the dietary treatment Table . SignifiP < 0.001; Fig. P < 0.05) or trends thereof (P < 0.1) with no region × treatment interaction. The PUFA content was generally higher in DL-Met and DL-HMTBA-fed animals compared to L-Met feeding across intestinal regions but particularly obvious in the two jejunal segments (PJ and MJ) and partly in ILE.The PUFA pathway was most prominently influenced by the dietary treatment (P = 0.026), 1 out of 12 monohydroxy fatty acids showed a treatment main effect (P < 0.05) and 4 showed a region × treatment interaction or trends thereof , with sphingosine and hexadecasphingosine (d16:1) being affected by a region × treatment interaction  was primarily based on higher abundance of γ-/β-tocoperol and α-tocotrienol in pigs supplemented with DL-Met and DL-HMTBA compared to L-Met  to S-methylcysteine sulfoxide  across regions of the small intestine. The dipeptide γ-glutamylcitrulline showed higher contents in all intestinal regions of DL-HMTBA-supplemented pigs, whereas γ-glutamylmethionine was raised in L-Met-supplemented pigs in the proximal small intestine.As dietary amino acid supplementation was the imposed treatment, the amino acid super pathway was looked at more closely. Out of 184 metabolites in the amino acid super pathway, 25 showed treatment effects, region × treatment interaction or trends thereof , NLRP3 (NLR family pyrin domain containing 3), IL1β (interleukin 1β), IL8 (interleukin 8), IL18 (interleukin 18) and TGFβ (transforming growth factor β).Despite several regional differences, there were no main effects of diet and no interactions between tissue × diet on inflammation-related genes  were lower in pigs fed diets supplemented with DL-HMTBA compared to pigs supplemented with DL-Met and, especially, L-Met. However, the lower levels of Met sulfoxide and Met sulfone cannot be translated linearly into lower oxidative stress in the small intestine of DL-HMTBA-fed pigs. Albeit there could be support of such postulate from few studies that suggest particular benefits of DL-HMTBA for the antioxidative capacity of the body31, there is disagreement in the literature. More recent research indicates that there are no consistent differences in antioxidant potential between DL-HMTBA and DL-Met when sufficient Met + cysteine is provided in the diet50. Other studies have even observed beneficial effects of either DL-Met or L-Met supplementation on antioxidant status compared with DL-HMTBA53. Thus, the higher levels of Met sulfur oxidation products (which may include both D-Met and L-Met sulfur oxidation products) in pigs supplemented with DL-Met and L-Met could be seen as a consequence of higher Met turnover in the intestinal epithelium during absorption. Vice versa, a decreased turnover of Met in the small intestine of pigs fed DL-HMTBA may explain the observed trend for higher abundance of alternative sulfur oxidation products (S-methylcysteine sulfoxide) in their small intestinal segments. As such, the shift from Met sulfur oxidation products to S-methylcysteine oxidation products in pigs fed DL-HMTBA bears similarities to the shift from the dipeptide γ-glutamylmethionine to γ-glutamylcitrulline; both phenomena appear due primarily to a lower turnover of Met in the intestinal epithelium of DL-HMTBA-fed pigs.Met is well known for its antioxidative capacity54. The oxidation products of PUFA would be monohydroxy fatty acids, which were increased in the distal small intestinal segments of pigs supplemented with L-Met. However, the altered metabolites in the monohydroxy fatty acid pathway, mainly included 2-hydroxyl products of saturated fatty acids which are intentionally produced by the body for synthesis of mammalian sphingolipids56. It was shown that 2-hydroxy fatty acids are highly abundant in intestine of developing rats, constituting the major fatty acid fraction in intestinal glucosylceramides57. Ceramides, a subclass of sphingolipids, are known for their inflammatory response signaling58 and induction of apoptosis59. It was also shown that ceramides containing monohydroxy fatty acids induce stronger pro-apoptotic signaling than non-hydroxy ceramides60. Importantly, however, the ceramide pathway was not significantly altered in our study  lower concentrations of PUFA in epithelial cells of all small intestinal segments, (2) higher levels of monohydroxy fatty acids in the distal small intestinal segments (MJ and ILE) and (3) lower levels of certain tocopherol metabolites across all small intestinal epithelia. As PUFA are highly susceptible to oxidation, their reduced levels in pigs fed the L-Met diet might indicate