This paper presents research on the impact of fly ash addition on selected physical and mechanical parameters of concrete made with slag cement. Experimental tests were carried out to measure the migration of chloride ions in concrete, the tightness of concrete exposed to water under pressure, and the compressive strength and tensile strength of concrete during splitting. Six series of concrete mixes made with CEM IIIA 42.5 and 32.5 cement were tested. The base concrete mix was modified by adding fly ash as a partial cement substitute in the amounts of 25% and 33%. A comparative analysis of the obtained results indicates a significant improvement in tightness, especially in concrete based on CEM IIIA 32.5 cement and resistance to chloride ion penetration for the concretes containing fly ash additive. In the concretes containing fly ash additive, a slower rate of initial strength increase and high strength over a long period of maturation are shown. In accordance with the presented research results, it is suggested that changes to the European standardization system be considered, to allow the use of fly ash additive in concrete made with CEM IIIA 42.5 or 32.5 cement classes. Such a solution is not currently acceptable in standards in some European Countries. Research on low-emission concrete, also called green concrete, is focused on waste recycling ,2 and thFly ash (FA) is a by-product of the coal dust combustion process in heat plants and power plants, and after passing the certification process, it can be used as an additive to concrete. The use of FA and blast furnace slag as an additive to cement and concrete mixtures allows for waste recycling, reduces the cost of producing building materials, reduces the carbon footprint of concrete, and has a positive effect on selected physical properties of concrete.Numerous experimental tests on concrete mixes have shown a significant improvement in the tightness of concrete after the application of FA additive. The positive effect of the addition of FA to cement and concrete mixes is closely related to the properties, quantity, and quality of the FA used . SignifiIn , GolewskIt is worth noting that fly ash can be obtained from various sources of waste recycling. Rutkowska et al.  studied To date, the conducted studies have focused on the properties of concrete with fly ash additive based on CEM I cement. The EN 206+A1:2016-12 standard  allows tstandard  does notIncreased stable strength and very good dynamics of strength increase over long periods of maturation;Low heat of hydration ;High resistance to the action of corrosion factors, especially sulfates ;Lower water and salt absorption ;Resistance to chloride penetration and reinforcement corrosion ;Low capillary porosity ;Good workability of the mix;Stability of volume;Resistance to the destructive action of alkali from cement in concrete with reactive aggregate ;Light color, allowing for its use in the production of architectural concrete.CEM III cement has the following properties:Van Noort  indicateBecause there is a significant amount of blast furnace slag in proportions from 35% to 65%, CEM III cement can be classified as an ecological cement binder, with a low carbon footprint and high recyclability properties. Slag is a by-product in the metallurgy industry resulting from the smelting of iron ore in a blast furnace. Because of its hydraulic properties, blast furnace slag is a valuable cement component. The use of blast furnace slag in cement allows the amount of Portland clinker to be reduced, which is favorable for ecological reasons. The production of Portland clinker is a highly energy-intensive process that has a negative influence on the environment .w/c = 0.45, and the minimum content of CEM I or CEM IIA cement is 320 kg/m3.The main areas of application of concrete with CEM III binder are foundation slabs, massive foundation footings, diaphragm walls, and structural elements of underground tanks. This is due to the need to provide low concrete shrinkage, resistance to sulfate aggression, or a low heat of hydration. Foundation slabs are designed in exposure class XD3 because of their susceptibility to corrosion due to chloride. According to the standard EN 206+A1:2016-12 , for expImproved workability and stability of the concrete mix;Reduced effects of water release from concrete—less “bleeding”;Increased tightness.Properties resulting from the use of fly ash additive to concrete include:These characteristics are extremely important when making foundation slabs.In , Bijen dThe purpose of this research is to demonstrate how the use of fly ash as an additive constituting more than 25% by weight of CEM IIIA N LH HSR NA cement with strength classes 32.5 and 42.5 affects the physical and mechanical properties of concrete; in particular, its tightness and the migration of chloride ions.3 for the XD3 exposure class. However, for concrete with additives, the minimum cement content was reduced to 270 kg/m3. Simultaneously, the standard [It should be noted that currently, in some countries, such as Poland, Denmark, and UK, the use of fly ash for concrete based on CEM III cement is not allowed by the standard  and its tandards . In the tandards , the minstandard  allows tThe results of the tests carried out in this work indicate a positive influence of fly ash additive on the properties of concrete made by using CEM IIIA LH HSR NA cement of strength classes 32.5N and 42.5N. The results of experimental tests that measured strength properties and resistance to water and chloride ion penetration for concrete made of CEM IIIA LH HSR NA cement with strength classes 32.5N and 42.5N, with and without fly ash additive, were analyzed. The experimental results show that it is possible to obtain concrete based on slag cement with fly ash additive with very good tightness and resistance to chloride corrosion without impairing concrete’s strength properties. The tests were carried out for the addition of fly ash, constituting 25% and 33% of cement weight. The justification for the use of such an amount of fly ash additive is the guidelines of standard EN 206+A1:2016-12 , for theThe samples for testing were prepared for three series of concrete mixes, using CEM IIIA 42.5N cement and three series of concrete mixes made using CEM IIIA 32.5N cement. The series of concrete mixes differed in the amount of fly ash additive substituted for slag cement. Slag cement CEM IIIA 42.5 and CEM IIIA 32.5;Fly ash;Fine aggregate—pit sand with a maximum size of 2.0 mm;Coarse aggregate—natural gravel with a minimum and maximum size of 2.0 and 8.0 mm;Lignosulfonate- and naphthalene-based admixture;Polycarboxylate-based admixture;Pure laboratory pipeline water.For testing, the following materials were used:CEM IIIA 42.5N LH HSR NA and CEM IIIA 32.5N LH HSR N slag cements  were used for testing the concrete series, denoted by CM1 and CM2, respectively. The physical parameters, mechanical parameters, and chemical properties of these cements, made available by the producer, are presented in As an additive to concrete, fly ash originating from hard coal combustion in the Kozienice CHP plant located in central Poland (Kozienice) was used. The used FA has pozzolanic properties, consists mainly of spherical grains, and is certified in the 1+ system, meeting the requirements of the EN 450-1 standard .Fineness: category N, below 12%;Loss on ignition: category A, below 5%.FA was characterized by the following parameters:3S2;Mullite A2;Quartz SiO2O3;Hematite Fe3O4.Magnetite FeThe detailed results of the chemical and phase composition  of fly aThe compounds contained in the phase composition are listed in order of decreasing percentage share of FA weight.w/c = 0.45 without the addition of fly ash. Next, the composition of reference concrete mixes was modified by adding fly ash, with the simultaneous reduction of cement and sand aggregate and the assumption of unchanged The tests were carried out for six series of concrete mixtures made using CEM IIIA 42.5N cement , and using CEM IIIA 32.5N cement . The reference concrete mixes CM1-0 and CM2-0 meet the requirements of exposure class XD3 and were designed assuming the value of standard , only foThe consistency of the concrete mix was tested using the falling cone method, in accordance with EN 12350-2 .nssmD was used, determined in accordance with [2 solution. The value of the nssmD coefficient was determined on the basis of the voltage applied, the temperature of the anolyte measured at the beginning and end of the test, as well as the depth at which chloride ions penetrated, which was measured on an axially split sample. The study was conducted within 48 h.The chloride ion migration coefficient in hardened concrete was determined according to the method presented in the NT BUILD 492 standard . The tesnce with . The chlThe value of the chloride ion migration coefficient in the transient state was calculated according to the NT BUILD 492 standard , using tU—value of voltage used (V);T—average value of the initial and final temperature of the anode liquid (°C);L = 50 mm—sample height;t—duration of the test (h).Concrete water tightness testing for samples of all series was carried out after 120 days of maturation. The tests were performed on cubic samples with a side dimension of 150 mm, whose side surfaces were protected with waterproofing resin. The samples were exposed to pressurized water. For this purpose, an apparatus for testing the water permeability of concrete 55-C0246/6  was used. The device consists of a steel frame with clamps with a hydraulic system, valves, a water pressure indicator, and transparent measuring burettes. The apparatus is adapted for simultaneous testing of up to six cubic samples, and enables water supply at a maximum pressure of 10 bar. The water pressure was increased every 24 h by 0.2 MPa, in the range of 7.4–8.0 MPa. The final pressure, corresponding to a water resistance of 80 m, was maintained for 24 h. The samples were then split, and the depth of water penetration into the concrete was measured using an electronic caliper. From the results of measurements of the depth of water penetration into concrete, the average depth of water penetration into concrete was calculated for the tested samples:mw—average depth of water penetration into concrete (mm);iw—maximum depth of water penetration into concrete in the ith sample;n—number of samples tested.Cubic samples with side dimensions equal to 100 mm were tested to measure the concrete compressive strength. The tests were carried out for concrete recipes CM1-0, CM1-25, CM-33 and CM2-0, CM2-25 and CM2-33 in subsequent periods of concrete maturation from 1 to 234 days. Samples were stored in accordance with the standard [Concrete tensile strength at splitting was tested in accordance with the standard . Six cubct,splf—tensile strength of concrete during splitting (MPa);F—splitting force (N);d—dimension of the sample side (mm).The results of consistency tests presented in A comparative analysis of the obtained results of the chloride ion migration test for the three analyzed recipes indicates a significant increase in resistance to the penetration of aggressive chlorides into concrete in which FA additive was used. The measurement parameters used during the testing are shown in The category of resistance to the penetration of chloride ions with the designation “good” was obtained, both in the case of concrete without the addition of FA, i.e., CM1-0, and in the case of concrete CM1-25, in which FA was used in the amount of 25% of the cement weight. However, comparing the average values of the chloride ion migration coefficient, one can notice a clear reduction in its value, in the case of concretes