Historically, studies with amino acids have focused on protein synthesis and accretion, especially with eggs and meat, whereas less importance has been given to their secondary functions on the metabolism. However, certain amino acids, such as arginine, methionine, and cysteine are precursors for other essential molecules in the immune defense, antioxidant system, cell signaling, and gene expression, and can act as regulators in the growth and development of the animals. Because poultry are subjected to stressful conditions throughout their lives, the use of these amino acids and their secondary functions could beneficiate their general health. This review describes the metabolism of arginine, methionine, and cysteine and how they modulate different tissues, especially during challenging conditions. Arginine supplementation has been shown to modulate musculoskeletal health development, reduce fat accretion, and improve the antioxidant system. Moreover, methionine and cysteine could improve the bone development and have a potential in mitigating the negative effects caused by heat stress. Understanding how these amino acids can ameliorate stressful conditions may provide novel insights about their use as nutritional strategies to modulate the health status of chickens.Eimeria infection. The use of arginine has been shown to reduce abdominal fat pad in ducks and increase lean tissue and bone mineral density in broilers. Additionally, the sulfur amino acids have been shown to improve bone development and are beneficial during heat stress. The use of L-methionine increased the cortical and trabecular bone mineral densities, in laying hens. Moreover, the dietary inclusion of these amino acids could reduce the damage caused by Eimeria spp. infection by regulating the antioxidant system and cell repair. Understanding how these amino acids can mitigate stressful conditions may provide us novel insights of their use as nutritional strategies to modulate the health status of chickens.Amino acids such as arginine, methionine, and cysteine are the precursors of essential molecules that regulate growth and health, being classified as functional amino acids. This review describes the metabolism of arginine and the sulfur amino acids and how they modulate, directly or indirectly, different tissues. Emphasis is placed on their effects in supporting health during challenging conditions, such as heat stress and  Over 700 amino acids (AA) can be found in nature; however, only 20 of them have been recognized as building blocks for proteins in cells ,2. HistoArginine, an essential amino acid for birds , has beeMethionine is also classified as an essential and functional AA . BecauseEimeria spp. infection and heat stress.Although much research in mammals has been performed to evaluate the roles of Arg and TSAA as regulators of key metabolic pathways to improve health, growth, and development, the research addressing the functional properties of these AA in chickens remains scarce. Furthermore, the requirements for Arg and TSAA for broilers and laying hens available in the literature considerably vary and are usually extrapolated from studies performed under comfort conditions. However, different stressors, such as environmental temperatures and intestinal diseases, could impact the availability and metabolic needs of these AA ,19,20, bArginine (Arg) is an essential amino acid (AA) required by animals to support protein synthesis, growth, and biological functions . For mamHowever, unlike mammals, the urea cycle in poultry is not entirely functional as they use uric acid as the main nitrogenous waste form . Tamir and Ratner  investigArginase is found in microphages, liver, and kidney, where it has its highest activity ,26. OrniNOS catalyzes the conversion of Arg into NO and citrulline, and it is believed that this is the only physiological path for NO production with Arg as the only substrate for all the isoforms of NOS . BecauseStudies in poultry ,8,10,15 Arginine was shown to act as a secretagogue for insulin  and growAdditionally, the bone development and health could be improved through the actions of GH, IGF-I, and NO following Arg supplementation. In vitro, Arg supplementation directly stimulated IGF-I at both mRNA and protein levels and increased collagen expression and synthesis in mouse osteoblast-like cells . It has The metabolism of Met and Cys, the TSAA, is directly related to the pathway of other molecules, such as homocysteine, taurine, glycine, glutathione (GSH), and polyamines, as previously reviewed in the literature ,54,55. IThe L-Met, coming from the diet or muscle catabolism, enters the transmethylation step, which begins with the activation of L-Met to S-adenosylmethionine (SAM). After SAM donates its methyl group to an acceptor , it becomes the coproduct S-adenosylhomocysteine (SAH) , which iThe remethylation process ultimately results in the de novo synthesis of L-Met, through the addition of a methyl group to homocysteine. The transsulfuration pathway, which only occurs in the liver, kidney, intestine, and pancreas, consists of the conversion of homocysteine and serine to Cys . It is aSeveral antioxidant and modulatory functions have been attributed to taurine , which cBecause Met can be irreversibly converted to Cys, the Cys need can be met by supplementing Met, while the requirement for Met can be satisfied only by Met supplementation to the diet . Thus, i14C-DL-Met in intact small intestine samples from birds subjected to cyclic chronic HS and thermoneutral conditions. Both studies observed a decrease in DL-Met uptake in HS samples, suggesting a shift from the energy sodium-dependent (ESD) transporters to the energy sodium-independent (ESI) transporters. Therefore, the changes in digestion and absorption due to HS could alter the TSAA use by the birds, leading to different requirements.Several potential stressors have been identified to occur during the birds’ lives, such as temperature stress either by cold  or heat Moreover, the HS ultimately leads to cellular oxidative stress in poultry, and the TSAA supplementation could mitigate the negative effects of HS by improving the antioxidant capacity. A study conducted by Del Vesco et al.  showed tAnother important economic and welfare issue faced by the poultry industry is related to bone health. A survey conducted with end-of-lay hens reported that approximately 29% of laying hens had at least one broken bone during their lifetimes in Europe . MoreoveCastro et al.  have repOver the years, the acknowledgement of the importance of nutrition to the gastrointestinal tract (GIT) health and function has increased, and Arg and TSAA were pointed out for their essential influence on GIT protection and development. In adult rats and adult humans, 38–40% of the luminal absorbed Arg is catabolized in a single pass and utilized by the intestinal mucosa, whereas the remaining 60% enters the circulation intact ,90. AccoThese findings suggest a high intestinal metabolism of dietary Arg and Met, and that the GIT might have its own requirement for these AA, which should be considered when considering their supplemental levels. This requirement could be related to energy generation, protein incorporation, or polyamine, Cys, and GSH synthesis . MoreoveEimeria, is one of the most important parasitic diseases in poultry production. It is commonly accepted that there are seven species of Eimeria which can infect chickens [E. acervulina, E. maxima, and E. tenella. The Eimeria reproductive stages take place in the intestinal epithelium, causing severe tissue damage, disruption of digestion and nutrient absorption, inflammation, and increase in oxidative stress [Coccidiosis, caused by a protozoa of the genus chickens ; howevere stress ,97,98,99e stress .Eimeria infection in broilers is related to a decrease in feed intake, body weight gain, feed efficiency, and increase in mortality [Eimeria on performance was not exclusively linked to anorexia [E. acervulina, E. maxima, E. tenella, and an Eimeria spp. pool, respectively, at a constant feed intake. Therefore, we can assume that, in this kind of challenge, the nutrient utilization can be reduced, and the nutrients absorbed are being redirected to maintain the health status rather than growth.The performance loss observed during an ortality . A meta-anorexia . The weiEimeria spp. life cycle takes place in the enterocytes, inflammation, cell death, and sloughing are commonly observed in the intestine of the birds [E. avervulina infection, leading to flattened and thickened villi, with ruptured enterocytes, elongated crypts, and higher turnover rate [Eimeria infection has been reported by Russel and Ruff [Eimeria infection, the digestibility and absorption of nutrients are impaired.Since part of the he birds . The intver rate ,102. Morand Ruff , Allen [and Ruff , and Adaand Ruff . ConsequE. necatrix [E. acervulina [E. maxima [E. acervulina. The authors also reported that the negative effect of the infection on the AA digestibility was greater during the first 2 days post-infection than in later periods. Rochell et al. [E. acervulina . The authors observed a linear and quadratic decrease for Arg and Met digestibility, respectively, with increased oocyst dose. The AID reduction was of approximately 2 percentage units for Arg and Met compared to the uninfected birds.A reduction in L-Met uptake by different intestinal sections, in vitro, was observed in birds challenged with necatrix , E. acerervulina , and E. . maxima  at 7 day. maxima  observedl et al.  studied 0, + and L was down- and up-regulated, respectively, in birds challenged with E. Maxima. The b0, + system, which is a Na+-independent AA transporter, mediates the inward transport of Cys, Arg, and lysine in exchange with neutral AA [+-independent AA transporter, mediates the inward transport of large, neutral, branched or aromatic AA in exchange for the efflux of other AA such as isoleucine, leucine, and Met [0, + system and increase in Met efflux, there is a decrease in intracellular pool of Arg, Met, and other essential AA. The authors hypothesized that this change in transport gene expression may be part of the defense mechanism against the infection, once the decrease in intracellular essential AA would limit the energy and protein production in the cell, leading to inhibition of Eimeria spp. replication and potentially cell death. Therefore, it is possible that the Arg and TSAA requirement might be increased due to the intestinal damage and changes in the digestion and absorption processes.Additionally, Paris and Wong  observedutral AA , whereas and Met . The L s and Met . Therefo2−), hydrogen peroxide (H2O2) and hydroxyl radical (·OH) [The reactive oxygen species (ROS) are a group of free radicals generated during the physiological oxygen metabolism  and are al (·OH) . At biolal (·OH) ,116,117.al (·OH) ,119. Recal (·OH) , in ratsal (·OH) . Becauseal (·OH) ,122.Eimeria spp. infection model, the supplementation of L-Met was able to increase the GSH levels in the liver of broilers compared to the deficient diet, ameliorating the negative effect of coccidiosis on the antioxidant system [E. coli could be oxidized with little effect on its catalytic effect. Kim et al. [Recent studies have demonstrated the antioxidant properties of Met in poultry and its potential in alleviating oxidative stress. The Met supplementation (5.9 g/kg) in broilers resulted in higher SOD activity in the serum, decreased hepatic malondialdehyde content at 7 days of age, and decreased GSH:GSSG ratio in the liver at day 21 . In 16-wt system . In addit system ,127. Thet system ,127. Thet system  observedm et al.  also demPutrescine, spermine, and spermidine are the most common polyamines, which are small aliphatic polycation molecules widely distributed in nature. The formation of these polyamines in birds is dependent on both Arg and Met ,130. It Eimeria acervulina challenge dosage, and, when the highest dose was used (1.0 × 106 sporulated oocysts), this reduction was off 22% compared to noninfected birds. Additionally, the authors have also observed that this plasmatic Arg reduction was accompanied by a 95% increase in plasmatic ornithine. Two theories were provided by the authors to explain these findings: (1) the reduction in plasmatic Arg of infected birds may have been related to increased demand of Arg for NO production, which is a key mediator of the immune response to coccidiosis [Rochell et al.  found a cidiosis ,133; andcidiosis . In thisEimeria spp. infection, and the supplementation of these amino acids could help to improve the intestinal health during and after the infection. Thus, future studies should address the supplementation of arginine and total sulfur amino acids as nutritional strategies in improving health, especially in birds subjected to stressful conditions including HS and disease infection.The supplementation of arginine and the total sulfur amino acids has been shown to improve the musculoskeletal development, the intestinal health, and the antioxidant system of poultry. The mechanism by which these amino acids modulate general health is mainly associated to their function as precursors of key metabolic molecules, such as nitric oxide, creatine, and glutathione, and as secretagogues for growth hormone and IGF-I. Furthermore, the digestion and absorption of both arginine and the total sulfur amino acids were shown to decrease in an