higher oxidative stress lower co7, was not altered.This is further supported by the changes in the tocopherol metabolism pathway. Although reduced levels of tocopherols could indicate higher oxidative stress, changes were restricted to low-abundance tocopherol metabolites whereas α-tocopherol, which is known as the dominating tocopherol metabolite in enterocytes62. Each of these steps can have an impact on its intraepithelial concentration.As an alternative explanation for the lower levels of epithelial PUFA in L-Met-supplemented pigs, it should also be considered that those lower levels might simply result from altered lipid digestion and absorption. Such postulate has partial support from parallel decreases in several long-chain fatty acids, monoacylglycerols and lysophospholipids in small intestinal epithelia of pigs supplemented with L-Met compared to DL-Met that were indicated by individual group comparisons but did not penetrate to overall significance on ANOVA or pathway enrichment analysis absorbed by the apical sodium dependent bile acid transporter (ASBT), predominantly, in ILE65. Besides active transport, deconjugated primary or secondary BA may pass the intestinal barrier passively in the small intestine and colon65. The latter challenges epithelial cell homeostasis; thus, deconjugated secondary BA have been associated with increased risk for colon cancer, because deconjugation of BA and synthesis of secondary BA occurs primarily in the large intestine67. Nonetheless, anaerobic and facultative anaerobic bacteria may perform deconjugation and hydroxyl group oxidation of conjugated BA already in the small intestine69. Thus, dysbiosis can have a major impact on secondary BA metabolism. It was shown in people and dogs that individuals with active inflammatory intestinal diseases display an increased fecal content of primary BA because of decreased small intestinal absorption and decreased microbial conversion of BA71. It is thus perceivable that the antibacterial effects of DL-HMTBA demonstrated in a porcine intestinal model in vitro72 and in chicken’s cecum in vivo73 may delay small intestinal production of secondary BA. This could explain the observed shift of secondary, especially deconjugated, BA in DL-HMTBA-supplemented pigs with lower levels in MJ and higher levels in ILE. One alternative explanation might be that HMTBA alters the active uptake of conjugated and/or the passive uptake of unconjugated BA in the distal small intestine.Bile acids could influence PUFA digestion and absorption as Rohrl et al.75.The observed changes in bile acid metabolism appear, in principle, compatible with a delayed metabolism of sphingomyelins to sphingosines, where a similar shift in sphingosine metabolites from MJ (lower levels) to ILE (higher levels) was observed in the DL-HMTBA-fed group. It has been shown previously that the release of sphingosine from sphingomyelins by intestinal alkaline sphingomyelinase is strictly dependent on the presence and species of BA13. Histidine can be decarboxylated to histamine, which is well known for its involvement in inflammation and a variety of diseases78. The enrichment pattern of the histidine metabolism pathway was not very clear and included region × treatment interactions for two histidine degradation products (imidazole lactate and cis-urocanate), indicating that dietary Met sources directly influence histidine related metabolites in selected intestinal segments. One very consistent finding was that histamine and its degradation product, 1-methylhistamine, tended to be specifically enriched in DL-Met-supplemented animals. The intestine is rich in mast cells as a primary source of histamine79, possibly implying that epithelial mast cells or their histamine production was stimulated by a DL-Met-containing diet. Alternatively, it has been shown in pigs that the colonocytes take part in clearing histamine from blood, most likely via organic cation transporter 1, followed by biotransformation by histamine N-methyltransferase (to 1-methylhistamine) and diamine oxidase80. Although those previous results were obtained in colon, basolateral organic cation transporters81 and both catabolic enzymes are also present in the small intestine83. Their stimulation by a DL-Met-containing diet could thus explain higher intraepithelial concentrations of histamine and 1-methylhistamine. In support of such postulate, supplementation of DL-Met was shown to induce increased absorption of L-Met in the same pigs as used in the present study23.As mentioned earlier, histidine metabolism was the only amino acid metabolism pathway where