containing FA additive. The average value of the coefficient obtained for CM1-25 concrete is 26% lower than the value of the coefficient obtained for CM1-0 concrete, while for CM1-33 concrete containing the largest amount of ash (33% of the cement weight), the coefficient value is as much as 43% lower. In addition, CM1-33 concrete was classified in the highest category of resistance to chloride ion penetration, defined as “very good”. The reduction in the chloride ion migration coefficient for concrete samples containing FA additive is directly related to the average depth of chloride ion penetration. In CM1-0 concrete, the depth of ion penetration is almost double that of CM1-33 concrete.One of the results obtained for the CM1-33.3 series was rejected because of microcrack induction.During the tests, no water permeation through the concrete sample was found in any case. The results in mw determined for concrete with the addition of less ash (CM1-25) is 10% smaller than the value determined for concrete without the addition of ash (CM1-0), and with the maximum ash addition (CM1-33), it is smaller by as much as 19%. In turn, in concretes made on the basis of CEM IIIA 32.5 cement, the addition of FA causes a decrease in the average value mw by 68% for CM2-25 concrete and by as much as 87% for CM2-33 concrete compared with the average value mw obtained for concrete without FA addition, i.e., CM2-0.The mean value of the water penetration depth The results of the compressive strength tests are summarized in Among CM2 concretes with long-term maturation, i.e., more than 90 days, the strength of CM2-33 concrete is observed to increase in relation to CM2-25 concrete by 4%, as shown in According to the results of this research, it can be stated that FA, as an addition to concrete based on slag cement, causes a decrease in the strength of concrete, after both the early and the long-term maturation of concrete. However, the use of FA additive in amounts above 30% in concrete based on CEM IIIA 32.5 cement does not rapidly cause a reduction in concrete compressive strength, as was observed in the case of concrete based on Portland cement .The impact of FA addition on the development of compressive strength after long-term concrete maturation was also analyzed. The results of this analysis are shown in Considering the concrete maturation time, which was 120 days, it can be concluded that the influence of the FA addition on the tensile strength of concrete made from slag cement is consistent with the results obtained for concrete based on Portland cement .The conducted tests indicate the usefulness of using type II additive in the form of FA for concrete containing blast furnace slag cement. The presented test results indicate that the addition of FA to concrete mixtures based on slag cements leads to an increase in concrete tightness and resistance to chloride aggression. Both these parameters are particularly important when making such structural elements as foundation slabs. The use of tight concrete resistant to the penetration of adverse and aggressive external factors is extremely important, not only for concrete durability but also for protecting reinforced concrete structures against corrosion. Limitation in the penetration of chloride ions is particularly important for protecting reinforcement steel against corrosion. Corrosion caused by chlorides is extremely dangerous because of its initially asymptomatic course. Consequently, it causes the formation of permanent damage to the reinforcement, which can lead to the sudden destruction of the structure. The phenomenon of corrosion caused by chlorides is additionally enhanced by moisture. For the analyzed concrete recipes, after using FA additive, a significant increase in the resistance of concrete to the penetration of chloride ions and water was observed. An increase in these parameters was observed with an increase in the amount of FA used. The results of the conducted tests indicate a significant reduction in the penetration of concrete by water after the application of FA additive for concretes using CEM IIIA cements, of both classes 42.5 and 32.5. The relationship between the depth of penetration of water and the depth of penetration of chloride ions into concrete was determined. Significantly limiting the depth of water penetration into concrete based on CEM IIIA 32.5 and 42.5 slag cements by using FA additive will significantly reduce the penetration depth of chloride ions, which is particularly important in the case of structures that are designed for exposure class XD3 and exposed to substantial chloride aggression.After a short maturation time, the FA addition significantly reduced the concrete’s compressive strength in both CM1 and CM2 concretes. With the elongation of maturation time, an increase in the strength of concrete containing FA additive was observed. Moreover, in the second phase of maturation, from day 56, the CM2-33 concrete based on CEM IIIA 32.5 cement and containing a maximum of 33% FA additive was found to have higher strength than the CM2-25 concrete containing 25% FA additive. Comparing the results of the tests, it can be seen that concrete based on CEM III 32.5 cement with the addition of FA has a much greater resistance to water penetration and a greater reduction in strength in the initial maturation phase than concrete based on CEM III 42.5 cement with the same percentage of FA additive. In concrete with CEM III 32.5 cement, neither compressive strength nor tensile strength at splitting decreases proportionally to the increase in the FA additive content, as is observed with concrete with CEM IIIA 42.5 cement. The test results also indicate the usefulness of using FA as an additive to concrete based on CEM IIIA 32.5 cement, since there is a significant improvement in concrete tightness. Nevertheless, it should be remembered that the strength of concrete is reduced during the initial time of concrete maturation, which must be considered when designing it. In light of the test results obtained, changing the national standardization systems to allow the addition of fly ash to concretes based on CEM IIIA 42.5 and 32.5 slag cements is an issue worth considering.The following conclusions were made from the results obtained during the research.The addition of fly ash increases the tightness and resistance to the penetration of chloride ions of concrete made of slag cement.Fly ash reduces the depth of penetration of water under pressure into concrete based on CEM IIIA 32.5 slag cement, to a greater extent than that based on CEM IIIA 42.5 cement. The addition of FA reduced the depth of water penetration under pressure by 19% for CM1-33 concrete, and by as much as 87% for CM2-33 concrete, compared with reference concretes that did not contain fly ash.mw is proportional to the decrease in the average depth of the penetration of chloride ions into concrete m,totx. The proportionality coefficient for this reduction is in the range The relationship between the resistance of concrete to water penetration under pressure and resistance to chloride ion penetration is demonstrated. A comparison of the test results obtained for concretes CM1-0, CM1-25, and CM1-33 indicates that the effect of FA addition on reducing the mean value of the water penetration depth under pressure into concrete FA addition to concrete based on slag cement reduces compressive strength during the initial and long-term maturation times. In the case of concrete based on CEM IIIA 42.5 cement, the reduction in compressive strength after the application of FA additive in amounts of 25% and 33% by the weight of cement is similar and does not exceed 5%. This applies to both the standard maturation period, i.e., after 28 days, as well as the extended maturation period, i.e., after 190 days. Concrete based on CEM IIIA 32.5 slag cement after using FA additive is characterized by a significant reduction in compressive strength, which after 28 days of maturation, is 23% (series CM2-25). After the long maturation period of over 120 days, the reduction in compressive strength of concretes containing FA additive does not exceed 10%.The use of FA additive for concretes based on slag cement in amounts of up to 33% by weight of cement is beneficial. The conducted research shows that the FA addition of 33% compared with the addition of 25% by the weight of cement in concrete based on CEM IIIA 42.5 cement does not significantly affect compressive strength and, in the case of concrete based on CEM IIIA 32.5 cement, even causes an increase in this strength. These observations refer to both standard and long maturation periods. The increased addition of fly ash significantly increases the tightness of concrete and its resistance to the penetration of chloride ions.k factor at XD1-3 concrete exposure for CEM IIIA cement classes as well.Consideration should be given to allowing the use of the


S. aureus VBNC states at low temperatures were investigated. Different acidity and nutritional conditions were then applied to food products to control the VBNC state formation. Four different concentration levels of each factor  were selected in a total of 16 experimental groups. Nutrition showed the highest influence on the VBNC state formation S. aureus, followed by acid and salt. The addition of 1% acetic acid could directly kill S. aureus cells and inhibit the formation of the VBNC state with a nutrition concentration of 25, 50, and 100%. A propidium monoazide-polymerase chain reaction (PMA-PCR) assay was applied and considered as a rapid and sensitive method to detect S. aureus in VBNC state with the detection limit of 104 CFU/mL.A Viable but non-culturable (VBNC) state is a bacterial survival strategy under reverse conditions. It poses a significant challenge for public health and food safety. In this study, the effect of external environmental conditions including acid, nutrition, and salt concentrations on the formation of  VBNC Esal cells . Furtheral cells . TherefoThe traditional detection method for foodborne microbes is culturing-based. However, in the VBNC state, bacteria remain metabolic activity but below detection levels, indicating the ability to cause false negative detection by culturing-based method . Thus, fS. aureus at low temperature (4 and −20°C) to obtain a better understanding of the conditions of the formation of the VBNC state in food systems and to enable us to control it. A PMA-PCR assay was applied to detect the VBNC cells of S. aureus.This study aimed to investigate the effect of nutrition, acid, and salt concentrations on the viability and culturability of S. aureus ATCC25923, which was maintained as glycerol stock and stored at −80°C before use. The strain was streaked on tryptic soy agar (TSA) plate and incubated at 37°C for 24 h to recover. A single colony was then inoculated into 2 mL of TSB and incubated at 37°C with 150 rpm for 12 h prior to further experiments.The stain used in this study was 7 CFU/mL. To avoid the effects of continuous freeze-thawing, the induction system was separated into multiple 1.5 mL centrifuge tubes. Subsequently, the tubes were placed at 4 and −20°C, respectively, to induce the VBNC state.The bacterial culture was inoculated into TSB with 1:100 dilution and was incubated until it reached the exponential phase according to the growth curve (data not shown). The exponential phase culture was centrifuged at 5,000 × g for 10 min and the cells were washed with 1 × phosphate buffer solution (PBS). The washed culture was resuspended in induction groups  to a finS. aureus cells, the plate counting method was applied to identify the culturable cell number. The induction culture was serially diluted with 0.9% NaCl and inoculated on TSA followed by incubation at 37°C for 24 h. When culturable, the cell number was < 1 CFU/mL for 3 days, and the cells were considered to be non-culturable  combined with fluorescence microscopy was used to determine whether the non-culturable cells were in the VBNC state following the manufacturer’s instructions.To determine the culturability of lturable . In addi7 CFU/mL with a total volume of 30 mL and stored at 4 and −20°C, respectively. The culturable cell number was measured by plate counting after 3 days . Then, the final induction group was stored at 4°C for 3 days and the culturability and viability were identified by plate counting method and LIVE/DEAD® BacLightTM bacterial viability kit, respectively.Twenty-five grams of Cantonese rice cake  was added to 225 mL of 0.9% NaCl and determined as a 100% food sample medium. Accordingly, 25 and 50% food sample medium were prepared with sterilization. 