The design of new host–guest complexes represents a fundamental challenge in supramolecular chemistry. At the same time, it opens new opportunities in material sciences or biotechnological applications. A computational tool capable of automatically predicting the binding free energy of any host–guest complex would be a great aid in the design of new host systems, or to identify new guest molecules for a given host. We aim to build such a platform and have used the SAMPL7 challenge to test several methods and design a specific computational pipeline. Predictions will be based on machine learning (when previous knowledge is available) or a physics-based method (otherwise). The formerly delivered predictions with an RMSE of 1.67 kcal/mol but will require further work to identify when a specific system is outside of the scope of the model. The latter is combines the semiempirical GFN2B functional, with docking, molecular mechanics, and molecular dynamics. Correct predictions  are contingent on the identification of the correct binding mode, which can be very challenging for host–guest systems with a large number of degrees of freedom. Participation in the blind SAMPL7 challenge provided fundamental direction to the project. More advanced versions of the pipeline will be tested against future SAMPL challenges. Supramolecular chemistry has experienced enormous growth in recent years. Supramolecular processes, and host–guest systems in particular, are studied both from a fundamental perspective and for their possible applications –4. By imSeveral years of SAMPL  challenge have shown interesting approaches to compute the binding free energy of host–guest complexes, with a relatively large range of methods and performances –8. In thIn any computational strategy for structure-based design, an important step is the prediction of probable conformations of the receptor-ligand complex –15. As aAt this stage, more important than the actual predictions, participation in the SAMPL7 was extremely useful to test several ideas in blind mode, resulting in the design of an automatic pipeline that will be evaluated in subsequent SAMPL challenges and eventually become publicly available.Docking is used to generating a first guess of the Host–Guest structure. We chose ADV for our assessment for several reasons. It (i) is faster and generally performs better than AutoDock itself, (ii) is freely available and competitive with commercial tools , 22.bind, the predicted binding energy in kcal/mol (‘score’ =  − ΔGbind). To obtain the maximum number of poses, we set num_modes to 20. Three different box sizes are used, all of them cubic and centered in the host cavity: one big box (edge length 10 Å) that allows completely blind docking, and two small boxes  restricted to the expected binding site. For each box, the top-scored solution is extracted. Additionally, for the smallest box, the extraction is followed by a steepest descent [Docking was performed using AutoDockVina v. 1.1.2 . Input c descent  and conj descent  minimizaHost and guest preparation The antechamber and tleap programs from the AMBER 18 package [ package  are used package . For the package  in a cubMinimization The system is minimized with GROMACS [ GROMACS –34 usingAll molecular dynamic (MD) simulations were set up in two steps:Production simulations were run also with GROMACS in the NPT ensemble with temperature control using V-rescale thermostat  at 300 Kextreme. Optimization, followed by hessian calculations were performed. The resulting geometries were verified as true minima by checking that no imaginary vibrational frequencies remained. The temperature was set to 298.15 K for assessing the thermostatistical corrections.We used the xtb program package (version 6.1)  to calcuFor the GDCC-7 dataset in SAMPL7, we decided to test a machine learning approach, taking advantage of pre-existing data. All OA or TEMOA host–guest systems from previous SAMPL challenges (SAMPL4 to SAMPL6) were collected, reconstructed from 2D to 3D, and optimized with the GFN2B method. Then, we used the CORINA web-platform  to compuThe descriptors of the dataset are reduced using the R software  with difIn order to predict the binding free energy, several machine learning models using regression are used: neural network , knn 4848, polynRRHOT) calculated following a coupled rigid-rotor-harmonic-oscillator approach, and the solvation contribution (Gsolv) calculated by the implicit solvation model GBSA.The Gibbs free energies of the optimized geometries were calculated as the sum of the Electronic Energy (E), which includes the D4 dispersion correction, thermostatistical corrections . The variation in free energy was as large as 10 kcal/mol for the different geometries, which confirmed the importance of conformational sampling. The overall lowest energy structure was defined as a reference for free energy calculation. Though the degrees of freedom of the guests are much reduced, we use a similar protocol for consistency  produced complexes with negative binding energy (scoring), but the guest only formed surface interactions with the host. This led us to test two additional docking conditions where the docking space is progressively reduced. The resulting docking geometries have positive scores, indicative of conformational clashes, but in this case, the guest inserts into the host cavity. Three to five different binding modes were selected for each docking protocol. Minimization with Chimera , a molecular mechanics (MM) minimization of the docking solution is performed with MOE and CHIMERA, thus removing any potential clash between host and guest. For some particular systems (G08 and G10) the MM minimization was deemed insufficient to attain a relaxed complex. In those cases, docking was followed by 200 ns of MD simulations. Even then, it failed to generate any binding mode where the guest is embedded into the cavity of the cyclic host. Further adding to our problems, the sulfonate groups tended to form unrealistic interactions after minimization with xtb. In some cases, the sulfonates were even inserted into the host pocket, which is largely hydrophobic, instead of remaining solvent-exposed, as expected for a negatively charged group Fig. . This inContrary to what was observed with the trimer-trip host–guest systems of previous editions, we had to conclude that a better method was necessary to generate correct binding modes for the SAMPL7 test set. Our method should allow for host flexibility in order to allow guest embedding with reasonable geometries. On the other hand, it was clear that the implicit solvation model implemented in xtb was falling short for ionic systems, and explicit solvation would be necessary for the conformational sampling stage. Both requisites pointed to MD simulations as an optimal solution, which we proceeded to implement and test.In what we refer as Spontaneous association MD (SaMD), the host and the guest are simulated in a TIP3P water box, starting from a dissociated configuration (distance ≥ 8 Å), extending the simulation until binding is observed. For the linear guests G01, G02, and G05, SaMD successfully completed the inclusion process, which proceeded in two steps: (i) rapid formation of surface contact between host and guest, leading to stable interactions; and (ii) a small opening of the host system, enabling the entry of the guest into the host cavity and formation of a stable complex Fig. . The secThis result indicates that starting from metastable host conformations may be a general strategy to accelerate SaMD and generate valid host–guest geometries.Notably, the binding mode of the guests inside the host is very dynamic, with fast rotations and frequent sliding movements that are only limited by the resistance of the charged group of the guest to enter the hydrophobic core of the host. As expected, the ionic groups rarely form direct contacts. Instead, they preserve their solvation shells. Overall, these results suggest that SaMD is an optimal and feasible strategy not only to obtain a bound conformation of the host–guest complex but also to capture the rich conformational diversity of the bound state. Unfortunately, between the setting up and testing of this protocol and the computational cost of the MD simulations, it was impossible to complete all these calculations by the challenge deadline. Posterior analysis confirms that correct identification of the binding mode through SaMD improves the quality of the binding free energy predictions (see next section).For each complex, we extract 5 to 10 different binding modes generated with the above-described protocols. These geometries are then individually minimized at the xtb-GFN2B semi-empirical level, and only those yielding a true minimum  are considered. The lowest energy complex is considered as the true minimum, except for a few cases where visual inspection identified issues with the corresponding geometry, always related to inadequate screening of charges by the implicit solvation method, such as those shown in Fig. For guest G18 and G19 we could not find a correct binding mode SaMD, and the docking results gave positive binding energy. As both protocols failed for these two cyclic guests (presumably due to their large volumes) we desisted from making predictions for them.We can see in Fig. In Fig. For GDCC prediction, as there was an important amount of pre-existing data from previous challenges, we decided to try an orthogonal approach-based ML. The dataset includes 35 compounds in total, belonging to three classes of host systems that are similar in structure and chemical composition: OA, TEMOA, and exoOA Fig. . The binThe GDCC-7 dataset to be predicted this year consisted of 8 guest compounds (4 charged and 4 non-charged) binding to two related host systems. After the model has been optimized, it takes only 10 s to calculate the free binding energy of the 8 guests in the 2 hosts. With RMSE and MAE values of 1.67 kcal/mol and 1.21 kcal/mol, respectively, the overall performance is rather satisfactory, especially by comparison with the thermodynamic-based approach. It is worth noting that the four negative guests are not predicting well, which can be explained by the limits of the model imposed by the composition of the training set: since the least favorable binding free energy value is − 3.73 kcal/mol, the model can’t predict more positive values. Even then, the hierarchy between the guest values is respected (G4 < G3 < G2). There is no experimental value for G1, so it has not been considered for this analysis. If we apply the same analysis to