diet-induced alterations were enriched in the present study. Indeed, Met is involved in histidine metabolism, as S-adenosylmethionine is a methyl group donor for the production of 1- or 3-methylhistidine and 1-methylhistamine77 and PUFA are also closely related to inflammation as they are precursors of lipid mediators like eicosanoids84. To elucidate a possible relevance of these metabolite changes for inflammatory status, we complemented the metabolomics analysis with qRT-PCR experiments to determine the expression of genes that are related to different inflammatory pathways. The main outcome of the qPCR experiments was that mRNA expression of all investigated genes but IL1β differed among intestinal segments. However, no gene expression differences were observed between feeding groups, suggesting either similar or no effects of the three dietary supplements on intestinal inflammatory status.The alterations in histamine metabolism and the earlier discussed alterations in PUFA and secondary BA metabolism could potentially imply diet-induced changes in inflammatory status. Histamine is a key facilitator of the inflammatory responseThis unique study on the effects of three relevant Met supplements on intraepithelial metabolite concentrations revealed only minor diet-dependent changes of the overall metabolome in epithelia of four small intestinal segments. However, in-depth analysis showed coordinated changes in the PUFA, monohydroxy fatty acid and tocopherol metabolism pathways in pigs supplemented with L-Met. This may suggest either increased oxidation of PUFA and/or decreased absorption of PUFA. The latter requires further investigations as none of these options was clearly supported by changes in other oxidative stress- and lipid digestion-related metabolites. Furthermore, pigs supplemented with DL-HMTBA showed a pronounced shift of secondary BA and sphingosine metabolites from MJ to ILE, possibly indicating altered microbial BA metabolism and/or epithelial transport. Importantly, the observed changes of some potentially inflammation-related metabolites (PUFA and BA), as well as the observed changes in histidine/histamine metabolism in DL-Met-supplemented pigs, had no measurable impact on the inflammatory status of the intestinal epithelia as evidenced by similar expression of several inflammation-related genes.All experiments involving pig handling and treatments were approved by the local authorities responsible for animal care and use approval, the ‘Landesamt für Gesundheit und Soziales Berlin’ (LaGeSo Reg. No. T 0264/15), which included assessment by the advising ethics committee (Tierversuchskommission Berlin). All methods were performed in accordance with German and European guidelines and regulations regarding animal protection and welfare. The study is reported according to the ARRIVE guidelines.23 and are available with open access under https://doi.org/10.1093/jn/nxaa115. In brief, 27 Danbred × Piétrain male castrated pigs were purchased from a commercial farm. Pigs were kept in stainless steel frames with concrete floor (1.9 m × 1.9 m) at the Institute of Animal Nutrition, Freie Universität Berlin, Germany. They were ~ 10 wks old and weighed ~ 25 kg at the start of the trial. The trial was performed in three consecutive runs with 9 pigs per run randomly allocated into 3 groups. Groups received a diet  based on corn, soybean meal, barley, and peas, which met requirements of the National Research Council (NRC)85 and contained sufficient concentrations of all amino acids except standardized ileal digestible Met + cystine (0.46%). To provide adequate standardized ileal digestible Met + cystine (0.67%), diets were supplemented with one of the following Met supplements: 0.21% DL-Met, 0.21% L-Met or 0.31% DL-HMTBA. DL-HMTBA was provided at a higher dietary concentration to account for its lower bioefficacy (~ 70% of Met)1. During the entire pre-feeding period, piglets had ad libitum access to water and feed.Details on the animal trial, sample size and experimental diets have been published previously86.Animals were killed after being on the diet for at least 10 d. Euthanasia was performed at 4 h after the morning feeding that was provided at 6.00 a.m. Pigs were sedated using 20 mg/kg bodyweight of ketamine hydrochloride  and 2 mg/kg bodyweight of azaperone . Pigs were then killed by intracardial injection of 10 mg/kg bodyweight of tetracaine hydrochloride, mebezonium iodide and embutramide . Euthanasia by drug administration was preferred over conventional slaughter by captive bolt stunning or electroshock and exsanguination as the latter