2 mL of S. aureus in the VBNC state was diluted to the final concentrations of 106, 105, 104, 103, 102, and 10 cells/mL using the diluting solution, respectively. The PMA reagent was used at the concentration of 5 μg/mL. Subsequently, the detection samples mixed with PMA were incubated in the dark at room temperature for 10 min before the tubes were placed horizontally on ice exposed to a halogen lamp (650 W) at a distance of 15 cm for 15 min to complete the combination of DNA and PMA , 2 μL of DNA template and added up to 25 μL with nuclease-free water. A mixture with 2 μL of nuclease-free water was used (instead of DNA) as a negative control. The protocol of PCR assay was as followed: 5 min denaturation at 95°C, 32 cycles of amplification at 95°C for 30 s, 52°C 30 s, 72°C for 35 s and final inactivation at 72°C for 5 min. The PCR products were detected by electrophoresis on 1.5% agarose gels and observed under UV light.The PCR assay was performed at a total volume of 25 μL. The reaction system consists of 12.5 μL 2 × Taq PCR MasterMix , 3 μM each of forward and reverse primers . These results showed that the key conditions for the VBNC state formation of S. aureus were adequate nutrition with strong acid at 4°C, insufficient nutrition with weak acid at 4°C, and oligotrophic system at −20°C.In summary, under low temperature (4°C) and strong acidity with sufficient nutrition (medium concentration ≥ 50%), S. aureus was able to enter into VBNC state under strong acid with adequate nutrition but not under strong acid that lacked nutrition, indicating with the treatment of strong acid, nutrition plays an important role during the formation of VBNC state. By comparing induction groups 1 and 2, under weak acid, it took cells in group 2 a shorter time to enter into VBNC state, demonstrating that weak acid may have an active contribution to the formation of VBNC state. Due to the salt-tolerant property of S. aureus, salt concentration had no significant effect. Under insufficient nutrition (medium concentration ≤ 50%) with strong acid [concentration of acetic acid (v/v) ≥ 0.7%] and high salt concentration (≥10%) without nutrition, the cells died within 3 days. Therefore, nutrition had the strongest effect on the formation of the VBNC state, followed by the concentration of acetic acid and salt.S. aureus cells with higher ATP concentration would enter into the VBNC state instead of dying. Similar phenomenon has been found in the VBNC L. monocytogenes  or superoxide dismutase (SodA) encoded by katA and sodA gene was present. The changes on the expression of genes rendered cell hypersensitive to seawater with a high concentration of salt at 4°C  conditions, S. aureus lost culturability and viability in 3 days, while low salt concentration with nutrition (medium concentration ≤ 25%) and 0.7%, 1.0% acetic acid (induction group 4 and 5), S. aureus remained culturability when stored at 4°C for 3 days. Among all, only the cells in induction group 5 stored at −20°C were non-culturable, indicating that eliminating VBNC state formation only by acetic acid treatment is not sufficient. S. aureus died at low nutrition, high salt, and strong acidity, indicating salt concentration can restrain the formation of the VBNC state However, given the low salt concentration in the food processing and storage of rice product, only acid treatment is less effective in eliminating S. aureus and its VBNC state , NC state .S. aureus could enter into VBNC state with strong acid treatment, the elimination of VBNC state by different nutrition conditions with strong acid were studied. Under low temperature (4 and −20°C), all cells were dead within 3 days in groups with no nutrition and strong acid . S. aureus may enter into VBNC under the treatment of some nutrition and low salt with strong acid. These results indicated that in low salt and strong acid environment, the VBNC state of S. aureus cannot be eliminated by only reducing nutrients. Thus, the control of VBNC state formation can be achieved by changing nutrition concentration in combination with other treatments. One way of eliminating the VBNC state of S. aureus, which could be applicable in food processing or used when cleaning equipment, would be to provide a condition of no nutrition with acid  assay was applied and considered to be a rapid and sensitive method for detecting S. aureus in the VBNC state, with the detection limit of 104 CFU/mL.This study investigated the impact of three elements including nutrition, acid and salt concentrations in food systems on the VBNC state formation of The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.JL and KW conceived of the study and participated in its design and coordination. T-YH, YM, YC, FS, RP, and JC performed the experimental work. CB, LY, and LC analyzed the data. JL prepared and revised this manuscript. All authors reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BDNF gene (rs6265) is associated with impaired activity-dependent release of brain-derived neurotrophic factor (BDNF), resulting in reduced synaptic plasticity, impaired glutamatergic neurotransmission, and morphological changes. While previous work has demonstrated Val66Met effects on magnetic resonance spectroscopy (MRS) markers of either glutamatergic metabolism (Glx) or neuronal integrity (NAA), no study has investigated Val66Met effects on these related processes simultaneously. As these metabolites share a metabolic pathway, the Glx/NAA ratio may be a more sensitive marker of changes associated with the Val66Met SNP. This ratio is increased in psychiatric disorders linked to decreased functioning in the anterior cingulate cortex (ACC). In this study, we investigated the correlation of the Val66Met polymorphism of the BDNF gene with Glx/NAA in the pregenual anterior cingulate cortex (pgACC) using MRS at 3 Tesla (T)  and 7 T . In both cohorts, Met carriers had lower Glx/NAA compared to Val homozygotes. Follow-up analyses using absolute quantification revealed that the Met carriers do not show decreased pgACC glutamate or glutamine levels, but instead show increased NAA compared to the Val homozygotes. This finding may in part explain conflicting evidence for Val66Met as a risk factor for developing psychiatric illnesses.The Met allele of the Val66Met SNP of the  BDNF) is a key molecule in neural plasticity that is involved in neuronal cell growth and differentiation, but also apoptosis2. In the mature synapse, it plays an important role in neurotransmission and synaptic plasticity4. In humans, a common single-nucleotide polymorphism (SNP) of the BDNF gene on chromosome 11p14.1 (196 G > A) results in a substitution of valine  with methionine (Met) at codon 66 . The Val66Met polymorphism has attracted considerable attention in psychiatric research, as the 66Met allele impairs intracellular trafficking and (activity-dependent) release of BDNF into the synaptic cleft6, even in heterozygous carriers7. The polymorphism is associated with the course and development of various neurological and psychiatric disorders10.The brain-derived neurotrophic factor  and hippocampus18. In humans, the Met allele is associated with reduced hippocampal and prefrontal volume22. This suggests that gross morphological changes related to BDNF Val66Met can be detected with magnetic resonance imaging (MRI) in humans.BDNF release is tightly linked to glutamatergic neurotransmission. BDNF facilitates glutamate release, influences excitatory synapse formation, and increases NMDA receptor and AMPA receptor subunit phosphorylation. These processes enhance long-term potentiation (LTP)25.Glx represents the sum of glutamate and glutamine concentrations. Previous work has studied NAA or Glx separately. Left hippocampal Glx (Met homozygotes only26) and NAA were reduced in Met carriers27. The distribution of metabolites like NAA-as well as Glx-as a function of BDNF Val66Met genotype may be subject to regional differences, as in the anterior cingulate cortex (ACC) NAA levels are increased in Met carriers28. Given the shared metabolic pathways of these molecules30 and the correlation of these metabolites often reported in healthy participants33, the ratio of Glx/NAA may be a more sensitive marker of dysregulation due to Val66Met.As Val66Met affects both glutamatergic neurotransmission and synaptic structure, its effects may be better characterized when both these targets are assessed simultaneously. One way to do so is by means of magnetic resonance spectroscopy (MRS). MRS can non-invasively quantify local in vivo concentrations of metabolites such as N-acetylaspartate (NAA) and Glx. NAA is a mitochondrial metabolite often reported as a proxy for neuronal integrity and viability38. In both disorders, decreases in gray matter volume have been reported for the ACC, especially for the pregenual part (pgACC)42. Since decreases in hippocampal and prefrontal gray matter volume have been shown for the Met allele of the Val66Met SNP, a reduced gray matter volume in the pgACC might be linked to the Val66Met SNP as well. The pgACC is a region implicated in the pathophysiology of several psychiatric disorders43, but that has not been investigated in the context of the Val66Met SNP. Investigating Val66Met effects on the Glx/NAA ratio in the pgACC could therefore provide insight into downstream functional consequences of the polymorphism that could indicate the Met allele as a protective or a risk factor.The Val66Met polymorphism has been associated with psychiatric disorders like schizophrenia or major depressive disorder (MDD), either as a risk factor or as a protective factorBDNF Val66Met polymorphism on an index of glutamatergic functioning and neuronal integrity in the pgACC. To this end, we used MRS to measure pgACC levels of Glx as a ratio over NAA. For a more robust assessment of the effect of the Val66Met SNP, two independent cohorts were investigated at 3 T and 7 T, respectively. We hypothesized that because the Val66Met polymorphism affects neurodevelopment as well as glutamatergic neurotransmission, Met carriers would show lower Glx/NAA compared to Val homozygotes. We expected that this would be primarily due to reduced concentrations of glutamate-related metabolites since specifically in the pgACC a decrease in Glx has been associated with MDD45. A decrease in NAA, on the other hand, has been associated with schizophrenia46. The increase in NAA in the ACC has been suggested to be just a compensatory mechanism for a deficit in BDNF regulation speaking for a decreased risk to develop schizophrenia and for a deficit in hippocampal NAA28. We tested this hypothesis in the second cohort, where the higher field strength of 7 T allowed us to perform exploratory analyses of absolute concentrations of NAA, as well as the major contributors of Glx (glutamate + glutamine), considering their better separation at ultra-high field.Here, we investigate the effects of the 2 = 0.016, P = 0.899, D = 0.408, cohort 2: N = 98, χ2 = 2.110, P = 0.146, D = 2.296).Descriptive statistics of the sample used in the main analyses are summarized in Table BDNF Val66Met carriers had altered Glx/NAA compared to Val homozygotes, we performed a linear model analysis with Glx/NAA as the outcome variable and BDNF genotype as predictor, controlling for the effect of age and gray matter proportion in the MRS voxel. Data from one participant was classified as an outlier and removed . The assumption of normality of model residuals was met . The linear model revealed a significant effect of BDNF genotype, F = 6.756, P = 0.016, η2partial = 0.230  or gray matter proportion (P = 0.093). When testing for the directionality of the BDNF genotype effect, a t-test for unequal variances revealed that the Glx/NAA ratio was higher in Val homozygotes  compared to Met carriers , t (12.246) = − 2.191, P = 0.049, 95% CI .To test whether notype, F,22 = 6.747. We excluded one data point that was classified as an outlier . Model residuals were normally distributed . There was a significant effect of BDNF genotype on pgACC Glx/NAA, F = 4.597, P = 0.035, η2partial = 0.050  and pgACC gray matter proportion (P = 0.384). We observed a significant main effect of gender (P = 0.026). To further explore this, we ran unplanned follow-up analysis adding the interaction term BDNF group*gender to the model. This revealed no significant interaction effect, P = 0.999.To test whether reduced Glx/NAA could be reproduced in a larger cohort measured at the ultra-high field strength of 7 T, we performed a linear model analysis as described above, additionally controlling for gender since metabolite concentrations may vary as a function of sexlx/NAA, F,92 = 4.5BDNF genotype. This revealed that Val homozygotes had significantly higher Glx/NAA  compared to Met carriers , t(71.593) = − 2.394, P = 0.019, 95% CI .Analogous to the analysis of cohort 1, we performed a t-test of unequal variances to test for the directionality of the main effect of P-values of the main effect of BDNF genotype on pgACC Glx/NAA from two independent cohorts using Fisher’s method revealed converging evidence for a reduction of pregenual Glx/NAA in Met carriers, χ2(4) =14.943, P = 0.005.Combining the BDNF Val66Met effects on aspects of excitatory neurotransmission and proxies of neuronal integrity, we capitalized on the increased signal dispersion at ultra-high field strength of 7 T in cohort 2. In post-hoc analyses, we performed absolute quantification of NAA, Glu, and Gln and modeled BDNF effects on these metabolites separately, controlling for the influence of age and gender. Voxel tissue composition was taken into account during the absolute quantification procedure . For all analyses reported in this section, the assumption of normality of residuals was met .To tease apart the F = 0.606, P = 0.438, η2partial = 0.004, Fig. F = 0.696, P = 0.406) or gender  = 2.261, P = 0.136). Gln concentration showed no main effect of genotype  = 0.016, P = 0.901, η2partial < 0.001, Fig. F = 0.071, P = 0.790), but a significant main effect of gender, F = 6.159), P = 0.015, η2partial = 0.067), such that male participants had higher Gln concentrations  compared to female participants . Including the interaction term of genotype*gender in the model revealed no significant interaction effect  = 0.308, P = 0.581).For the analysis of Gln, four data points were removed because of insufficient data quality. For the analysis of both NAA and Gln, four data points were classified as outliers and subsequently removed. For the analysis of Glu, two outliers were removed. Glu concentration revealed no significant main effect of genotype  = 6.356, P = 0.013, η2partial = 0.068  = 7.807, P = 0.006, η2partial = 0.068. To further explore this, we included the interaction term BDNF*gender in the linear model. This resulted in a no longer statistically significant main effect of BDNF group  = 0.012, P = 0.912) but a significant interaction effect, F = 5.598, P = 0.020. When followed up with separate linear models for each gender, men showed a significant main effect of genotype, F = 10.585, P = 0.002  = 0.018, P = 0.893.In the analysis of NAA, we found a significant main effect of gender, 002 Fig. , such thBDNF Val66Met polymorphism on measures of excitatory neurotransmission in relation to neuronal integrity in the pgACC, a region of key importance in the pathophysiology of mood disorders such as MDD. The results from the independent cohorts provide converging evidence for a reduction of Glx/NAA in Met carriers. This is in line with our hypotheses. Follow-up analyses of the metabolites comprising the ratio revealed that, contrary to our primary expectation, this effect is due to an increase in NAA rather than a decrease in Glx. To our knowledge, this is the first study assessing the influence of this SNP on glutamatergic and NAA metabolism in the pgACC. These findings were strengthened by the fact that the two independent cohorts reported here were measured at different field strengths (3 T vs 7 T), used different MRS acquisition schemes (PRESS vs STEAM), and used male-only versus male and female participants, respectively.In two independent samples of healthy participants, we investigated the effects of the 48, has a role in energy metabolism, osmoregularity, and myelin formation, and is frequently described as a marker of neuronal viability23. NAA synthesis is coupled to the capacity of mitochondria for ATP synthesis50. Glu is not only the main excitatory neurotransmitter, it has roles in energy metabolism as well51. NAA and Glu are linked mainly through the tricarboxylic acid (TCA) and glutamate-glutamine cycles24. Although the precise role of NAA in the brain is debated, one proposal suggests that NAA serves as a reservoir for glutamate synthesis30. This assumption is based on a cycle converting NAA into aspartate and acetate by aspartoacylase in astrocytes and oligodendrocytes. Aspartate and acetate are then converted to oxaloacetate and acetyl CoA, important constituents of the TCA cycle. In the mitochondria, these intermediates are combined to form citrate. Citrate is oxidized to alpha-ketoglutarate, which is then converted to glutamate via transamination. Glutamate can also re-enter the TCA cycle for ATP production24.Given the shared metabolic pathways of NAA and Glu, the ratio of Glx/NAA may be a more sensitive marker of (dys)regulation in related metabolites. NAA is highly abundant in the brain, where it is predominantly found in neurons. It is most highly concentrated in the mitochondria of pyramidal glutamatergic neurons13C-MRS study showed that the rate of glutamate labeling from 13C-glucose infusions was tightly coupled with NAA synthesis52. In healthy participants, studies have found positive correlations between NAA and Glx in the cerebellum31, hippocampus33, pgACC32 but conflicting results for the dorsal ACC53. In MDD patients, pgACC NAA and Glx were more strongly correlated in patients compared to healthy control participants32. Patients with schizophrenia, in contrast, showed a decoupling of NAA and Glx in the hippocampus33 and dorsolateral PFC53. In summary, the equilibrium between glutamate and NAA related metabolism may be disturbed in different classes of psychiatric disorders.Consistent with this metabolic cycle, some studies have shown correlations between MRS measures of local NAA and glutamatergic metabolites in healthy controls, that were disrupted in patients. A 54. Patients with MS had elevated ratios of glutamatergic metabolites to NAA in white matter compared to healthy controls which correlated with symptom severity54 and longitudinal brain volume loss55. While BDNF Val66Met has not been associated with disease risk or severity in MS58, a protective role for the Met allele has been suggested with respect to preserving grey matter integrity60 and maintaining memory network function62 in MS patients. This same increase is also seen in patients with disorders linked to decreased prefrontal and hippocampal volume such as schizophrenia and MDD when compared to healthy control participants63. In the hippocampus of patients with schizophrenia33 and post-traumatic stress disorder64, Glx/NAA and Glu/NAA respectively were elevated, which could be due to excitotoxic effects of excessive glutamate. In the ACC, NAA/Glx was reduced in patients with bipolar disorder compared to healthy controls and the ratio was normalized after a lamotrigine treatment65. In depressed youth with current suicidal ideation, pgACC Glx/NAA was higher compared to depressed youth without current suicidal ideation and compared to healthy control participants63. Similarly, another study found that medial prefrontal Glx/NAA (but not Glu/NAA) was higher in patients with MDD compared to healthy participants66. The Val66Met polymorphism has been linked to an increased risk for the development of MDD34 and schizophrenia35, although there is conflicting evidence36. Some studies report that the Met allele is a protective factor against development of schizophrenia37. Others report that Val66Met is not a risk factor for developing MDD38 or only a risk factor in males67. In general, an increased ratio of glutamatergic metabolites to NAA is associated with poorer mental health outcomes. A decrease in Glx/NAA in Met carriers hints at a potential protective effect of this genotype, but the mechanisms through which this may arise are unclear. One rather straight-forward explanation might be a higher overall availability of BDNF in Met carriers, which has been reported for BDNF serum levels and suggested to reflect a compensatory increase in constitutive secretion of BDNF68. Alternatively, or more likely additionally, a potential modulation of BDNF signaling via the p75 neurotrophin receptor by BDNF Val66Met might be considered a candidate mechanism5. The p75 receptor has been implicated in neuroinflammatory processes71, and a recent study suggests that higher serum BDNF 66Met carriers may be associated with lower concentrations of the pro-inflammatory cytokine TNFa72. However, the sample size in that study was relatively small, and future studies will be required to follow up on this intriguing possibility.Our finding of reduced Glx/NAA in Met carriers is particularly interesting, since studies investigating psychiatric and neurological disorders associated with tissue damage such as multiple sclerosis (MS) have reported an overall increase of Glx/NAA or Glu/NAA compared to healthy participants11 and reduced glutamatergic neurotransmission18, but on the other hand at any point in time, a proportion of glutamate serves metabolic roles and it appears that vesicular, neurotransmitter glutamate cannot be detected with MRS74. Subtle changes in neurotransmitter glutamate could therefore have gone undetected in our sample. Another possible reason may be that there is a dose-dependent effect of BDNF Val66Met on glutamatergic measures. Reduced Glx was previously reported in the hippocampus and posterior medial frontal cortex26 but only in Met homozygotes. Only one participant in cohort 1 carried two Met alleles, and no participants in cohort 2 were Met homozygotes. The relative rarity of homozygotic Met carriers may have impaired the detection of possibly subtler, dose-dependent effects of the Met-allele on the glutamatergic metabolites76. Moreover, both cohorts were young healthy participants and it may be that the effects of Val66Met are age-related and more relevant in older age78.To further investigate the effects of Val66Met on pgACC metabolism, we capitalized on the better signal