every subgroup (based on the positive or negative charge and the host they are interacting with) we obtain an almost perfect hierarchical prediction. The only exception is the OA-G7 complex, which was predicted lower than OA-G6 due to the fact that OA-G7 has been underestimated  while OA-G6 have been predicted very close to his experimental values .In fact, all systems, except for the four negative compounds interacting with exo-OA, are predicted within 1 kcal/mol of the experimental values Fig. . For theThe participation in SAMPL7 allowed us to test two orthogonal approaches to calculate host–guest binding free energies, identifying in each case strengths and limitations that will be considered for the final design of an automated platform.The thermodynamic-based approach is absolutely general and can be used, in principle, on any host–guest system. The use of an advanced semiempirical basis set (GFN2B) to calculate energies and thermostatistical corrections offers increased performance relative to MM approaches with a moderate computational cost (1–2 h on a single CPU) and eliminates the dependency on small-molecule force-fields, which are often inaccurate , 56. HowThe use of knowledge-based methods can be highly advantageous when there is sufficient pre-existing data. Contrary to protein–ligand complexes, where a large body of data exists, host–guest systems cannot benefit from massive training sets. Thus, we were particularly interested in examining the suitability of machine learning approaches, with a particular concern on the risk of overfitting. The results obtained on the GDCC system are really encouraging and motivate us to build a database of host–guest systems, with their corresponding binding free energies, and train both general and host-specific models. Two critical aspects that will be explored are the use of other molecular descriptors to improve the predictions and the introduction of selection criteria to decide when a particular system is within the scope of the model.Overall, the participation in SAMPL7 has allowed us to design an automatic pipeline to compute binding free energies for any Host–Guest system. We currently implementing and improving the protocol, that will be tested in subsequent SAMPL editions.See Table See Table


MET alterations in 474 advanced non‐small‐cell lung cancer (NSCLC) patients by nCounter, an RNA‐based technique. We identified 3% with METΔex14 mRNA and 3.5% with very‐high MET mRNA expression, a surrogate of MET amplification. MET alterations identified by nCounter correlated with clinical benefit from MET inhibitors. Quantitative mRNA‐based techniques can improve the selection of patients for MET‐targeted therapies.We studied  MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA‐based technique, together with next‐generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT–PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very‐high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very‐high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very‐high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA‐based techniques can improve the selection of patients for MET‐targeted therapies.MET inhibitors have shown activity in non‐small‐cell lung cancer patients (NSCLC) with  FISHfluorescence in situ hybridizationHShistoscoreIHCimmunohistochemistrynCnCounterNGSnext‐generation sequencingNSCLCnon‐small‐cell lung cancerRT–PCRreverse transcription polymerase chain reaction1MET) gene has recently emerged as an actionable target, particularly in non‐small‐cell lung cancer (NSCLC) [MET activation, which increases cell proliferation, survival, invasion, and metastasis. MET amplification has been described in 1–6% of newly diagnosed NSCLC tumors and constitutes a frequent mechanism of acquired resistance in EGFR‐mutant (EGFR‐mut) NSCLC patients treated with tyrosine kinase inhibitors (TKI) [MET exon 14 alterations in donor and acceptor splicing sites—including point mutations, indels, and whole‐exon deletions—lead to the exclusion (skipping) of MET exon 14 at the RNA level (MET∆ex14), which has been described in 3–4% of patients with advanced NSCLC [Aberrant activation of the mesenchymal–epithelial transition ( (NSCLC) , 2. Mult (NSCLC) , 4 can lrs (TKI) . MET exoed NSCLC , 8.MET gene in NSCLC and mutations leading to MET∆ex14 were first reported in 2005 and 2006, respectively [MET∆ex14 as a potential therapeutic target in advanced NSCLC [MET inhibitors in patients with MET∆ex14 and MET amplification [MET∆ex14 [Amplification of the ectively , 10, whied NSCLC , 12. Sinfication , 16, 17,MET∆ex14 , and novMET∆ex14 .MET gene copy number variations in the clinical setting are fluorescence in situ hybridization (FISH) and next‐generation sequencing (NGS), whereas for MET∆ex14 detection, both NGS and reverse transcription polymerase chain reaction (RT–PCR) are commonly used [MET testing [MET status in advanced NSCLC and the fact that most reports evaluating the performance of different techniques have focused on a single MET alteration.The most frequent technologies used to assess nly used , 23, 24. testing  have beeMET alterations in particular types of tumors [MET alterations. To this end, we screened a large cohort of NSCLC patients and compared the nCounter results with those obtained by standard techniques. Our results indicate that multiplex, RNA‐based techniques such as nCounter have the potential to become the technology of choice to select patients for MET‐targeted therapies.The NanoString nCounter™ Analysis System is a high‐throughput, quantitative transcript‐based hybridization technology that allows for the simultaneous analysis of the expression of hundreds of target genes  and can f tumors , 29, 30,f tumors , 31. Her22.1MET alterations. Samples were collected from 10 participating hospitals (Supporting Information) with prior full informed patient consent and approval from the corresponding ethical committees. All advanced NSCLC patients arriving to our institutions and having biopsies available with sufficient tumor tissue were offered to participate in the study, which was conducted in accordance with the principles of the Declaration of Helsinki. FFPE slides (4 µm) were obtained by standard procedures and stained with hematoxylin and eosin. A pathologist determined the tumor area and evaluated the percentage of tumor infiltration. RNA was extracted with a high purity FFPE RNA isolation kit , while the GeneRead DNA FFPE Kit or the QIAamp DNA FFPE Tissue Kit  was used for DNA extraction from FFPE samples, according to the manufacturer’s instructions. DNA and RNA concentrations were measured by Qubit . Three cell lines  were used for validation purposes. The Hs746T cell line, harboring METΔex14, was purchased from the American Type Culture Collection. E98 is a patient‐derived astrocytoma cell line with amplification of MET gene. EGFR‐mut PC9 cells were obtained from F. Hoffman‐La Roche Ltd  with the authorization of Dr. Mayumi Ono . All cell lines were cultured in RPMI medium with 10% fetal bovine serum under standard conditions and counted after trypsinization. Pellets from a minimum of five T‐75 flasks were used to generate FFPE blocks.A total of 474 formalin‐fixed, paraffin‐embedded (FFPE) tumor samples from patients with NSCLC were tested to identify 2.2MET was performed with the ZytoLight® SPEC MET/centromere 7 (MET/CEP7) Dual Color Probe  according to manufacturer’s instructions. Three positivity criteria for MET amplification were used as follows: (a) a ratio (r) MET/CEP7 ≥ 2; (b) gene copy number (GCN) per cell ≥ 6; (c) ≥ 5 copies in ≥ 50% of cells; (d) or ≥ 15 copies in > 10% tumor cells. These three criteria have been employed for patient stratification in clinical trials of anti‐MET therapies (Table FISH for es Table . Immunos2.3® QIAact Lung DNA UMI Panel (Qiagen) or OncomineTM Solid Tumour (OST) DNA Panel (Thermo Fisher Scientific), according to the manufacturer’s instructions (Table DNA NGS was performed with the GeneReadns Table . Both pa® QIAact Lung RNA Fusion UMI Panel (Qiagen) according to the manufacturer’s instructions. This panel targets a fusion‐specific and splicing variants  software was employed to align the read data and call sequence variants, which were imported into the Qiagen Clinical Insight Interpret (QCI‐I) web interface for data interpretation and generation of final custom report. In the case of the OST panel, 10 ng of purified DNA was used as a template. Libraries were pooled at 20 pM and, following ion spheres, sequenced using the Ion Personal Genome Machine (Thermo Fisher Scientific). The Ion Reporter Server (Thermo Fisher Scientific) was used to align the read data to the human reference genome and call sequence variants. The MET gene copy numbers by NGS were assessed using the QCI‐A and QCI‐I software. Copy numbers provided by QCI‐I were selected according to the tumor infiltration of the sample, as assessed by an expert pathologist. The GCN ≥ 6 was chosen as a threshold based on the previous experience of our laboratory. During the validation of the NGS panel prior to its implementation in the clinical setting, this threshold had been found to show the highest correlation with FISH MET/CEP7 > 2.Qiagen Clinical Insight Analyze (2.4METΔex14 was amplified using HotStart Taq polymerase (Qiagen) in a 20 µL reaction and visualized in agarose gels. Primers used were located in exons 13 and 15, sequences were as follows: forward (exon 13) 5′‐TTTTCCTGTGGCTGAAAAAGA‐3′ and reverse (exon 15) 5′‐GGGGACATGTCTGTCAGAGG‐3′. Amplification generated a 246‐bp band for wild‐type (wt) MET RNA and a 106‐bp band for METΔex14. Positive samples were confirmed by bidirectional Sanger sequencing of RT–PCR products, using the big‐dye 3.1 sequencing kit .RNA was converted to cDNA using M‐MLV retrotranscriptase (Thermo Fisher Scientific) and oligo‐dT primers, and 2.5MET‐wt and METΔex14 target sequences. Detailed sequence information for the MET gene target regions is provided in Table ACTB; proteasome 26S subunit ATPase 4, PSMC4 and mitochondrial ribosomal protein L19, MRPL19), positive and negative controls. All processes of hybridization, capture, cleanup, and digital data acquisition were performed with nCounter Prep Station® and Digital Analyzer®  according to the manufacturer’s instructions. Reporter counts were collected with the nsolver analysis software version 2.6. Samples were considered not evaluable if the geometrical mean (geomean) of counts corresponding to the housekeeping genes was lower than 100. Counts from MET probes were normalized in two steps, as described [MET expression values. Two cutoff values were used for log‐MET results; (a) the mean plus standard deviation (SD) was used to define cases with moderately (mod.)