initiates profound sympathetic activation with release of adrenaline, noradrenaline and cortisol together with histaminetunica muscularis externa was mechanically removed. Samples of the tunica mucosa were snap-frozen in liquid nitrogen and subsequently stored at − 80 °C for metabolomics analyses. Tunica mucosa samples for qRT-PCR were preserved in RNAlater  and stored at 4 °C overnight, followed by storage at—20 °C until analyses.After killing, a mid-line incision was made, and samples were obtained from DUO, PJ, MJ and ILE. The A complete set of sample aliquots (100 mg to 300 mg of each sample) were shipped to Metabolon Inc.  on dry ice, where samples were kept at − 80 °C until analysis by ultrahigh performance liquid chromatography and tandem mass spectroscopy (UPLC-MS/MS). As samples were analyzed in Metabolon’s semi-automated work flow without any prior communication on possible outcomes, sample blinding was not required. The proprietary technology of Metabolon Inc. includes addition of several recovery standards during sample preparation for quality control purposes. After protein precipitation with methanol, extracts were divided into four fractions, of which two were used for two separate reverse phase (RP) UPLC-MS/MS methods with positive ion mode electrospray ionization (optimized for either hydrophobic or hydrophilic compounds), one for an RP-UPLC-MS/MS method with negative ion mode electrospray ionization and one for hydrophilic interaction liquid chromatography (HILIC)/UPLC-MS/MS with negative ion mode electrospray ionization. Analyses were run on Waters ACQUITY UPLC  and Thermo Scientific Q-Exactive  high resolution/accurate mass spectrometer. MS was combined with heated electrospray ionization (HESI-II) and an Orbitrap mass analyzer operated at 35,000 mass resolution.Following chromatographic mass spectral analyses, raw data was extracted, peak-identified and quality control-processed using Metabolon’s hardware and software. Identification of compounds was achieved by comparison to Metabolon’s compound library that includes retention time/index, mass-to-charge ratio and chromatographic data . Biochemical identifications were based on (1) retention index with a narrow retention time/index (RI) window of the proposed identification, (2) accurate mass match to the library ± 10 ppm and (3) MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores were based on a comparison of the ions present in the experimental spectrum to the ions present in the library spectrum containing ~ 5,300 commercially available purified standard compounds. Peaks were quantified using area-under-the-curve. A data normalization step was performed for studies spanning several analytical days. Each compound was corrected in run-day blocks by registering the medians to equal one and normalizing each data point proportionately.Statistical analysis was run by Metabolon Inc. as part of the provided service. Analyses included log transformation of data and imputation of missing values prior to repeated measures ANOVA with the fixed factors region , treatment  and the two-way region × treatment interaction. Collection cohort was incorporated as a random effect.P ≤ 0.05, with tendencies towards significance defined at 0.05 < P ≤ 0.10.Metabolites that showed significant treatment effects or trends towards a treatment effect and metabolites that showed region × treatment interactions were incorporated into a pathway enrichment analysis based on Metabolon’s pathway map using the chi square test implementation of base R. No multiple-testing correction was performed to increase sensitivity over specificity for the pathway enrichment analysis. Results were considered significant at Principal component analysis using the prcomp function from base R was performed to identify and visualize similarities in data set structures among data of different regions and treatments.The Nucleospin RNA kit  was used for RNA extraction according to the manufacturer’s instruction. RNA quality was checked using lab-on-a-chip electrophoresis by Agilent 2100 Bioanalyzer . Four samples were excluded because of poor RNA quality (RIN < 6). All other samples had satisfactory RNA quality (RIN ≥ 6). Purity of RNA was verified by NanoPhotometer P330, Version 1.0 IMPLEN  with absorption ratios ≥ 2.0 at 260/280 nm and 260/230 nm. NanoPhotometer P330 was also used to determine RNA concentration.2O .cDNA was synthesized from a 1,000 ng/μl RNA template using iScript cDNA Synthesis Kit and iCycler iQ . A -RT (reverse transcriptase) sample was prepared without enzyme mix to be used as negative control in