dispersion at 7 T by computing absolute quantified values for Glu, Gln and NAA. In contrast to our hypothesis, reduced Glx/NAA in Met carriers was unlikely to be due to glutamatergic differences, as we found no significant difference in glutamatergic (Glu or Gln) levels between the two genotypes. This is striking, because the Val66Met SNP is associated with reduced BDNF signaling27 and lacking increase after training for a difficult task79. Given the evidence for reduced neuronal integrity in Met carriers in the hippocampus, our finding of increased NAA in Met carriers in the pgACC is particularly remarkable. However, an earlier study at 3 T similarly reported an NAA increase in Val66Met Met carriers in the ACC28. Our result therefore strengthens their finding and speaks for region-dependent effects. Gallinat et al. suggest that higher pgACC NAA may protect from developing psychiatric illnesses such as schizophrenia and bipolar disorder28. These disorders are associated with an altered ratio of Glx/NAA65 as well as a decoupling between glutamatergic metabolites and NAA53. In MDD and schizophrenia, research suggests a decrease in NAA concentrations as the disease progresses84. Taking into account the aforementioned shared metabolic pathways of glutamate and NAA, a relative increase of NAA in the pgACC of Met carriers may serve to readily and adaptively provide glutamate when necessary to maintain normal neuronal functioning85. The molecular pathways or gene–gene interactions that link the effects of Val66Met on BDNF release to an increase in NAA remain to be investigated.An intriguing finding is that of a significant increase in NAA in Met carriers. Apart from reduced spine density and other morphological abnormalities, Met carriers  were previously shown to have reduced NAA in the hippocampal formation34, specifically in males67. One explanation for discrepant results may be related to gene-environment interactions, as another meta-analysis found that the association between the Met allele and depression risk might be primarily found in individuals with high levels of life stress or childhood adversity86. Although Met carriers and Met/Met homozygotes showed larger cortisol responses to acute stress88, gender specific findings seem to be more controversial as some studies reported an attenuated cortisol response in male90 and others in female91 Met carriers. Cognitive performance was reported to improve from physical activity in male Val homozygotes only92, while the decline in executive functioning and processing speed over time is lowest in female Met carriers93. In an exploratory analysis, we found an interaction between genotype and gender such that male but not female Met carriers showed increased NAA in the pgACC. In healthy subjects, pgACC NAA has been shown to not differ significantly between males and females47. Assuming male Met carriers are more likely to develop MDD, we may have investigated a sample of healthy, resilient males with compensatory higher NAA levels. Further, a compensatory increase in NAA in Met carriers may not be necessary in females, as ovarian hormones such as estrogen increase BDNF mRNA levels in the cingulate cortex in rodents94. Hormone cycle specific explanations need further investigation, as we did not control for that in the current study.Meta-analytic evidence suggests that the Met allele confers increased risk for developing MDD95, and dorsolateral prefrontal areas97 when compared to Val homozygotes. For the ACC, there is not much evidence for such a reduction. Montag et al. found greater GM volume in the ACC in Val homozygotes compared to Met carriers, but lowest ACC GM volume in carriers of two different polymorphisms98. BDNF Val66Met carriers were also shown to have greater GM volumes in temporal and superior frontal areas99. GM volume differences of the Val66Met polymorphism seem to be region specific. In addition, the relative increase in pgACC NAA in Met carriers may play a role in prevention of cortical GM reduction resulting from excitotoxic effects. The absence of a difference in GM proportion between the two genotypes is therefore not surprising.Whereas earlier studies reported differences in the ratio of gray matter (GM) volume between Met carriers and Val homozygotes, we did not observe a significant difference in GM tissue proportion in the pgACC voxel in neither of the two cohorts. In previous studies, Met carriers showed reduced GM volume in the hippocampusBDNF Val66Met on pgACC NAA levels. This should be addressed in future studies. Further, we did not include the hippocampus as a control region since our hypothesis concentrated on the pgACC. Further, the cohorts differed in the GM ratio, with lower values in cohort 2. This might be related to the better contrast due to the higher spatial resolution in the 7 T images. It was shown previously that more accurate segmentation at higher field strength led to lower estimates of cortical thickness100. Last, we report measures of NAA and not the commonly used tNAA . Glx/tNAA and Glx/NAA were strongly correlated at 3 T (r(25) = 0.954, P < 0.001, 95% CI = ) and 7 T (r(96) = 0.903, P =  < 0.001, 95% CI = ). Therefore, we have repeated these with tNAA instead of NAA. At 3 T, the effect of BDNF Val66Met genotype remained significant (F(22) = 6.150, P = 0.021, η2partial = 0.221). At 7 T, the main effect of BDNF genotype was no longer significant, F(92) = 2.839, P = 0.100. Strikingly, there was also no longer a significant main effect of gender, F(92) = 2.036, P = 0.157. In the analysis of absolute levels of tNAA, there were no significant main effects of BDNF genotype (F(90) = 2.746, P = 0.101) nor of gender (F(90) = 3.413, P = 0.068). Taken together, these findings suggest that the better separation of NAA and NAAG at 7 T enables us to identify an effect of BDNF Val66Met that can be attributed to NAA and not to glutamate-related metabolites.Although our study has many strengths, it should be considered in the context of some limitations. Due to limited previous literature and reported effect sizes, we did not calculate a priori sample sizes. Post hoc power calculation showed that for Cohort 1 power was 82%, and for Cohort 2 61%. For cohort 1, we did not perform an absolute quantification of NAA due to lower signal dispersion at 3 T. Therefore, we could not check for the effect of NAA we observed in the 7 T sample. Due to the specification to optimally resolve Gln from Glu in the study with 7 T, the contribution of Gln to the measured Glx may not be substantially suppressed as the 3 T PRESS (80 ms) sequence. Our results have to be interpreted with caution. As the gender groups in our 7 T sample were not balanced, we cannot draw definitive conclusions about a male-specific effect of BDNF Val66Met polymorphism in two independent samples. Although many psychiatric and neurological disorders are associated with an increase in Glx/NAA, Met carriers of the BDNF SNP showed a decrease in this ratio. Against our expectations, follow up analyses suggest that glutamatergic metabolism is not affected by the polymorphism. Instead, Met carriers showed a relative increase in NAA in the pgACC. Future research should test whether this increase protects the region from effects of psychiatric disorders such as schizophrenia or MDD.To sum up, we found an altered ratio of Glx/NAA in carriers of the 101. All participants were screened for prior and current neurological or psychiatric illnesses using the German version 5.0.0 of the Mini International Neuropsychiatric Interview (M.I.N.I.)102. All study procedures obtained the approval of the ethics committee of the University of Magdeburg, Faculty of Medicine, and conformed to the Declaration of Helsinki. All participants received detailed information on the study and provided written informed consent to participate.We investigated two independent samples of healthy subjects. Cohort 1, measured at 3 T, consisted of 30 healthy men (mean age = 29.00 ± 5.55). Cohort 2 measured at 7 T comprised 98 healthy participants . Some participants of cohort 2 were recruited as the control group of a patient study. This cohort of healthy subjects was previously used in a published analysis on the association between glutamatergic disbalance and harm avoidanceBDNF val66met polymorphism (NCBI accession number: rs6265) was amplified using the primers BDNF_rs6265_f (forward): 5′- GCA TCC CGG TGA AAG AAA GCC CTA AC-3′ and BDNF_rs6265_r (reverse): 5′- GCC CCT GCA GCC TTC TTT TGT GTA AC-3′, and standard Taq polymerase (Qiagen). The resulting PCR products were digested with the PmaCI isoschizomer Eco721 (ThermoFisher Scientific), yielding two allele-specific amplicons (398 + 278 bp) for the more common Val allele, and the entire region (676 bp) for the less common Met allele. DNA fragments were separated on a 2.5% agarose gel stained with Midori Green  and visualized under UV light.Whole–blood samples were collected from participants in EDTA–coated tubes  and stored at 4 °C. Genomic DNA was extracted from whole blood using the GeneMole automated system  according to the manufacturer’s protocol. Genotyping was performed using PCR followed by allele–specific restriction analysis. Briefly, the DNA fragment on chromosome 11p14.1 containing the 3). These anatomical scans were used for accurate placement of the pgACC voxel according to an established protocol of anatomical landmarks, as described by Dou et al.103. MRS spectra were acquired in the bilateral pgACC (20 × 10 × 20 mm3)  sequence was used with the following parameters: TE = 80 ms, TR = 2 s, 256 averages, band width = 1200 Hz, acquisition time for one image = 853 ms104.With cohort 1, we conducted all measurements using a 3 T MAGNETOM Trio MRI scanner with an 8-channel head coil . Prior to MRS measurements, we acquired a high-resolution structural magnetization-prepared rapid gradient-echo (MPRAGE) T1-weighted scan (echo time (TE) = 4.77 ms, repetition time (TR) = 2.5 s, inversion time (TI) = 1.1 s, flip angle = 7°, bandwidth = 140 Hz/pixel, acquisition matrix = 256 × 256 × 192, isotropic voxel size = 1.0 mm105. We used a measured basis set that included sixteen metabolites: Creatine (Cr), Glutamate (Glu), Myo-Inositol, Lactate, N-Acetylaspartate (NAA), Phosphocholine (PCh), Taurine, Aspartate, γ–Aminobutyric acid (GABA), Glutamine (Gln), Glucose (Glc), Alanine, N–acetylaspartylglutamate (NAAG), Phosphocreatine (PCr), Guanidinoacetate , and Glycerophosphocholine. The default simulated macromolecules were included in the modelling. The analysis window was set to range from 4.3 ppm to 0.5 ppm. Eddy current correction was performed based on the water signal (LCModel parameter DOECC = T) and water suppression was performed (DOWS = T). The attenuation factors of water (ATTH2O) and metabolites (ATTMET) were assumed to be 0.2905 and 0.4079, respectively. The standard deviation of the expected value for zero-order phase correction (SDDEGZ) and first-order phase correction (SDDEGP) were set to 0.5 and 0.2, respectively. For our analyses, we used a joint measurement of glutamate and glutamine (Glx), expressed as a ratio to NAA. We used conventional criteria to ensure sufficient spectral quality. That is, metabolite values with Cramér Rao lower bound (CRLB) estimates of fitting error > 20%106, FWHM > 12 Hz, or SNR < 8 were excluded from further analyses. An exemplary spectrum is shown in Fig. The spectra were analyzed using LCModel version 6.1.03) . Before MRS measurements, we acquired an MPRAGE T1-weighted scan . MRS voxel placement in the pgACC conformed with procedures described for cohort 1 (20 × 10 × 15 mm107. We used a measured basis set that included Creatine (Cr), Glutamate (Glu), Myo-Inositol, Lactate, N-acetylaspartate (NAA), Phosphocholine (PCh), Taurine, Aspartate, γ-Aminobutyric acid (GABA), Glutamine (Gln), Glucose (Glc), Alanine, N-acetylaspartyl-glutamate (NAAG), Phosphocreatine (PCr), Scyllo-inositol, Acetate, Succinate, Phosphorylethanolamine, Glutathione (GSH), Citrate, and Glycerophosphocholine. The default simulated macromolecules were included in the modelling. The analysis window was set to range from 4.0 ppm to 0.6 ppm. Eddy current correction was performed based on the water signal (LCModel parameter DOECC = T) and water suppression was performed (DOWS = T). The attenuation factors of water (ATTH2O) and metabolites (ATTMET) were assumed to be 0.67 and 0.69, respectively. The chemical shift of the singlet used for scaling (Cr) was set to 3.0 ppm. To account for uncertainty in the referencing between in vitro (the simulated basis set) and in vivo measurements, the default SD of shift (DESDSH) and the expectation of 1/T2 (DEEXT2) were set to 0.01 and 12.0, respectively. Additional changes in the SD of shift (ALSDSH) from the new default SDSH were applied for NAA (0.004), NAAG (0.004), Glc (0.025), and PCh (0.025). Lactate, Scyllo-inositol and Acetate were omitted (CHOMIT) from the basis set used for fitting. Figure 108. In exploratory analyses, we capitalized on the increased signal dispersion that ultra-high field provides, and calculated the absolute concentrations of Glu, Gln, and NAA. During the absolute quantification procedure, corrections were applied for the proportions of gray matter, white matter, and cerebrospinal fluid in each MRS voxel. For more details on the absolute quantification procedure, see Giapitzakis et al.109.The data was fitted using LCModel version 6.3.0 Glc 0.02, and PChwww.neuro.uni-jena.de/vbm/) and expressed the gray matter (GM) proportion in the voxel as the segmented GM within the voxel divided by the total volume of the voxel.To account for differences in tissue composition within each participant’s MRS voxel, we segmented each individual MRS voxel using voxel-based morphometry (VBM) as implemented in the CAT12 toolbox  with RStudio IDE Version 1.1.383 . Before the actual statistical analyses, we verified that the allele frequency distribution of the rs6265 was at Hardy–Weinberg equilibrium.After excluding datasets that did not meet our MRS quality assessment (see above), the cohort 1 sample used in subsequent analyses consisted of 27 participants , and the cohort 2 sample consisted of 98 participants , unless otherwise specified in the exploratory analyses.Differences in covariates  were analyzed using Welch independent sample t-tests and Chi-square tests.BDNF genotype  on pgACC Glx/NAA values at 3 T and at 7 T, we computed a linear regression model with Glx/NAA as the dependent variable and BDNF genotype as fixed factor. As covariates, we included gender , age, and proportion of GM determined with VBM. The choice of our nuisance covariates was based on previous work suggesting that metabolite concentrations may vary as a function of age111, sex112, and voxel tissue composition113. We assessed normal distribution of residuals using Shapiro–Wilk tests of normality, and homogeneity of variances using Levene’s test. Outlier detection and removal was done using boxplots  below the first quartile or above the third quartile were considered outliers)114. To assess the directionality of significant main effects identified by the linear model, we performed Welch’s t-tests for unequal variances, testing for differences of Glx and NAA levels between BDNF genotype groups.To investigate the effect of P-values115 to test the global null hypothesis that BDNF genotype, corrected for covariates, has no effect on pgACC Glx/NAA levels.Because we addressed the same question in cohort 1 and cohort 2, and study samples were independent, we used Fisher’s method of combining Using the 7 T data from cohort 2, we performed post hoc analyses of Glu, Gln, and NAA, using the same statistical approach as in the analyses of Glx/NAA. The goal of these additional analyses was to investigate the metabolite specificity of our findings. Based on post hoc results, we performed exploratory analyses to investigate potential effects of gender in our data.post-hoc analyses, we used a Bonferroni correction for the three metabolites (effective threshold: ⍺ = 0.017).The significance level was set at ⍺ = 0.05. To control for multiple comparisons in our


Immunoglobulin G4-related disease (IgG4-RD) is a novel disease concept of unknown cause that is characterized by abundant infiltration of IgG4-positive cells, mass-forming lesions, and elevated serum IgG4 levels. The infiltration of IgG4-positive plasma cells and lymphocytes causes swelling, inflammation, fibrosis, and obliterative phlebitis in multiple organs. On the other hand, IgG4-RD occurring in the ureters has rarely been reported. To our knowledge, this is the first report of laparoscopic partial ureteral resection for IgG4-related focal retroperitoneal fibrosis in a ureter with suspected colon cancer recurrence.A 72-year-old man with a history of sigmoid colon cancer visited Shiga University of Medical Science Hospital for regular follow-up in December 2019. Enhanced abdominal computed tomography revealed a mass involving the left ureter. Furthermore, fluorine-18 fluorodeoxyglucose positron emission tomography showed significant accumulation of fluorodeoxyglucose uptake in the same region. Due to the possibility of colon cancer recurrence, a laparoscopic excisional biopsy with partial ureteral resection was performed. Histologically, IgG4-positive plasma cell infiltration exceeding 10 cells per high-power field and a high ratio of IgG4-positive/IgG-positive cells exceeding 40% were observed. The postoperative serum IgG4 level was 384 mg/dL. With the application of these findings to the diagnostic algorithm in the comprehensive diagnostic criteria for IgG4-RD, the mass-forming lesion was diagnosed as definitive IgG4-related focal retroperitoneal fibrosis.IgG4-RD should be considered in the differential diagnosis of retroperitoneal lesions. Moreover, laparoscopic surgery may be useful for making the diagnosis in difficult-to-biopsy cases. Immunoglobulin G4-related disease (IgG4-RD) is a novel systemic disease of unknown cause that is characterized by IgG4-positive plasma cell and lymphocyte infiltration histologically and elevated serum IgG4 levels. This disease, first described by Hamano et al. , affectsA 72-year-old man visited Shiga University of Medical Science Hospital for regular follow-up of sigmoid colon cancer in December 2019. He had a medical history of undergoing an appendectomy at age 10 and laparoscopic sigmoidectomy for sigmoid colon cancer (pStage I) at age 70. The histopathological diagnosis of the colon cancer was moderately differentiated tubular adenocarcinoma and tumor invasion to the submucosa with venous invasion. His father died of gastric cancer. Enhanced abdominal computed tomography (CT) revealed a mass involving the left ureter that had grown slowly since the previous CT. He underwent further examinations for the diagnosis. A physical examination revealed no abdominal pain or palpable mass. Additionally, no enlarged lymph nodes were palpable in the cervical, axillary, or inguinal regions. Laboratory data included elevated lactate dehydrogenase (239 U/L), creatinine (1.14 mg/dL), and carbohydrate antigen 19-9 (41 U/mL) levels. Other serum biochemical parameters and tumor markers were within the normal ranges. Urine cytology showed no atypical cells (class II).Enhanced abdominal CT showed a 30-mm soft-tissue mass involving the left ureter in front of the bifurcation of the left common iliac artery but no enlarged lymph nodes, hydronephrosis, or other abnormal findings Fig. . The masDue to the possible lymph node recurrence or peritoneal seeding of colon cancer indicated by the slow growth and significant FDG uptake, we performed a laparoscopic excisional biopsy. Under general anesthesia, the patient was placed in the lithotomy–Trendelenburg position. First, a ureteral stent was inserted in the left ureter radiologically using a guide wire. The endoscopist stood to the upper right of the patient, the surgeon stood near the surgeon’s right hand, and the assistant stood opposite the surgeon. A transumbilical skin and fascia incision was performed. The peritoneum was opened, and a 12-mm trocar was introduced. Under pneumoperitoneum, a 12-mm trocar was placed in the lower right abdomen, and 5-mm trocars were placed in the bilateral lower and upper abdomen. We dissected and mobilized the left-sided colon using the medial-to-lateral approach in Toldt’s space. After recognizing that the left ureter was involved in the mass-forming lesion in front of the bifurcation of the left common iliac artery, we mobilized the left ureter throughout the whole circumference of the mass-forming lesion Fig. . After rMacroscopically, an elastic and soft mass similar to a swollen lymph node with a lumen structure was detected Fig. . A histoIgG4-RD is a novel disease characterized by the abundant infiltration of IgG4-positive cells, mass-forming lesions, and elevated serum IgG4 levels. This disease was first described by Hamano et al., who reported elevated IgG4 levels in patients with autoimmune pancreatitis . ClinicaThe diagnosis of IgG4-RD is based on clinical and histological features. In Japan, the Ministry of Health, Labour and Welfare released the Comprehensive Diagnostic Criteria for IgG4-RD in 2011 . In our On the other hand, secondary retroperitoneal fibrosis caused by drugs, radiation, bleeding, malignancy, and infection had to be considered as a differential disease in the preoperative diagnosis. In our case, these factors were excluded based on symptoms, medical history, and examination findings. The ureteral tumor was also excluded because there were no abnormal findings on the preoperative ureterography or malignant cells in the urine cytology. However, in our case, it was difficult to completely distinguish malignant tumors from benign lesions. No specific symptoms associated with the mass-forming lesion were observed, but colon cancer recurrence was possible considering the slow growth and significant FDG uptake. Therefore, we performed laparoscopic resection to enable the diagnosis and treatment. Retrospectively, serum IgG4 level should be measured before the preoperative diagnosis since IgG4-RD is a differential disease. Although performing a biopsy for the definitive diagnosis is often difficult when retroperitoneal fibrosis is close to various organs, laparoscopic surgery enabled minimally invasive excisional biopsy in this case.IgG4-RD treatment remains controversial. Yoshino et al. reported that corticosteroid therapy was effective for retroperitoneal fibrosis with elevated serum IgG4 level, and clinical symptoms improved as serum IgG4 level decreased . HoweverOn the other hand, Wallace et al. reported that a history of malignancy might be associated with subsequent IgG4-RD development . One posMoreover, Hamano et al. reported the probability of developing IgG4-RD including autoimmune pancreatitis during the clinical course of IgG4-related retroperitoneal fibrosis . Our patTo our knowledge, the published literature concerning IgG4-RD occurring in ureters is rare, with only 13 cases found globally in our survey. All cases including ours are summarized in Table Here, we reported a case of IgG4-related focal retroperitoneal fibrosis in a ureter with suspected colon cancer recurrence that was resected by laparoscopic surgery. IgG4-RD should be considered in the differential diagnosis of retroperitoneal lesions. Moreover, laparoscopic resection could be useful for enabling the diagnosis of difficult-to-biopsy cases.