‐elevated MET mRNA, (b) the mean plus two SD for very‐high MET mRNA levels. Regarding METΔex14 testing, log‐ratios were obtained dividing the normalized counts of the METΔex14 probe by the normalized counts for the MET‐wt probe. The cutoff for METΔex14 positivity was established as the average log‐ratio of the sample cohort plus 2 SD. Samples with no counts for the METΔex14 probe were directly considered negative.Total RNA was hybridized with a custom‐designed mixture of biotinylated capture tags and fluorescently labeled reporter probes (Elements Chemistry) that included, among others, probes for escribed , and sub2.6MET‐wt) tested negative. Next, using FFPE blocks prepared by spiking different numbers of MET‐dependent cells (Hs746T and E98) in a suspension of PC9 cells, we established that 1 mm2 of a 4‐µm section with a minimum of 10% of Hs746T cells was required for METΔex14 detection, whereas 30% of E98 cells were needed to detect high levels of MET mRNA expression by nCounter. In similar experiments, RT–PCR detected METΔex14 mRNA in mixtures with 0.1% mutant tumor cells. Finally, we performed repeatability studies using FFPE tumors from advanced NSCLC patients. In the case of METΔex14 detection by nCounter, four positive and 20 negative samples were analyzed in two independent experiments showing a 100% concordance. Regarding MET mRNA expression, 29 samples run in two independent experiments revealed concordant results in the classification for 28 of them .Using frozen pellets from Hs746T or E98 cells, we found that 5000 cells and 25 ng of RNA were sufficient for successful detection of spliced transcripts or very‐high MET mRNA levels, respectively. In contrast, pellets containing 500 000 of PC9 cells  had geomean of housekeeping gene counts below 100 and were excluded from the study. The remaining 422 evaluable samples, corresponding to 405 patients, were mostly stage IIIB/IV adenocarcinomas Table , Table 11% had ge3.2METΔex14 vs. the MET‐wt probes. A sample was considered positive if the log‐ratio was above a threshold value, established as the mean plus two times the SD of all samples analyzed  were identified, with a majority of nonsmoking females and a median age of 70 years (Table N = 4) or stabilization of the disease (N = 1) by RECIST criteria , mainly deletions in the acceptor splice‐site region (Table PIK3CA mutations while concomitant MET amplification by GCN (6–10) was identified in only one sample (20%). However, this case was negative (< 2) by MET/CEP7 ratio. Sufficient tissue to perform MET IHC was available for four of the 13 patients; MET staining was intermediate in three cases and strong in one . According to our classification algorithm (see Section 2), 15/422 samples (3.5%) presented very‐high MET mRNA levels  had MET amplification by FISH or NGS . Five patients with very‐high MET mRNA were treated with MET‐TKIs, all of them showed partial responses by RECIST criteria. Interestingly, one was negative by FISH  and epidermal growth factor receptor (EGFR), fusions in ALK receptor tyrosine kinase (ALK), and METΔex14 the most prevalent  and only three discordant samples, all of them positive by nCounter and negative by DNA‐based NGS. Regarding nCounter and RT–PCR, there was a fair agreement  and all the discordant cases (N = 11) were negative by nCounter and positive by RT–PCR. Six of those discordant samples had been analyzed by DNA‐based NGS; mutations associated with METΔex14 were not detected in any case . A moderate to substantial agreement was observed if the nCounter very‐high cutoff was employed, with the highest agreement for the FISH ratio MET/CEP7 ≥ 2  and the 14 discordant cases had moderately elevated MET mRNA but did not show copy number gains , 19 intermediate (HS 100–220), 21 weak (HS 50–99), and 17 were very weak‐negative (HS < 50). In general, a fair agreement was observed between IHC and nCounter expression levels, with IHC positivity observed among cases with both mod‐elevated and very‐high mRNA levels , but not in cases with EGFR and KRAS mutations or ALK rearrangements. Next, we compared MET mRNA expression in samples at baseline vs. progression. Overall, there were no significant differences in log‐MET values between the 306 basal and the 52 progression samples included in the study. When classified by drivers, we did observe a significant increase in MET mRNA levels in tumors at progression vs. baseline only in EGFR‐mutant samples. However, a subpopulation of samples at progression with high MET mRNA expression was apparent not only in EGFR mutant, but also in ALK‐positive rebiopsies.Finally, we compared ers Fig. . Baselin4MET alterations—amplification and exon 14 skipping—in a large cohort of 474 NSCLC samples using of a RNA‐based technology  is in line with the literature [METΔex14 transcripts by nCounter (N = 13) were also detected by RT–PCR; however, a large subset of samples positive by RT–PCR (N = 11) tested negative by nCounter. Five of these discordant samples could be submitted to an orthogonal RNA‐based NGS, which confirmed them as negative. In addition, most of them were found to harbor known drivers, particularly KRAS. Although RT–PCR is used in some laboratories to identify MET∆ex14 transcripts, these results suggest that it might not be the most adequate technique for this purpose. Low level of METΔex14 transcripts can emerge by splicing ‘mistakes’ in the cell without translating in any genomic alteration with oncogenic relevance [Our prevalence of terature , 7, partterature , 33. In elevance , 35, 36.MET∆ex14. However, this technique has been reported to detect only 63% of literature‐described splicing mutations associated with METΔex14 [METΔex14‐positive cases previously missed [METΔex14 by nCounter that were submitted to DNA‐based NGS, three (37.5%) tested negative for mutations associated with METΔex14 splicing sites. One of these discordant patients was treated with a MET‐TKI and derived clinical benefit  comprised a majority of males, former, or current smokers with a median age of 58 years. Interestingly, very‐high MET mRNA expression closely correlated with MET gene amplification by NGS or FISH and was mutually exclusive with METΔex14. Therefore, it is not surprising that the phenotype of the patients with very‐high MET mRNA agrees with the characteristics previously reported in patients with high MET amplification  [MET mRNA cases treated with MET inhibitors achieved partial responses; the majority of case baseline (7/9) did not harbor any detectable driver. In contrast, the cohort of patients with mod‐elevated MET mRNA expression did not correlate with any specific phenotype, being distributed independently of gender and smoking habits, and known drivers were detected in most cases. Although the biological oncogenic significance of MET amplification in NSCLC has been controversial [MET being a true driver in patients with very‐high expression mRNA levels. In contrast, we believe that MET is unlikely to play a clinically relevant role in tumors with mod‐elevated expression levels.In addition to the presence of or ≥ 10) , 39, 40.oversial , our resMET∆ex14‐positive NSCLC. Overall response rates of 32% have been reported for crizotinib, a nonselective inhibitor, compared to 46–68% and 40–55% in first or subsequent lines for the selective inhibitors capmatinib, tepotinib, and savolitinib [MET‐amplified patients are significantly worse, with overall response rate (ORR) 20–47% [MET/CEP7 ratios or GCN) that have been used as eligibility criteria.Several trials have recently evaluated the efficacy of MET‐TKIs in olitinib , 13, 14.) 20–47% , 40, 42.MET expression levels in NSCLC here presented provides a unique opportunity to advance in the understanding of the processes underlying MET biology. For instance, our RNA‐based assessment allowed the identification of amplification‐negative tumors with very‐high MET expression levels and also the relatively infrequent cases with gene amplification that did not express MET mRNA. Remarkably, one of the patients with very‐high MET mRNA levels but negative by FISH  was treated with a MET inhibitor achieving a partial response. These observations suggest that FISH positivity might be capturing a heterogeneous group regarding dependence on aberrant MET signaling and that RNA‐based techniques could improve the performance of FISH for patient selection. This, in turn, may help to explain the inferior response rates observed with MET‐TKIs in FISH‐amplified tumors. In order to improve outcomes to MET inhibition in this setting, it would be important to further investigate the efficacy of MET‐TKI in those tumors with gene amplification that do not express MET mRNA, and the real clinical value of high expression levels in amplification‐negative tumors where MET inhibition therapy could be considered.Our study can shed some light on this relevant issue, and the characterization of METΔex14, recent studies have suggested that MET protein expression is required for clinical benefit from MET‐TKIs [METΔex14 patient in our cohort who exhibited stable disease to MET‐targeted therapy had low levels of mRNA by nCounter, whereas all treated patients with mod‐elevated levels had partial responses. Whether mRNA expression levels can assist to predict outcome in patients with METΔex14 is currently unknown but also merits further investigation. One of the limitations of our study derives from the fact that MET is known to be expressed not only in tumor cells but also in normal epithelial [MET mRNA levels by nCounter and MET IHC staining in tumor cells indicated that, in most samples, the contribution of noncancer components to the nCounter results was not significant.Regarding MET‐TKIs . Interesithelial , dendritithelial . In cons5MET in a large cohort of advanced NSCLC and have validated the use of nCounter to identify METΔex14 in this malignancy. Our work also provides useful insights into the biology of MET as a driver in NSCLC, supporting MET very‐high mRNA expression as a surrogate of amplification, and suggesting the relevance of MET mRNA levels in patients responding to MET‐TKIs. Our results support the use of mRNA‐based techniques for multiplex, accurate, and reliable assessment of MET alterations in order to select patients for MET‐targeted therapies.We have comprehensively characterized CT has received fees for consultancy/advisory roles from Pfizer, Novartis, MSD, Roche, AstraZeneca, and Takeda, and research funding from Pfizer and Novartis. CC has received travel grants from MSD, Pierre‐Fabre Oncology, fees for consultancy/advisory from Boehringer Ingelheim, Roche, Pfizer, and Boehringer Ingelheim. LLV has received fees for consultancy/advisory roles from AstraZeneca and Roche. SV has received fees for consultancy/advisory roles from Bristol‐Myers Squibb, Roche, MSD, Abbvie, Ose Pharma, and Merck. AAH has received fees for consultancy/advisory roles from Bristol‐Myers Squibb, Roche, MSD, and Lilly. MGC has received fees for consultancy/advisory roles from Bristol‐Myers Squibb, Roche, AstraZeneca, and Pierre‐Fabre Oncology, and research funding from AstraZeneca. AP has received fees for consultancy/advisory roles from Pfizer, Lully, Nanostring Technologies, Amgen, Oncolytics Biotech, Daiichi Sankyo, PUMA, Bristol‐Myers Squibb, Novartis, and Daiichi Sankyo, and research funding from Pfizer, Amgen, Roche, Novartis, and Daiichi Sankyo. NR has received fees for consultancy/advisory roles from MSD, BMS and Pfizer, and research funding from Pfizer, Novartis, and Ministry of Health, Instituto de Salud Carlos III, Spain. CA, RR, RRe, AGC, EM, NV, SC, SM, AA, IS, EA, SR, APR, IM, AFC, RP, CS, MMG, MDL, AMB, EG, WPJL, RR, LMM, and MAMV have declared no conflicts of interest.CA, CT, RR, RRe, AGC, EM, MAMV, and NR conceived and designed the study. CA, CT, RR, RRe, AGC, EM, SM, AA, EA, SR, MAMV, and NR conducted the experiments, acquired, and analyzed data. CC, NV, SC, SM, LLV, IS, IM, SV, AFC, RP, CS, MMG, MDL, AAH, AMB, MGC, EG, WPJL, RRo, LMM, and AP provided clinical samples and collected clinical information. NR, CT, CA, and MAMV wrote the manuscript. All authors reviewed and/or revised the manuscript.https://publons.com/publon/10.1002/1878‐0261.12861.The peer review history for this article is available at Fig. S1. Heatmap displaying the molecular and clinical characteristics samples with METΔex14 discordant results between RT‐PCR and nCounter.Fig. S2.MET mRNA expression levels by nCounter in samples harboring different driver alterations.Table S1. MET amplification criteria used in clinical trials of MET inhibitors.Table S2. Description of the NGS panels used in the study.Table S3. Description of the nCounter codeset used in the study and probes design for MET wild type and METΔex14.Table S4. Characteristics of the samples with valid results, N (%). Fusions were determined by nCounter, mutations by NGS.Table S5. Results for MET exon 14 alteration splice‐site regions and mutation types.Table S6. Concordance of nCounter with RT‐PCR and NGS for METexΔ14 status.Table S7. Concordance of the nCounter categorization using the cut‐off for very high MET levels with IHC, FISH and NGS.Table S8. Concordance of the nCounter categorization using the cut‐off for moderately elevated MET levels with IHC, FISH and NGS.Click here for additional data file.