qRT-PCR. The thermal cycler protocol was run according to iScript cDNA Synthesis Kit instructions . After reverse transcription, cDNA was diluted 1:10 with ddHCASP1, NLRP3, IL1β, IL8, IL18 and TGFβ. The following unregulated housekeeping genes were used for normalization: GAPDH  and ACTB (β-actin). The unregulated expression of the used housekeeping genes was verified statistically  was used. Run method included 40 amplification cycles . Thresholds were manually set to 1,500.qRT-PCR was performed using iQ SYBR Green Supermix Kit  in assay volumes of 15 μL with 5 μL cDNA. Reactions were carried out in 384-well plates  with three replicates per reaction. For negative control, ddHt) values were normalized to the mean value of the two housekeeping genes and calibrated to the pool sample using the ΔΔCt method88. Calibrated normalized relative quantity expression data was used for statistical analysis. 19 Outliers were identified using a Grubbs test89 at a significance level of 0.05 and removed from the dataset. Five values were excluded due to operational errors (n = 7–9). Sigma Plot 11.0  was used for statistical analyses. A two-way ANOVA with the fixed factors intestinal tissue region , dietary treatment  and the two-way region × treatment interaction was performed, followed by Student-Newman-Keul’s posthoc test. Results were considered significant at P ≤ 0.05.Cycle threshold (CSupplementary Figure 1.Supplementary Table 1.


In this study, an investigation of the shear behavior of full-scale reinforced concrete (RC) beams affected from alkali–silica reactivity damage is presented. A detailed finite element model (FEM) was developed and validated with data obtained from the experiments using several metrics, including a force–deformation curve, rebar strains, and crack maps and width. The validated FEM was used in a parametric study to investigate the potential impact of alkali–silica reactivity (ASR) degradation on the shear capacity of the beam. Degradations of concrete mechanical properties were correlated with ASR expansion using material test data and implemented in the FEM for different expansions. The finite element (FE) analysis provided a better understanding of the failure mechanism of ASR-affected RC beam and degradation in the capacity as a function of the ASR expansion. The parametric study using the FEM showed 6%, 19%, and 25% reduction in the shear capacity of the beam, respectively, affected from 0.2%, 0.4%, and 0.6% of ASR-induced expansion. The durability of concrete is an important consideration for reinforced concrete (RC) structures. One of the major concerns is the degradation of concrete from alkali–silica reaction (ASR), which is a slowly occurring chemical process between the hydroxyl ions available in the pore water of concrete and the aggregate containing reactive silica. The chemical product, named ASR gel, is highly expansive in the presence of moisture. The gel expansion develops an internal pressure and induces micro-cracking, which degrades the mechanical and physical properties of the concrete . As micr3 and 75 kg/m3 of hooked-end steel fibers. All beams were 250 mm wide, and each group included three beams each with 500 mm, 1000 mm, and 1500 mm height and an effective depth 60 mm less than the beam height. The shear span for each beam was three times the effective depth of the beam. The results showed that the modified compression field theory and disturbed stress field model accurately predict the shear behavior of different size beams with and without fibers. Wu and Hu [The shear failure of RC beams is relatively less understood; particularly, for damaged infrastructure components. Sucharda  presenteu and Hu  presenteu and Hu  presenteu and Hu . Schmidtu and Hu  presenteASR, being one of the most prevalent damage mechanisms in RC structures, deserves further investigation with regards to its influence on the shear capacity of RC beams. This study aims to fill this gap of scarce data and analysis on the shear capacity of ASR-damaged beams. First, a comprehensive literature review has been performed to synthesize the available data in terms of the reduction in concrete mechanical properties as a function of ASR expansion. Then, the results of two full-scale shear tests on a RC beam have been presented in terms of maximum shear capacity, crack maps and widths, and strains in the reinforcement. The two shear spans had the same geometry and reinforcement details such that the repeatability of the experiments could be verified. The results from the second shear test in which the beam was pushed to ultimate strength and failure have been used to validate a finite element model (FEM), which was later used to simulate the long-term structural behavior of the beam affected from ASR. The reduction in the mechanical properties of the concrete was estimated using relationships obtained from data synthesized from the literature and then used as inputs to the FEM in a parametric study. The reduction in the shear capacity of the beam was predicted as a function of the concrete ASR expansion. Numerous studies have reported the effect of ASR on the mechanical properties of concrete; particularly, on the compressive and tensile strength, and Young’s modulus. However, the effect on the compressive strength is disputed among researchers ,18. MostA comprehensive experimental program was performed by the authors of this study on the effect of ASR on the mechanical properties of concrete using accelerated aging . A 0.8% Sanchez et al.  preparedEsposito et al.  studied Fan and Hanson  performe3 of NaOH pallets were added to the concrete mixtures to accelerate ASR. To accelerate aging, the specimens were cured in an environmental chamber at 50 °C and 100% relative humidity for 8 months. Compression and splitting tension tests were performed at different ages on 75 mm diameter cores drilled from the specimens. It was observed that the expansion was significantly lower in the restrained directions while the volumetric expansion remained relatively constant. Fifteen percent less volumetric expansion for the biaxially stressed specimens was measured compared to the unstressed specimens. The splitting tensile strength for the unrestrained non-reactive specimens increased by 60% while the reactive concrete showed a 50% reduction. The increase in the strength was attributed to the continuing hydration of the anhydrous cement in the samples. No statistically meaningful change was observed in the compressive strength while an average of 20% reduction was observed in the elastic modulus. The confinement increased the compressive strength while the elastic modulus was similar both in restrained and unrestrained samples.Gautam et al.  performe3 were used in the concrete. Additional Na2SO4 and K2SO4 were added to the mixture to increase the alkali content to 7 kg/m3. After curing, the specimens were kept in water at 38 °C to accelerate the chemical reactions. Expansions in 100 × 100 × 500 mm prisms and 150 × 300 mm cylinders were measured using a digital Vernier scale. The reduction in the compressive strength was 59% after 12 months at 1.6% expansion. The losses in the split tensile and direct tensile strength were found to be 60% and 80%, respectively.Ahmed et al.  investig3. The specimens were stored at 38 °C in a high humidity environment according to the Canadian CSA A23.2-14A Standard [Sargolzahi et al.  evaluateStandard . The com−1 NaOH solution at 38 °C for 3.5 months. The alkali content  of the reactive concrete was 5.25 kg/m3. Ten prisms, 75 × 75 × 300 mm, with reference studs for expansion measurement and 40 cylinders, 100 × 200 mm, for mechanical tests were prepared. The maximum expansion in the reactive and non-reactive concretes was measured as 0.19% and 0.02%, respectively. While the difference between compressive strength for the reactive and non-reactive mixtures was insignificant, a 13% reduction in the elastic modulus was observed.Saint-Pierre et al.  applied 2O by mass of cement, respectively. The high-alkali mixture was prepared by dissolving NaOH pellets in the mixing water. Non-reactive coarse limestone and fine granitic aggregates were used for both concretes with a water-to-cement (w/c) ratio of 0.41. The specimens were kept at 23 °C and 100% relative humidity until testing. Prisms 50 × 75 × 350 mm in size and 100 × 200 mm cylinders were tested at various ages up to 180 days. The results revealed the difference between the properties of concrete samples made from high-alkali and low-alkali mixtures. At an age of six months, the high-alkali concrete specimens showed 12% lower compressive strength, 7% lower modulus of elasticity, 15% lower direct tensile strength, 14% lower split tensile strength, and 3% lower modulus of rupture compared with the low-alkali concrete specimens.Smaoui et al.  evaluate2O equivalent content of the mixture to 1.25%. Cylinders with measurements of 160 × 320 mm were tested for mechanical properties. The samples were kept at 38 °C for 12 months in sealed conditions. The elastic modulus of the reactive concrete was 20% lower at 12 months compared to the 28-day value and 27% lower compared to the non-reactive concrete. The tensile strength was 3% higher after 12 months compared to the 28-day value but 9% lower compared to the non-reactive concrete of the same age. The reactive concrete also had a 10% increase in compressive strength after 12 months but the difference with the non-reactive concrete was indistinguishable.Multon et al.  performe2O equivalent of 5.25 kg/m3. The cylinders were initially wrapped with plastic to prevent moisture loss and then stored at 38 °C and 95% relative humidity. In the El Paso sand concrete, the compressive strength and elastic modulus showed a 13% and 45% reduction, respectively, after one year of aging and 0.6% expansion compared to their 28-day values. The New Mexico gravel concrete showed a 10% increase and a 19% reduction in the compressive strength and elastic modulus, respectively, after one year of aging and 0.85% expansion.Giannini and Folliard  used El More than 50 data points for elastic modulus, and compressive and tensile strength were extracted from the literature, as listed in The experimental program and the results are described in detail in a recent study by the authors . Since tConcrete with a w/c ratio of 0.52, a slump of 190 mm and an average 28-day strength of 27.5 MPa was designed as shown in To obtain the mechanical properties of the concrete, standard material tests were performed at 28 days and 210 days after casting, the latter of which being when the beam was tested. SPi, for deflection measurements under the loading points. Additionally, the strain gauges installed on the stirrups and longitudinal reinforcement are shown in A full-scale RC beam, 6.4 m in length with a rectangular cross-section 0.6 m deep and 0.3 m wide, was fabricated. The volumetric ratio of the gross longitudinal reinforcement was constant at 4.2% over the length of the beam, while a light transverse reinforcement with a volumetric ratio of 0.13% was provided in the two shear spans with a span-to-depth ratio of about 2.9, as shown in The test setup consisted of the components shown in The tests were conducted under quasi-static loading. As described in  in detaiAlthough both tests resulted in a shear failure, since during the second test the beam was pushed to the complete failure, for brevity, the results of the second shear test are presented in the following. As mentioned earlier, a FEM is created for the shear failure of the RC beam using ATENA 3D . The selA bilinear model with strain hardening, CCReinforcement, was used to model the steel reinforcing bars. The input parameters were derived from the uniaxial rebar testing for different steel reinforcements in the beams as presented in The geometry of the model is based on the dimensions of the beam presented earlier. The model is composed of three parts: the concrete beam, reinforcement, and the steel plates for the supports, loading plate and the clamps. The concrete was meshed with iso-parametric 3D linear brick elements see . To modeThe load was simulated by applying a 25.4 mm prescribed deformation, for a complete analysis, at increments of 0.01 mm displacement in the negative Z, vertical, direction. The supports were free to rotate about the transverse axis of the beam and they were restricted to move in the vertical direction. Surface springs were placed under the supports to represent the behavior of elastomeric pad of the supports shown in The Newton–Raphson method with the line search optimization approach and tangent type stiffness was used for solving the governing equations. The line search strategy adjusts the displacement increments to optimize the required work and reduces the out-of-balance forces. The FEM is validated using the experimental data from the second test. Like the second shear test, the first test also resulted in shear failure but the shear failure of the second test included the ultimate shear load capacity and the load drop since the test continued to capture the entire failure. The primary step taken for validation is to compare the load versus deflection diagrams, which is shown in For further evaluation of the FEM, the crack patterns corresponding to the peak load are compared, as shown in The FEM was also validated by comparing the reinforcement strains. From four #3 stirrups, the ones close to the middle of the span showed a significant strain. Therefore, in FG is the fracture energy (N/m) and cmf is the mean compressive strength (MPa) [In this section, the validated FEM is used to evaluate the influence of ASR on the shear performance of the beam. This investigation is based on the degradation in the concrete mechanical properties due to ASR. The mechanical properties affected by ASR are chosen as the concrete compressive and split tensile strength, and elastic modulus. The equations extracted from the literature, as presented in th (MPa) . Three cAdditionally, for different ASR cases, The shear tests indicated that the nonlinear shear behavior governed the response of the beams with span to depth ratios of 2.9, a shear reinforcement ratio of 0.13%, and a longitudinal reinforcement ratio of 4.2%. This was confirmed based on the data from the strain gauges installed on the shear and longitudinal reinforcement.The major diagonal shear cracks were from the top corner loading point towards the bottom corner support. Some secondary diagonal shear cracks also occurred between the top corner loading point and the middle of the span at the bottom and between the bottom corner support and the middle of the span at the top.The major shear cracks were more concentrated in the middle of shear span than towards the ends. Similarly, the stirrups close to the middle of shear span yielded while the stirrups close to the loading point and support remained elastic throughout the test.According to the parametric study using the validated FEM, 0.2%, 0.4%, and 0.6% of ASR expansion induced 6.3%, 19.4%, and 25.4% reduction in the shear capacity of the beams.For ASR degradation less than 0.4% expansion, the maximum crack width at peak load decreased compared to the reference model but as the ASR degradation progressed, the maximum crack width at peak load increased. For the highest level of ASR degradation with 0.6% expansion, the maximum crack width at peak load slightly exceeded that of the reference model. This happened due to the significant reduction in the beam stiffness, which caused higher deflection at a much lower peak load.For ASR degradation less than 0.2% expansion, the stirrup strain at peak load is reduced compared to the no ASR case. However, for higher levels of ASR with 0.4% and 0.6% expansion, the strains of the stirrups exceeded the strains in the reference model. This is because the stirrup strain is related to the deflection. The deflection at peak load is smaller at the early stages of ASR but then it increases as the ASR further develops due to further reduction in the material properties. Early-stage ASR results in a slightly lower peak load and corresponding deflection. Higher ASR degradation considerably affects the beam shear performance and reduces the peak load and stiffness. This causes more deflection in the beam and higher stirrups strain.For different levels of ASR degradation, no significant variation in the top longitudinal reinforcement strain at peak load was observed. On the other hand, as the level of ASR degradation increased, the strain of the bottom longitudinal reinforcement at peak load decreased. Unlike the stirrup strains, which were correlated to the deflection at peak load, the strain of the bottom longitudinal reinforcement was correlated to the peak load.As a part of this study, a large-scale beam was constructed and tested to study its shear performance. The beam was designed to fail in shear prior to any flexural yielding. Two shear tests were conducted by applying a load to each shear span of the beam, with span-to-depth ratios of about 2.9 at each end. The shear tests were conducted under quasi-static loading. The acquired data were processed, and the experimental results, including shear load versus deflection diagram, reinforcement strains, crack maps and maximum crack width at peak load were used to validate a detailed FEM of the beam. The validated FEM was in turn used to study the effect of different levels of ASR degradation corresponding to 0.2%, 0.4%, and 0.6% of the concrete expansion. The reduced concrete compressive strength, tensile strength, modulus of elasticity, and fracture energy were calculated based on ASR expansion and used in the FEM. The following conclusions are made:The computational models in this study were developed to capture the degradation of beam shear strength as a result of degradation of the concrete mechanical properties from ASR. The relation between the concrete degradation and ASR expansion was derived from the literature and implemented in the models but the models did not consider the influence of the self-prestressing effect as a result of concrete expansion. Therefore, further research is needed to quantify the self-prestressing effect as a function of the ASR expansion, reinforcement size and ratio, and concrete properties, and if significant implement this effect in modeling the behavior of reinforced concrete members affected from ASR.