The reduction of the moisture content of concrete during the drying process reduces the concrete’s volume and causes it to shrink. In general, concrete shrinkage is a phenomenon that causes concrete volume to dwindle and can lead to durability problems. There are different types of this phenomenon, among them chemical shrinkage, autogenous shrinkage, drying shrinkage including free shrinkage and restrained shrinkage, and thermal contraction. Shrinkage-reducing admixtures are commercially available in different forms. The present study investigates the effect of liquid propylene glycol ether on mechanical properties and free shrinkage induced by drying at different water-cement (w/c) ratios. Furthermore, the effect of shrinkage-reducing admixtures on the properties of hardened concrete such as compressive and tensile strength, electrical resistivity, modulus of elasticity, free drying shrinkage, water absorption, and depth of water penetration was investigated. The results indicated that shrinkage reducing agents performed better in a low w/c ratio and resulted in up to 50% shrinkage reduction, which was due to the surface reduction of capillary pores. The prediction of free shrinkage due to drying was also performed using an artificial neural network. Concrete is the most widely used building material in the world, such that in some countries, the use of reinforced concrete is more common than steel structures ,2,3. TheIn addition to shrinkage reducing agents, the use of poly vinyl alcohol (PVA) fiber and volcanic ash are also effective in reducing shrinkage. These additives can increase the compressive strength of concrete, while reducing the surface stresses and strains associated with shrinkage. It should be noted that SRA reduces the tensile strength of concrete, its versatility and its compressive strength, which can be compensated for by using fiber ,11. TypiToday, the use of soft computing in civil engineering to predict experimental results has been extended due to its high accuracy. Many researchers in the field of concrete technology have used soft computing and artificial neural networks (ANN) in particular to predict concrete properties based on experimental results. Mechanical properties of different types of concrete such as compressive strength, tensile strength, elastic modulus, and flexural strength can be predicted using soft computing methods.The most important studies using artificial neural networks predict compressive strength in different concretes such as self-compacting concrete ,15,16,17In a study conducted by Bui et al. , the tenGiven that the effect of SRA on concrete shrinkage has not been extensively investigated in low, medium, and high water-cement (w/c) ratios, this study was conducted to investigate the effect of different w/c ratios on concrete containing SRA. For this purpose, six mix designs were considered and the effect of SRA on parameters such as free shrinkage, tensile compressive strength, Young’s modulus, electrical resistance, water absorption, and depth of penetration were investigated. Furthermore, an artificial neural network was applied to predict dry shrinkage of concrete based on the experimental results conducted in the study.3. The chemical composition of the constitutive elements of the cement used in the study is presented in 3. The size distribution of coarse and fine aggregate sand was in accordance with the standards ASTM C136 and ASTM C33 [The cement used in this study was Portland Cement Type II—in accordance with the ASTM C150 —with a sASTM C33 .In order to achieve the objectives of this study and obtain the mechanical properties of the intended mix designs, the concrete was manufactured with the desired mixing design. There are various tests to determine the properties of fresh and hardened concrete, some of which have been used in this study. For fresh concrete slump and air content and for hardened concrete compressive and tensile strengths, electrical resistance, modulus of elasticity, restrained drying, and water absorption, were obtained on the basis of different standards. As mentioned, the aim of this study is to investigate the effect of SRA on the drying-induced shrinkage of concrete in different w/c ratios. For this purpose, specimens in three low, medium, and high w/c ratios as well as control specimens were made. The concrete manufacturing process was as follows: Pouring sand into the mixer, adding gravel to the sand, adding cement to the aggregate, adding water to the mixture, adding SRA together with mix water which was kept from the previous steps, and, finally, adding superplasticizer with mix design water to the mixture. The concrete poured into the mold was kept for 24 h and then immersed in lime-saturated water at 23 ± 2 °C.The impact of SRA on the properties of fresh and hardened concrete was investigated in six mix designs classified in three groups. After fabricating the concrete with the intended mix design, the tests for fresh and hardened concrete were carried out. The results of the specimens and comparisons between them are presented in this section.In this study, two slump and air content tests were conducted based on the ASTM C143 and ASTM C231 standards in order to determine the effect of different percentages of SRA on the workability and air content of the manufactured concretes.As shown in A compressive strength test was performed on cubic specimens of 150 by 150 mm in accordance with EN 12390-3  at the aThe tensile strength test was performed on cylindrical specimens measuring 300 by 150 mm in accordance with the ASTM C496 [The electrical resistivity test was performed on 100 by 100 by 100 mm specimens according to ASSHTO T358 . Figure Young’s modulus testing was performed on specimens of 200 by 100 mm according to ASTM C469 at 28 days. A free-drying shrinkage test was performed in line with ASTM C157, according to which the specimens were stored in a chamber with a relative humidity of 50 ± 5 and 23 ± 2. Length of the specimens was also read at intervals of 1, 4, 7, 14, and 28 days, and of 8, 16, 32, and 64 weeks . Figure The water absorption test for concrete specimens was performed according to BS 188: Part 122 . Figure Many researchers have proposed models in various fields of study using soft computing methods ,57,58,59In this study, ANN is performed to propose a model to predict the dry shrinkage of the tested specimens. To create a network, a total of eight parameters, including the w/c ratio, type of admixture, weight of sand, gravel, and superplasticizer, were considered as input. The dry shrinkage of concrete specimens was considered to be the output parameter. The network properties used in this model are described in Different hidden nodes were examined (1 to 10) and their mean square error (MSE) value and the regression value for training, testing, validation, and the whole dataset, were obtained. The criterion for choosing the best network was having at least MSE , with the regression values being as close as possible to one. According to this criterion, in this study, the two-layer network was selected as the optimal network. The validation performance and training state and the values of the regressions are presented in The values predicted by the optimal network and its prediction errors are given in The network is selected in a way that the error values for training, validation, test, and all data are the minimum values. The other networks had shown a higher number of errors than this network. The purpose of using neural network in this study is to investigate the predicted results for shrinkage using limited available experimental data. As shown in The use of SRA reduced the slump compared to the control specimen, and the higher the w/c ratio, the greater the decrease of the slump.The use of SRA reduced compressive strength and tensile strength as compared to the control specimen. The higher the ratio of water to cement is, the greater the reduction in compressive strength. Using SRA caused a decrease in the Young’s modulus of the concrete and had a greater effect in a w/c ratio of 0.4.The use of SRA increased the electrical resistivity of the concrete, which has the same effect on the electrical resistivity of concrete in low, medium and high w/c ratios.Using SRA caused a decrease in the depth of penetration of water under pressure. It decreased less in a low w/c ratio compared to other ratios.Using SRA caused a decrease in the water absorption rate of concrete. It was observed that the effect of SRA on the reduction of water absorption of concrete is dependent on the immersion time of the concrete in water.The use of SRA caused a 50% decrease in free shrinkage induced by drying.Prediction of the dry shrinkage of specimens was performed using an artificial neural network, with low and acceptable mean error, indicating the high accuracy of ANN in predicting dry shrinkage based on experimental results.The present study investigates the effect of liquid propylene glycol ether on mechanical properties and free shrinkage induced by drying at different water-cement ratios. Furthermore, the effect of shrinkage-reducing admixtures on the properties of hardened concrete such as compressive and tensile strength, electrical resistivity, modulus of elasticity, free drying shrinkage, water absorption, and depth of water penetration was investigated. Based on the experiments, the following conclusions regarding the effect of SRA on different properties of concrete in low, medium, and high w/c ratios are drawn.