In addition, it has been observed that using both FOPC and TCA yields a similar performance to that observed using EPC, in light of the improved early strength development at low temperatures.This study examines effects of cement fineness and chemical admixtures of early strength agents on the early strength development of concrete. Three cement types were selected, namely ASTM type-I ordinary Portland cement (OPC), fineness ordinary Portland cement (FOPC), and ASTM type-III early Portland cement (EPC), and the mixing proportions of concrete were set by adding a triethanolamine-based chemical admixture to FOPC. The evaluation items considered in this study included raw material analysis, compressive strength, and maturity (D∙h). The time required for the development of concrete strength of 5 MPa in the three cement types was estimated and compared. The results revealed that using FOPC enhances the strength development of concrete owing to its higher fineness and SO Concrete forms a structural matrix by combining cement, water, fine aggregate, coarse aggregate, and admixture. The strength of concrete is developed by the homogeneous bonding force of each material, and the primary influential factor of the strength development is cement. Cement is condensed and cured as it stabilizes while releasing heat energy through its hydration reaction with water ,2,3. ConMoreover, the most economical and efficient process control is pursued in construction sites, which is closely related to the strength development period of concrete. Concrete remains in formwork until its strength development achieves sufficient structural stability, and each country has different criteria for compressive strength. These include the minimum time ,6,7 and As concrete is significantly affected by temperature during the hydration process of cement, its strength development tends to decrease at low temperatures. There are cases when strength development becomes impossible if the hydration reaction stops for a certain period. Considering these characteristics, many studies have reported on the early strength development of concrete at various curing temperatures ,11,12.Ordinary Portland cement (OPC) has been primarily used as a binder because it exhibits stable strength development at temperatures of 15 °C or higher. Its strength development, however, tends to be delayed at low temperatures. Consequently, cases in which strength development was examined using early strength cement and early strength chemical admixture to improve the strength development of cement through hydration have been continuously reported ,17,18.Frigione et al. ,20,21 re2O3-SO3 system due to the generation of various hydrates according to the Al2O3/SO3 and CaO/SO3 molar ratios. Lee et al. [3 content on the strength development of fine ordinary Portland cement (FOPC) at early ages.It has been observed that the hydration degree of cement increases with a reduction in its particle size. Klein and Metha  presentee et al. ,24 exami3 contents, demonstrates enhanced early strength development in the 13–15 °C temperature range. ASTM C150 [3 concentration in cement to ensure sufficient early strength of OPC.It has been observed that commercialized fine ordinary Portland cement (FOPC), which improved the OPC powder and SOSTM C150  specifieHewlett et al. ,27,28 prHowever, these extant studies mostly dealt with mortar-based materials and lacked consideration of quality control aspects, such as those pertaining to the economic efficiency of mixing, batch plant productivity, and in-field workability during application in construction sites. In addition, it is necessary to examine the early strength of various commercialized cements in order to ensure consistent quality in mass production and optimized mix design in construction sites.In this study, various binders, such as OPC, early Portland cement (EPC), and FOPC, were selected based on the C24  concrete mix, which is produced in ready-mixed concrete factories. Their influence on early strength development at different curing temperatures was examined. In addition, polycarboxylate (PC)-based superplasticizers with early strength agents were added and their influence on strength development was analyzed.3 and fineness of 330 m2/kg, FOPC with density equal to 3130 kg/m3 and fineness of 380 m2/kg, and ASTM type-III EPC with density equal to 3160 kg/m3 and fineness of 488 m2/kg were considered in this study.3, absorption: 0.79%) and crushed sand  were mixed and used. The fineness of the mixed fine aggregate was 2.84. For the coarse aggregate, crushed granite aggregate  was used.3) and sodium sulfate (Na2SO4) based on triethanolamine (TEA)  were used.As for chemical admixtures, a PC-based admixture (polycarboxylic acid group) and a PC-based admixture with added sodium nitrate .To establish concrete curing temperatures, the outdoor (average temperature = 12 °C), chamber (13 °C), and cast-in-place (slab-embedded specimens for outdoor curing) conditions were set as illustrated in 3, considering the water-reducing ratio of the PC-based admixture. Fine aggregate, washed sea sand, and crushed sand were mixed at a 4:6 ratio. As for the properties of concrete, the slump was set to 180 ± 25 mm and the air content to 4.5 ± 1.5% to secure sufficient workability for field construction.The compressive strength of concrete was calculated by measuring the maximum load at the target age using a 300 ton universal test machine after fabricating Ø 100 × 200 mm specimens in accordance with ASTM C873  and ASTM3 mock-ups were fabricated, and ø 100 × 200 mm cylinder molds were embedded using sleeves. The molds were fixed to the formwork using nails to minimize deformation due to their movement during concrete pouring.This method examines the compressive strength of concrete by simulating the site slab and reproducing the temperature history of the structure. In this study, 500 × 500 × 210 mmM(t) = the temperature–time factor at age t, shown as degree days or degree hours; ∆t = a time interval, shown as days or hours, aT = average concrete temperature during time interval ∆t in °C; and oT = datum temperature in °C.The hydration heat of concrete was measured by embedding K-type thermocouples in the center of each specimen. The maturity of concrete was calculated using Equation (1), based on the hydration heat measured in accordance with ASTM C1074 :(1)M(t)=3S (3CaO∙SiO2), C2S (2CaO∙SiO2), C3A (3CaO∙Al2O3), and C4AF (4CaO∙Al2O3∙Fe2O3) minerals. EPC contains more C3S crystal peaks compared to both OPC and FOPC. The amount of C3S calculated by Bogue’s equation [equation  on X-rayThe FOPC-based concrete exhibited slightly higher strength compared to OPC; the strength development effect was larger at temperatures higher than 22 °C than at lower temperatures. In the case of the concrete that used FOPC and TCA, the 5 MPa strength development occurred within 24 h at 13 °C. The strength development of the concrete that used FOPC and TCA further increased as the curing temperature increased, making it possible to develop 5 MPa within an 18 h.FOPC_TCA and EPC exhibited similar early strength performance within 24 h at low temperatures, but EPC showed higher strength after 72 h. At temperatures higher than 22 °C, FOPC_TCA and EPC could develop 5 MPa within 18 h. They exhibited similar strength after 72 h.3. Accordingly, five temperature histories and accumulated temperatures corresponding to the outdoor, chamber (13 °C), and cast-in-place  curing methods were determined.Results obtained in this study reveal that when using a small test specimen measuring ø 100 × 200 mm, temperature histories pertaining to each mixture are nearly similar in the ± 2 °C range of the outdoor and chamber curing temperatures. Therefore, this was suggested as the curing temperature for the outdoor and chamber samples (13 °C). When using the cast-in-place curing method, it was confirmed that the temperature history differs from the curing temperature corresponding to the outdoor method using a mock-up member measuring ø 500 × 500 × H210 mmThe maturity of concrete linearly increased over time at all curing levels. Similar maturity increase patterns were observed under both outdoor and chamber (13 °C) conditions. The cast-in-place curing condition exhibited significantly higher values compared to the outdoor and chamber (13 °C) conditions, suggesting that the influence of the temperature history of a structure can favor strength development. Among cement types considered in this study, EPC demonstrated the highest maturity, followed by FOPC and OPC. It has been confirmed that cases affected by the temperature history of a structure exhibit higher strength development compared to small-specimen conditions. As observed, EPC- and FOPC-based concrete mixtures demonstrated greater potential for early strength development.At a temperature higher than 20 °C, a linear relationship with the cement fineness was observed similarly to the under 13 °C curing condition, but the increment was larger. In addition, the compressive strength development rate significantly increased after 72 h, resulting in smaller differences from EPC. This tendency was more noticeable when TCA was mixed. These results confirmed that the effect of increasing the fineness increased as the temperature increased, and that the effect of adding TCA was excellent at low temperatures ,41,42.This difference in fineness is a major factor influencing the difference in calories due to cement hydration. 3 content for FOPC and EPC ranged from 3% to 4%, which is 107–129% higher compared to OPC. These results are similar to Mohammed’s report [3 content ranged from 3.0% to 3.2%. In addition, Lee reported that the early strength increased when the cement fineness was increased, while the SO3 content was increased to 3.1 [3 content of 4%.The SOs report , which fd to 3.1 ,24,25. IIn construction sites, the critical path of construction process management is the period of vertical formwork. The removal time of vertical formwork is based on ACI , BS EN 16 °C, 12 h), and Asian  standards  °C, 12 h,8,9. Thi3 content, exhibited the shortest elapsed time for 5 MPa development, followed by FOPC and OPC. As the temperature decreased, the time reduction effect became larger. In the case of FOPC_TCA, the elapsed time for 5 MPa significantly decreased to a level equivalent to that of EPC.EPC, which had the highest cement fineness and SOIn this study, the effects of the fineness and TEA-based chemical admixture on the early strength development of concrete were examined. The results are summarized as follows.1)When evaluating the compressive strength of concrete, it was observed that the use of FOPC increases the early strength development effect at high temperatures compared to lower ones. At a curing temperature of 13 °C, the development of compressive strength of FOPC_TCA was observed to increase significantly compared to FOPC. FOPC_TCA demonstrated lower overall compressive strength compared to EPC, but the overall difference observed over a 24 h period was negligible. With increase in curing temperature, the compressive strength development of FOPC_TCA was observed to increase significantly and tended to decrease the difference as compared to EPC.2)The evaluation of the maturity of concrete revealed that OPC exhibited the highest maturity to develop a concrete compressive strength of 5 MPa, followed by FOPC, FOPC_TCA, and EPC, respectively. The reduction rate of the maturity for 5 MPa concrete strength development compared to OPC was 45% when FOPC was used. FOPC_TCA (63%) and EPC (68%) exhibited similar values.3)There was a linear relationship between the cement fineness and the compressive strength regardless of temperature conditions, and it was found that higher temperatures had a larger influence on strength development. FOPC, with a higher fineness than OPC, showed a larger impact on strength development at high temperatures, but it is considered that the use of TCA will lead to a strength development rate equivalent to that of EPC, even at low temperatures.4)3 content ranged from 3% to 4%, which was 107% to 129% higher than that of OPC. As for the SO3/Al2O3 ratio, FOPC exhibited a 116.1% higher value and EPC a 132.2% higher value than that of OPC, indicating that they were effective in developing early strength.For FOPC and EPC, the SO3 content are higher than those of OPC. In addition, the use of both FOPC and TCA can achieve performance similar to that of EPC. because it can improve early strength development, even at low temperatures. Considering construction site productivity and economic efficiency, the use of FOPC is considered to have a marked effect compared to OPC, but its economic efficiency compared to EPC needs to be examined in more detail. The results of this study are expected to be used as the basic data for the performance design of concrete mixes to shorten construction periods on construction sites.The FOPC that was used in the scope of this study can contribute to improving the strength development of concrete, because its fineness and SO


Existing buildings, especially historical buildings, require periodic or situational diagnostic tests. If a building is in use, advanced non-destructive or semi-destructive methods should be used. In the diagnosis of reinforced concrete structures, tests allowing to assess the condition of the reinforcement and concrete cover are particularly important. The article presents non-destructive and semi-destructive research methods that are used for such tests, as well as the results of tests performed for selected elements of a historic water tower structure. The assessment of the corrosion risk of the reinforcement was carried out with the use of a semi-destructive galvanostatic pulse method. The protective properties of the concrete cover were checked by the carbonation test and the phase analysis of the concrete. X-ray diffractometry and thermal analysis methods were used for this. In order to determine the position of the reinforcement and to estimate the concrete cover thickness distribution, a ferromagnetic detection system was used. The comprehensive application of several test methods allowed mutual verification of the results and the drawing of reliable conclusions. The results indicated a very poor state of the reinforcement, loss in the depth of cover and sulphate corrosion. Periodic inspections of existing buildings, as well as renovation or modernisation works and even planned demolition works, require appropriate diagnostic tests ,2,3. In The significant and rapid development of technology in the field of measurement methods has recently contributed to the use of tools in construction diagnostics that allow destructive tests of elements and specimens as well as measurements with methods that are non-destructive or semi-destructive in their effects on building structures ,9,10,11.Carefully considered diagnostic tests allow verification of the data contained in historic project documentation or obtained on the basis of an analytical model ,3,7,12. However, not only should a well-prepared program of diagnostic tests account for an assessment of the existing condition, it should also provide a forecast for the durability of the structure in the future. This applies in particular to load-bearing structures operated in unfavourable environmental conditions, in which the design assumptions for ensuring adequate durability were incorrect ,14,15,16The comprehensive and systematised classification of test methods for the diagnosis of concrete structures can be found in .In the article, the authors also provided information on external and internal factors influencing the degradation of the structure, the degree of their impact and the optimal selection of research methods . Diagnostic tests can be divided into tests aimed at estimating the load-bearing capacity of structure elements and strength parameters of materials (very often destructive or semi-destructive tests) and tests aimed at assessing the durability of elements and the entire structure. The authors took into account the usefulness of using various methods of assessing the durability of structures depending on the degradation mechanisms and their impact on durability. Non-destructive methods (NDT) were analysed, i.e., those that do not violate the integrity of the structure in any way, and semi-destructive methods (SDT), i.e., those that require material sampling or that slightly disturb the integrity of the structure. The authors proposed the classification of physicochemical and biological diagnostic methods to assess the durability of RC structures and presented the research equipment .In the case of reinforced concrete structures, diagnostic tests primarily concern the assessment of the state of reinforcement and the concrete cover, which directly determines the durability of the structure ,18,19,20In reinforced concrete structures, concrete  is the basic and best protection for the reinforcement ,13,21,22Most often the deterioration is the effect of carbon dioxide in the air and changes in temperature and humidity of the environment, which leads to gradual carbonation of concrete and directly affects the loss of the protective properties of the cover in relation to the reinforcement ,22,23,24As a result of carbonation, the pH of the concrete gradually decreases, and the areas of neutralised concrete extend further and further into the cover, eventually reaching the passive layer. In areas where the pH of the concrete drops below ~11.8, the surface of the reinforcing steel is depassivated, which directly leads to the initiation of electrochemical corrosion of the reinforcement.On the other hand, in the case of seaside facilities located in the coastal zone (port and tourist buildings), exposed either to the direct influence of sea salt or to the so-called salt mist, as well as road structures , concrete surfaces, parking lots and open garages, due to the use of de-icing agents containing NaCl, the cause of degradation is the action of chlorides ,25,26,27On the other hand, mechanical damage to the concrete cover (caused by accidents or minor damage accompanying the use of the facility), although visible to the naked eye in the form of cracks and defects, paradoxically often does not indicate an advanced process of structural degradation .Nevertheless, the assessment of the load-bearing capacity of structural elements is a very important factor in the diagnostics of buildings. For this purpose, computational and experimental models are often used ,32,33. IUsually, diagnostics of reinforced concrete structure elements is carried out in several ways. One of them concerns the measurements of the thickness and continuity of the concrete cover and the location of the reinforcement. The second are electrochemical, non-destructive or semi-destructive tests, which indirectly allow one to determine and forecast the degree of concrete and steel corrosion advancement. Third is the testing of the material, i.e., fragments of concrete cover taken from the structure, on the basis of which the chemical composition of concrete and its microstructure are determined. Computational and experimental models are also used to assess the load-bearing capacity of structural elements.The applied research methods are quite known in the diagnostics of reinforced concrete structures. However, often the tests are performed selectively—they are limited to one type of method, which provides limited information. The authors would like to point out that only the knowledge and combination of several methods allows for the preparation of an appropriate research plan and a full assessment of the condition of the structure, especially if it concerns historic objects, in the diagnostics of which it is only possible to use non-destructive or semi-destructive methods, as in the described example. Additionally, the authors would like to draw attention to the measurement techniques used. While the ferromagnetic method used in the research can be successfully replaced by other methods (which are described in more detail in this chapter), the galvanostatic pulse method seems to be the most precise and easy to use compared to other commonly used electrochemical methods. X-ray analysis and phase composition analysis are also noteworthy as techniques allowing for very precise recognition of the effects of carbonation.The thickness of the concrete cover is one of the key factors determining the durability of reinforced concrete structures susceptible to aggressive external environment, because it determines the speed of penetration of aggressive substances that can damage the passive layer and initiate corrosion of the reinforcement ,13,15,29There are many test methods that allow determining the thickness of the cover and locating the reinforcement bars, their diameter and mutual position. Currently, a number of the methods in use are based on changes in electromagnetic waves penetrating through the tested reinforced concrete element medium. The tests can be performed using electromagnetic, radiological, ultrasonic, or radar methods. The precision of the obtained results as well as the effectiveness and efficiency of the research as a function of time depend on the research method used.One of the most accurate among those mentioned above is the radiological (radiographic) method, which uses ionising radiation (X-rays or gamma rays) ,38,39,40A more widely used alternative to radiographic methods are electromagnetic methods ,37, whicThe radar method can also be used to locate reinforcement in concrete. It uses the phenomenon of the penetration and reflection of an electromagnetic wave (in the range of 100 MHz–2 GHz) through a medium (reinforced concrete element) which, depending on the medium material type (concrete or steel), the wave penetrates into depth or is scattered or reflected ,48,49,50Impact-echo  and impuIn most cases, electromagnetic methods are effective and sufficient. In the tests described in this article, the PS 200 Hilti  scanner was used to locate the reinforcement and determine the cover parameters. This device has been on the market for a long time. It enables easy and quick measurement and subsequent analysis of the obtained results. The performed measurements allow one to estimate the cover thickness and the diameter of the reinforcement. The ability to perform surface scans and combine them is particularly useful. As mentioned earlier, this is not an accurate method, and any magnetic materials in the concrete, such as tie wire or other ferrous particles, as well as reinforcement corrosion products, will distort the results. However, it is still the fastest method of reinforcement detection and in most cases it is sufficient in the diagnosis of reinforced concrete elements .Some of the mentioned methods allow the detection of defects and discontinuities of the material that are not visible on the surface. The method of acoustic emission allows, among others, the recording of acoustic signals caused by defects in the microstructure of the material during the use of the structure . The advOn the other hand, defects in the form of scratches and cracks visible on the concrete surface can be diagnosed quite simply with the use of practical handy tools . To measMore and more often, a scanning electron microscope  is also used to analyse the defects in the microstructure of concrete (SEM). SEM tests are very accurate but require special preparation of samples taken from the structure; thus, SEM tests are associated with slight damage to the structure. Therefore, these tests should be classified as semi-destructive tests. On their basis, it is possible to identify the causes of cracks ,58.An inventory of cracks allows one to estimate the causes of damage, locate places of potential weakening of elements, identify possible corrosion of the reinforcement and indicate the areas from which material for laboratory tests should be taken.Electrochemical tests are particularly valuable because of the possibility of conducting them in the field with simultaneous assessment of the interaction of the current steel and concrete properties in real operating conditions. They make it possible to determine areas exposed to corrosion on the surface of elements as well as the degree of reinforcement corrosion advancement and to forecast its progression over time ,11,16. TAmong the above-mentioned electrochemical methods, the most reliable, and at the same time that which allows one to conduct measurements in the field in a relatively uncomplicated manner, are the measurements of the corrosion current density.Polarisation methods, depending on the method of initiating the disturbance, are three in number: the electrochemical impedance spectroscopy method ; the linear polarisation resistance method ; and the galvanostatic pulse method .In the case of diagnostic tests, it is worth using various measurement techniques simultaneously to verify the obtained results. It is particularly important to combine research with electrochemical methods with material tests and appropriate control opencasts.The research described in the article used the GP-5000 GalvaPulse™  set for measurements using the galvanostatic pulse polarisation method. The equipment allows one not only to measure the corrosion current density , but also to measure the stationary potential of reinforcement and the resistivity of the concrete cover. The scope of the research makes it possible  to determine the areas on the structure where the probability of corrosion is relatively higher than elsewhere. The obtained results are analysed in relation to certain criterion values, which in the form of tabulated data are attached to the apparatus . DependiA reinforcement covered in concrete is protected against corrosion by the steel passivation mechanism. However, it is only effective when the rebars are well-covered by concrete. Concrete may be affected by the environment and change its properties overtime. The carbonation process is considered to be very unfavourable. The effect of carbon dioxide that penetrates the concrete dissolves in the pore solution, resulting in the formation of carbonic acid. This acid reacts with the cement paste alkaline components, which results in the formation of carbonates and a lowering of concrete pH. When the pH drops below a certain level (pH < 11.8), the steel depassivates and in the presence of water and oxygen begins to corrode . CarbonaFPLPt  and maps of the distribution of values of all measured parameters were generated in the PsiWin program , The measurement results of all three parameters  was obtained for rebar no. 2 and two points of rebar no. 3. Moderate corrosion activity (icor = 5–15 μA·cm−2) was observed at four points of rebar no. 3 and five points of rebar no. 4. High corrosion activity was found only at one point of rebar no. 4, where the corrosion current density exceeded icor = 15–A·cm−2. At the same time, the results allowed estimating the corrosion rate in the tested rebars within the 33–57 μm/year range in rebar no. 2, 40–153 μm/year in rebar no. 3, and 68–182 μm/year in rebar no. 4, indicating a very poor condition of the floor slab reinforcement.In the floor slab, the analysis of the stationary potential of the reinforcement showed that the probability of corrosion in the selected area of the slab was very high. At ten measurement points, it exceeded E10 kΩ·cm . Measurest = −200 mV) was obtained at all four points of the rebar no. 1. But in rebar no. 2, the reinforcement stationary potential was much higher. At one point, rebar no. 2 indicated a 50% probability of corrosion, and at the other 3 points the corrosion probability was 95% (Est < −350 mV). The values of concrete cover resistivity in rebar no. 1 indicated the ambiguous probability of corrosion, from small  through medium  to large . In rebar no. 2, the occurrence of corrosion probability at all points was defined as large (Θ < 10 kΩ·cm). Measurements of corrosion current density of the column reinforcement were consistent with the measurements of the corrosion potential and resistivity of the concrete cover. At two points determining the corrosion activity in rebar no. 1, the measured values did not exceed icor = 2 μA·cm−2, which indicated that the activity was not predicted or negligible, and at the remaining two points, the values slightly exceeded icor = 2 μA·cm−2, which should be read as low corrosion activity. The rate of corrosion development in this rebar was estimated at 2.2–30.1 μm/year. However, at the points determining the corrosion activity in the rebar no. 2, the values of the corrosion current density were much higher, indicating a moderate  and even high corrosion activity in this rebar . The rate of corrosion development in rebar no. 2 can be estimated as 203.3 μm/year.The same analysis was carried out for two reinforcing bars in the load-bearing column. A low 5% probability of corrosion  of the removed concrete cover pieces revealed complete carbonation of the concrete cover .3): calcite, aragonite and vaterite are visible in the X-ray diffraction pattern in 3, portlandite peaks are still clearly visible in the sample. Analysis of the paste sample confirms the presence of a significant amount of aggregate dust fractions, which is represented by the intense peaks of dolomite and quartz appearing on the X-ray patterns. The presence of dolomite dust contributes to noticeable loss of sample mass above 600 °C. This is also associated with the presence of carbonation products. Gypsum and sylvite found in the paste indicate that the concrete may have been exposed to sulphate and chloride attack. The aggregate tested consists of dolomite, which is accompanied by quartz mixed with pyrite  and column (high probability of corrosion and high corrosion activity in one of the two bars).The carbonation test for in situ concrete showed total depassivation across the cover depth.Material tests confirmed the information on the degree of concrete carbonation in the tested elements. Concrete carbonation was accelerated by the destruction of the concrete structure as a result of sulphate corrosion.Performed research and analysis allowed the following conclusions to be drawn:The described tests still need to be supplemented with diagnostic tests to assess the condition of the structure, mainly in terms of its load-bearing capacity. These tests should include, among others, determining the strength parameters of concrete and steel, crack morphology and displacement measurements. Because of the historical nature of the object, non-destructive and semi-destructive sclerometric, acoustic, pull-off methods can be used in the research.


Ten weeks of metformin treatment in late middle age is efficacious for improving cognitive function in the old age. Improved cognitive function following metformin treatment was linked with antiinflammatory effects, the activation of adenosine monophosphate‐activated protein kinase (AMPK), the mammalian target of rapamycin (mTOR) signaling, and increased autophagy in the aged hippocampus. The results suggest that metformin therapy in middle age leads to better cognitive function in old age. Metformin, a drug widely used for treating diabetes, can prolong the lifespan in several species. Metformin also has the promise to slow down age‐related cognitive impairment. However, metformin's therapeutic use as an anti‐aging drug is yet to be accepted because of conflicting animal and human studies results. We examined the effects of metformin treatment in late middle age on cognitive function in old age. Eighteen‐month‐old male C57BL6/J mice received metformin or no treatment for 10 weeks. A series of behavioral tests revealed improved cognitive function in animals that received metformin. Such findings were evident from a better ability for pattern separation, object location, and recognition memory function. Quantification of microglia revealed that metformin treatment reduced the incidence of pathological microglial clusters with alternative activation of microglia into an M2 phenotype, displaying highly ramified processes in the hippocampus. Metformin treatment also seemed to reduce astrocyte hypertrophy. Additional analysis demonstrated that metformin treatment in late middle age increased adenosine monophosphate‐activated protein kinase activation, reduced proinflammatory cytokine levels, and the mammalian target of rapamycin signaling, and enhanced autophagy in the hippocampus. However, metformin treatment did not alter neurogenesis or synapses in the hippocampus, implying that improved cognitive function with metformin did not involve enhanced neurogenesis or neosynaptogenesis. The results provide new evidence that metformin treatment commencing in late middle age has promise for improving cognitive function in old age. Modulation of microglia, proinflammatory cytokines, and autophagy appear to be the mechanisms by which metformin facilitated functional benefits in the aged brain. We specifically tested whether long‐term MET treatment commencing in late middle age would maintain better cognitive function in old age. Our results demonstrated improved pattern separation, object location, and recognition memory function in mice receiving MET compared to age‐matched mice receiving no treatment. Improved brain function was associated with reduced incidence of microglial clusters, increased density of M2 microglia with no effects on neurogenesis or synaptic proteins in the hippocampus. Moreover, MET treatment diminished the concentration of proinflammatory cytokines, activated AMPK, modulated the mammalian target of rapamycin (mTOR) signaling, and increased autophagy in the hippocampus.22.1ad libitum. The experimental procedures conducted were approved by the Animal Care and Use Committee of Texas A&M University.Male C57BL6/J mice (n = 59) purchased from Jackson Laboratories were employed. Of these, 48 were in late middle age (18‐month‐old mice), and 11 were young adults (~3‐ to 4‐month‐old mice). The current study focused only on male mice due to sex differences in behavioral pattern separation ability, neurogenesis, synaptic plasticity, and transcription during the estrus cycle Foster, . All anin = 11–12/group) were interrogated with a series of neurobehavioral tests. Next, for studying the effects of long‐term MET treatment on cognitive function, two groups of mice were randomly assigned either to the control aged or aged‐MET groups . Animals in the aged‐MET group received MET via oral gavage  for 10 weeks. To neutralize the potential confounds associated with handling and oral gavage, we gave mock oral gavage (without administering the drug) to mice in the control aged group. The weight of animals was comparable between aged and aged‐MET groups at the commencement of the study , and at different time‐points after the commencement of treatment . The behavioral tests commenced in the 8th week of the treatment period and completed in the 10th week of the treatment period. The animals in control aged group were untreated, but behavioral tests were done in parallel to the aged‐MET group. The animals were euthanized for histological analysis immediately after the completion of behavioral tests. The animals in both groups were 21 months old at the time of tissue harvesting. Furthermore, to understand the molecular mechanisms underlying MET‐mediated beneficial effects, another cohort of mice  received MET  or no treatment for 2 weeks. The animals were euthanized 3 h after the last treatment, and the blood samples were collected from the heart for measuring blood glucose concentration. The brain tissues were harvested, snap‐frozen, and stored at −80° centigrade for various biochemical analyses. The investigators who performed the behavioral tests and biochemical analysis were blinded to group identification of animals or samples. The animal numbers for behavioral studies, and histological and biochemical studies were determined through power analysis using G*Power software.The experiments were performed following two weeks of acclimatization to the vivarium. First, to discern cognitive function status in late middle age, young adult and 18‐month‐old mice (2.2The pattern separation test (PST) employed in this study encompassed four consecutive trials (T1–T4), with each lasting 5 min and separated by an inter‐trial interval (ITI) of 30 min. See the supplemental file for details.2.3The object location test (OLT) also included three trials (T1–T3), with each trial lasting 5 min and trials separated by an ITI of 15 min. See the supplemental file for details.2.4The test comprised three trials (T1–T3), with each lasting 5 min and separated by an ITI of 15 min. See the supplemental file for details.2.5The animals were deeply anesthetized, perfused with 4% paraformaldehyde, the brain tissues were post‐fixed, and processed for cryostat sectioning following cryoprotection. Thirty‐micrometer thick coronal sections through the forebrain were collected serially, and every 15th section through the entire septotemporal axis of the hippocampus was processed for immunohistochemistry. See the supplemental file for details.2.6The overall morphology of IBA‐1+ microglia and GFAP+ astrocytes from aged and aged‐MET groups was measured by tracing the soma and processes using a semi‐automatic cell tracing system  linked to a Nikon microscope. See the supplemental file for details.2.7n = 4–5/group). Additional immunofluorescence studies comprised visualization of Syn+ presynaptic puncta and PSD95+ postsynaptic puncta, and p62+ structures using tissue sections through the hippocampus from the young adult, aged, and aged‐MET groups (n = 5/group). See the supplemental file for details.Immunofluorescence studies comprised detecting pAMPK expression among 4′,6‐diamidino‐2‐phenylindole (DAPI) neurons, and alternatively activated CD206+ M2 type microglia among IBA‐1+ microglia in the cerebral cortex and the hippocampus of aged and aged‐MET groups  the area fraction of Syn+ and PSD95+ puncta in randomly chosen 303 µm2 images (n = 5/subfield/group) using ImageJ; (b) the mean number of Syn+ and PSD95+ puncta in randomly selected 4 µm2 areas ; and (c) the cumulative number of putative synapses  in randomly selected 4 µm2 areas . The area fraction of p62+ structures in the CA3 subfield was also quantified in a blinded manner using Image J .For measuring changes in Syn+ and PSD95+ puncta density and putative synapses  in the stratum radiatum of CA1 and CA3 subfields, we employed 0.52.9We measured the effect of 2 weeks of MET treatment on the concentration of proinflammatory markers TNF‐α, IL‐1β, and MIP‐1α, mitochondrial complex I, oxidative stress markers MDA and protein carbonyls, pAMPKα, mTOR complex, and autophagy proteins beclin‐1, ATG5, and MAP1‐LC3B in the hippocampus of 18‐month‐old mice. See the supplemental file for details.2.10t test in the Prism software was used to compare two columns of data within groups in behavioral tests or the data between aged and aged‐MET groups. The Mann–Whitney U test was employed when standard deviations between groups were found to be statistically significant. One‐way ANOVA with Student–Newman–Keuls post hoc tests was employed for comparisons involving three groups. The values mentioned in the bar charts are mean ±SEM, and p < 0.05 was considered statistically significant.Two‐tailed, unpaired, Student's 3n = 11) and 18‐month‐old (n = 12) male C57BL/6J mice with a series of neurobehavioral tests. The tests evaluated the ability of animals for pattern separation, perceiving minor changes in the environment (a measure of location memory), and novel object recognition memory. Following this, two groups of 18‐month‐old male mice were randomly assigned either to the control aged or aged‐MET groups (n = 12/group) to investigate the effects of long‐term MET treatment for improving cognitive function. We administered MET orally  at a dose of 100 mg/kg  and the treatment continued for 10 weeks. In the last 3 weeks of MET treatment, animals were evaluated for pattern separation ability and location and recognition memories.We first determined the extent of cognitive dysfunction in late middle age by interrogating young adult  and aged mice receiving MET (aged‐MET), the distances traveled and the velocities of movement in T1 were comparable   but not in T3 , a behavioral test for recognition memory that examines the ability of animals to distinguish a new object from a familiar object. The first two trials involved the exploration of an empty open field apparatus (T1) and the exploration of two identical objects placed on opposite sides of the open field apparatus T2)  immunostaining. We examined whether 10 weeks of MET treatment in aged mice altered the distribution or morphology of microglia, one of the principal mediators of chronic inflammation in the aged brain. We first examined the occurrence of clustering of microglia, a pathological feature consistently seen in the aged hippocampus . Morphometric analyses of traced microglia suggested that MET treatment in late middle age modulated microglia into an M2 phenotype in the aged hippocampus. The M2 features of microglia in the MET‐treated group include the increased area occupied by individual microglia, increased total process length, and a higher number of nodes and endings   positive astrocytes in aged mice appeared reactive with thick primary and secondary processes, in comparison to mostly thinner processes in aged‐MET mice . The overall distribution and density of DCX+neurons seemed comparable between the aged mice and aged‐MET mice  in the dentate subgranular zone and the granule cell layer  and postsynaptic density protein 95 (PSD95) dual immunofluorescence. Representative images of Syn+ and PSD95+ puncta in the CA3 stratum radiatum of mice belonging to young, aged, and aged‐MET groups are illustrated in Figure 3.9n = 6/group). In comparison to untreated mice, MET‐treated mice displayed reduced concentration of tumor necrosis factor‐alpha  or no treatment for 2 weeks n = /group. I3.10n = 6/group). In comparison to untreated mice, MET‐treated mice displayed increased concentration of pAMPKα  and mTOR complex (mTORC1+mTORC2) in the hippocampus of 18‐month‐old mice receiving MET  or no treatment for 2 weeks n = /group. I3.11n = 6/group). The autophagy markers included beclin‐1, autophagy protein 5 (ATG5), and microtubule‐associated protein 1 light chain 3 beta (MAP1‐LC3B). In comparison to untreated mice, MET‐treated mice displayed increased concentration of beclin‐1 . The expression of p62 in cells is used as a reporter of autophagic activity because p62 delivers ubiquitinated cargoes for autophagic degradation and acts as a substrate during autophagic degradation  dual immunofluorescence (3.12n = 6/group). In MET‐treated animals, the blood was collected 3 h after the last MET administration. Although blood glucose concentration was reduced in MET‐treated animals by 13%, the reductions were not significant statistically . Thus, MET's beneficial effects on aged brain function do not appear to be mediated through better glucose metabolism.We measured non‐fasting blood glucose concentration in 18‐month‐old animals that received MET or no treatment for two weeks , the dorsal hippocampus , and both hippocampus and the perirhinal cortex . In all three cognitive tests, MET‐treated aged animals showed improved cognitive and memory function than untreated aged mice, which comprised enhanced proficiency for pattern separation and location and recognition memories.A progressive increase in the extent of neuroinflammation in the hippocampus is one of the hallmarks of aging (Filfan et al., We also examined whether MET treatment commencing in late middle age improved the extent of hippocampal neurogenesis or synaptic density in old age. Measurement of the number of newly born neurons and Syn+ and PSD95+ puncta in the hippocampus revealed no differences between untreated aged mice and MET‐treated aged mice, implying that 10 weeks of MET treatment is not efficient for enhancing the extent of hippocampal neurogenesis or inducing neosynaptogenesis in male C57BL/6 J mice. The neurogenesis results are consistent with a recent study showing that MET does not improve subventricular zone neurogenesis in male C57BL/6 J mice (Ruddy et al., MET treatment may have also modulated several other age‐related adverse effects such as neuronal dendritic regression, altered gene expression, and other factors affecting hippocampal plasticity and network activity underlying cognitive, memory function (Burke & Barnes, 5Our results in a mouse model of aging provide novel evidence that 10 weeks of MET treatment commencing in late middle age is efficacious for improving cognitive function in the old age. Additional analyses suggested that improved cognitive function following MET treatment is linked to antiinflammatory effects, the activation of AMPK, inhibition of mTOR signaling, and enhanced autophagy in the aged hippocampus. These results imply that MET therapy in middle age leads to better cognitive and memory function in old age.The authors declare no conflicts of interest.Concept: AKS. Research design: MK, SA, LNM, and AKS. Data collection, analysis, and interpretation: MK, SA, LNM, BS, RU, JJG, XR, and AKS. Preparation of figure composites: MK, SA, and AKS. Manuscript writing: MK and AKS. All authors provided feedback and edits to the manuscript text and approved the final version of the manuscript.Supplementary MaterialClick here for additional data file.