Staphylococcus aureus as formulated in the ESC guideline was found to be dissimilar to current Dutch practice, and was therefore made less restrictive. The recently adapted ESC diagnostic criteria for IE were endorsed, while the practical employment of the relevant diagnostic techniques was simplified in an adapted flowchart. In addition, the presence of a multidisciplinary, so-called ‘endocarditis team’ in tertiary centres was proposed as a quality indicator. An adapted flowchart specifically tailored to Dutch practice for microbiological diagnostic purposes was constructed. Lastly, the working group recommended the Stichting Werkgroep Antibioticabeleid  guidelines for IE treatment instead of the antibiotic regimens proposed by the ESC.Because the occurrence of infective endocarditis (IE) continues to be associated with high mortality, a working group was created by the Dutch Society of Cardiology to examine how the most recent European Society of Cardiology (ESC) guidelines for IE management could be implemented most effectively in the Netherlands. In order to investigate current Dutch IE practices, the working group conducted a country-wide survey. Based on the results obtained, it was concluded that most ESC recommendations could be endorsed, albeit with some adjustments. For instance, the suggested pre-operative screening and treatment of nasal carriers of  One of the oldest cardiac diseases, infective endocarditis (IE), remains one of the most fatal manifestations of heart disease . DespiteThe importance of IE is reflected in the frequent publication of new guidelines, for instance by the European Society of Cardiology (ESC) . In 2017Although widespread antibiotic prophylaxis for IE has long been considered effective, the policy for liberal use of antibiotic prophylaxis has gradually changed to more restricted indications. Of note, the 2008 guidelines of the National Institute of Health and Clinical Excellence (NICE) from the UK recommended that antibiotic prophylaxis should be abandoned completely . HoweverStaphylococcus aureus have more infections after cardiac surgery [S. aureus carriers only. The ESC guidelines recommend only the latter procedure. But the merits of the two methods are of course comparable, and this is—according to the survey—reflected by the concomitant use of both approaches for decolonisation of S. aureus in Dutch hospitals. Therefore, the text of the recommendation in Tab. 7 of the guidelines was (slightly) adapted as follows: ‘Preoperative screening and/or treatment of nasal carriage of Staphylococcus aureus is recommended before elective cardiac surgery in order to treat carriers’, while the last recommendation of the same table  was deleted.Nasal carriers of  surgery , and theBartonella spp. or Coxiella burnetii, fungi or fastidious bacteria may be in play and additional diagnostic testing may be required. Table 12 and Fig. 2 in the ESC guidelines refer to these circumstances. However, some microbiological tests included therein are not available in the Netherlands. The working group has therefore developed a flowchart adapted to Dutch practice  PET/CT or radiolabelled leucocyte single photon emission computed tomography (SPECT)/CT, as a ‘major’ criterion for IE. The currently applicable two major and five minor criteria for IE are described in Tab. 18F‑FDG PET/CT and cardiac CT are demonstrated in Fig. The diagnostic accuracy of the modified Duke criteria—which merge the presence of an infective syndrome and endocardial involvement, classically employing echocardiography—is only moderate, in particular in IE of a prosthetic valve , 11, 12.An important addition in the latest ESC guidelines is the recommendation to establish a multidisciplinary ‘endocarditis team’. Such a team, comprising—at least—a cardiologist, cardio-thoracic surgeon, infectious diseases specialist, microbiologist and radiologist/nuclear medicine physician should provide the expertise needed to treat complex IE patients. The guidelines refer—among other things—to the team approach adopted in France, with standardised medical therapy and uniform recommendations for surgical interventions that were found to improve outcome relative to earlier experience –18. A coStichting Werkgroep Antibiotica Beleid . Importantly, SWAB recently updated their guidelines for antibiotic treatment for IE, on the basis of an in-depth comparison of the most recent ESC and the AHA IE guidelines. In cases of discordance between the recommendations in these documents, SWAB guidance is based on a formal literature review on best current Dutch practice, taking into consideration national resistance patterns and dosing habits. For all these reasons, the working group recommended the employment of the SWAB guidelines for subsequent use in the Netherlands [There are major differences between European countries in the use of antimicrobial therapy and consequently in the antibiotic resistance patterns of pathogens. The Netherlands has the lowest rate of antibiotic use in Europe. The result is a stable level of antimicrobial resistance, whereas most countries experience increasing levels each year. European guidelines for antimicrobial therapy therefore cannot be simply adhered to but have to be tailored to individual countries. Recommendations for antibiotic therapy in the Netherlands are provided by the herlands .Heart failure resulting from valvular regurgitation or obstruction, uncontrolled infection and embolic events occurring under adequate antibiotic treatment constitute major complications of IE and may require surgical treatment , 21–24.


Our research focused on the influence of fillers obtained from crushed waste materials on the selected properties of concrete composites. The used waste materials were sourced from the production of ceramic tiles, ceramic pots, and sanitary ceramics. We evaluated concretes modified with the addition of 10% (by mass of cement) of different fillers. The properties, including the air content in the fresh concrete mix, consistency, compressive strength, and freeze-thaw resistance were examined. The evaluation of the freeze-thaw resistance was carried out by testing the concrete with the direct method for 150 cycles of freezing and thawing. The characteristics of the concrete porosity structure were assessed using automated digital image analysis. Concretes modified by coarse and fine fillers were characterized by different improvements in the mechanical properties and resistance to cycles of freezing and thawing. Composites with the addition of coarse fillers did not show any significant changes in comparison to the control concrete. An automated digital image analysis of the pore distribution in concrete proved to be an effective tool for the assessment of the freeze–thaw resistance of the concretes in question. Currently, a reduction of the volume of industrial waste and sustainable use of natural resources has become a necessity. The utilization of waste materials in the technology of mortars, concretes, or other cements and cement–like composites is one of key methods of environmental protection. This offers opportunities for the recycling and utilization of significant amounts of different types of waste materials . In certThe general conclusion of the research in question was that concretes with the addition of ceramic fillers have good workability and strength characteristics, comparable to ordinary concretes. In the research carried out by Siddique et al. , the resThe durability of concrete exposed to freeze-thaw cycles is not directly affected by the porosity but by the size of pores and their spacing . The uniTaking all the above facts into consideration, we decided to conduct research dedicated to studying the influence of varied waste ceramic fillers on the aeration and mechanical characteristics of concrete. We hypothesized that the addition of specific fine or coarse ceramic filler could shape the internal pore structure of concrete and transform it into a highly freeze-thaw resistant material. One must keep in mind that ceramic waste (both white and red) is commonly available. Up to 30% of the pottery that is produced is crushed, damaged, or dismissed during production . BuildinOrdinary tap water and Portland cement CEM I 42.5R  were used for the creation of all planned concrete mixes. The tap water characteristics were presented in a previous publication . The cheA mix of natural aggregates was used as the main aggregate. To obtain the optimal granular composition of the natural aggregate, we experimentally composed three aggregates . The sieve analysis of the aggregate mix was conducted in accordance with the requirements of the standard EN 933–1:2012 [Three different ceramic wastes were utilized in this research-namely waste from the production of ceramic pots, ceramic tiles, and sanitary ceramics was used for the creation of ceramic fillers. The procedure of the filler creation consisted of two stages. First, ceramic waste was ground using a basic ball mill. In this way, coarse fillers (CF) were obtained, characterized by a maximum particle diameter of 4 mm. Secondly, amounts of CFs were fed to a disintegrator. As a result of processing with the disintegrator, fine fillers (FF) were achieved. The maximum size of the particle diameter of all FFs was 0.5 mm. The ball mill used, disintegrator, and the procedure of the preparation of ceramic fillers were thoroughly described in previous publications ,21. The 3, pH equal to 5.0 ± 1.0, total chloride ion content less than 0.1%, and sodium oxide equivalent less than 2.0%. The admixture was dosed in the amount of 1.0% of the cement weight. This is a moderate volume of dosage as recommended by the producer.All mixes were modified by a commercially available superplasticizer based on sulfonated naphthalene polycondensate (lignosulfonate). This was characterized by a density of 1.155 ± 0.020 kg/dm3, respectively. The compositions of the cast mixes are summarized in All tested mixes were prepared harnessing the same procedure. First, the dry ingredients  were added to a mixer. They were mixed for 10 s. Subsequently, water with the diluted admixture was added to the mixer. All ingredients were mixed for 90 s. Ceramic fillers were utilized to partially substitute for the aggregate. Taking into account the previous experience of the research team with similar waste materials ,22,23,243 for concrete with ceramic tiles, no filler, sanitary ceramics, and ceramic pots, respectively. This indicates that the mixes were differentiated up to 0.12% of the volume in comparison to the reference concrete. We concisely adopted this approach (which does not influence the key results in any noticeable way) due to our experience with similar mixes as described in [During the research program, the natural aggregate was replaced with ceramic fillers by mass (0.54%). The volume of the created mixes was not constant; however, the differences were negligible. The volumes of the mixes were equal to 999.8, 1000.0, 1000.5, and 1001.2 dmribed in .The conducted research consisted of three main stages. The first stage was focused on testing the properties of the fresh concrete mix. During this part of the research, the consistency of the fresh mix and air content (Ac) were evaluated. For the consistency test, an ordinary slump procedure according to EN 12350–2:2011  was harnThe third stage of the research was dedicated to destructive tests. The compressive strength after 28 days of curing was tested according to EN 12390–3:2011  using thThe achieved properties of the fresh concrete mixes are presented in SEM images of the used ceramic fine fillers are presented in The results of the compressive strength test  of the reference concrete and the concretes modified by ceramic fillers are presented in A, pore distribution index A300 [A300 should be up to 0.20 mm and over 1.5%, respectively [The results of the image analysis of the cross–sections of hardened concretes are presented in index L¯ , and theL¯ [A300  were detectively ,29,33.The most favorable effect of the ceramic filler on the compressive strength was found in the case of using ceramic tiles FF and sanitary ceramics CF. The influence of all examined ceramic fillers on compressive strength was relatively small and reached up to 4%. This increase in compressive strength was negligible from the point of view of the strength class, which would be granted to the concretes in question. All tested concretes would be described by the strength class C 45/55 according to PN–EN 206:2014.Ceramic pots were characterized by a higher Al content compared with ceramic tiles and sanitary ceramics. Ceramic pots contain significantly less Fe in comparison to sanitary pots and ceramic tiles . The amounts of Na and K are similar in all three ceramics types in question. Taking into account the development of the compressive strength of the tested concretes see , the eleThe resistance to freeze–thaw cycles was improved for each modified concrete. All used ceramic fillers reduced the loss of compressive strength after 150 freeze–thaw cycles. The loss ranged from 5.3% to 15.2%. The delamination process was clearly slowed down. A300 ranged from 1.2% for the reference concrete to 2.3% for the concrete modified by the fine filler obtained from ceramic tiles. The second largest content of the micro–pores A300 was achieved by concretes modified by fine filler obtained from ceramic pots and sanitary ceramics. Both contents of micro–pores A300 values were equal to 2.0% for those concretes. The porosity of hardened concrete and the pore diameter distribution were also significantly influenced by the addition of ceramic fillers. The content of the micro–pores A300 > 1.5%, which was recognized by multiple researchers [All concretes with ceramic fillers except CF were characterized by earchers ,29,33 asThe effect of the addition of ceramic fillers (both fine and coarse) on the compressive strength was negligible.The addition of ceramic fillers increased the aeration of concrete.Concretes with ceramic fillers were characterized by higher contents of micro–pores in class 18 (with a diameter of up to 300 µm) in comparison to the reference concrete.The content of micro–pores directly influenced the resistance of the tested concretes to freeze–thaw cycles.The best effect on the resistance to freeze–thaw cycles was achieved by the addition of all three fine fillers that were considered in this study.Additional tests should be conducted using different ceramic fillers to confirm the achieved results.The possibility of reactions between the fillers and cement paste should be investigated in the future.The results obtained in the study lead to the following conclusions: