M12L8 metal–organic cages (MOCs), using the instant synthesis method, it is possible to kinetically trap and control the formation of interlocking M12L8 nanocages, giving rare M12L8 TPB-ZnI2 poly-[n]-catenane. The catenanes are obtained in a one-pot reaction, selectively as amorphous (a1) or crystalline states, as demonstrated by powder X-ray diffraction (powder XRD), thermogravimetric (TG) analysis and 1H NMR. The 300 K M12L8 poly-[n]-catenane single crystal X-ray diffraction (SC-XRD) structure including nitrobenzene (1) indicates strong guest binding with the large M12L8 cage , allowing its structural resolution. Conversely, slow self-assembly (5 days) leads to a mixture of the M12L8 poly-[n]-catenane and a new TPB-ZnI2 (2) coordination polymer , as revealed by SC-XRD. The neat grinding solid-state synthesis also yields amorphous M12L8 poly-[n]-catenane (a1′), but not coordination polymers, selectively in 15 min. The dynamic behavior of the M12L8 poly-[n]-catenanes demonstrated by the amorphous-to-crystalline transformation upon the uptake of ortho-, meta- and para-xylenes shows the potential of M12L8 poly-[n]-catenanes as functional materials in molecular separation. Finally, combining SC-XRD of 1 and DFT calculations specific for the solid-state, the role of the guests in the stability of the 1D chains of M12L8 nanocages is reported. Energy interactions such as interaction energies (E), lattice energies (E*), host–guest energies (Ehost-guest) and guest-guest energies (Eguest-guest) were analysed considering the X-ray structure with and without the nitrobenzene guest. Not only the synthetic control achieved in the synthesis of the M12L8 MOCs but also their dynamic behavior either in the crystalline or amorphous phase are sufficient to raise scientific interest in areas ranging from fundamental to applied sides of chemistry and material sciences.Here, we show that in a supramolecular system with more than 20 building blocks forming large icosahedral  Despite advances in the synthesis of catenanes6, the formation of catenanes made of interlocked MOCs is still very challenging7. Interestingly, the combination of properties of MOFs and MOCs11 can be achieved with poly-[n]-catenanes formed by the interlocking of large icosahedral M12L8 nanocages via the mechanical bond12. The long extension along one direction of the mechanical bond confers poly-[n]-catenanes the strength, stability and dynamic behavior of a metal–organic material similar to those observed in MOFs. As quoted by Stoddart, the strength of a mechanical bond is comparable to the weakest “participating” chemical bond in the sense that its cleavage will allow the system components parts to dissociate13.Poly--catenanes where icosahedral M12L8 nanocages with internal voids of ca. 2600 Å3 are interlocked by means of mechanical bonds. There are only four reported crystal structures of M12L8 poly-[n]-catenanes with TPB ligands, including different guests21 and six structures, without X-ray-determined guests, considering the analogous 2,4,6-tris-(4-pyridyl)pyridine (TPP) ligand22. However, to the best of our knowledge, there is no report of TPB-ZnI2 poly-[n]-catenane synthesized using nonconventional approaches such as instant synthesis or in the solid-state upon mechanochemical means23 under ambient conditions. It is known that small changes in one of the building blocks  constituting a MOC can result in different products, particularly when there are a large number of molecules and/or ions as starting components. Thus, replacing the halide in the metal node of a MOC can be sufficient to give different structures or different behavior and properties in a family of isostructural materials24.To date, most of the reported polycatenanes self-assembled from interlocked nanocages have used harsh conditions at high temperatures using solvothermal reactions under thermodynamic controlM12L8 nanocages, by using the instant synthesis method25, it is possible to kinetically trap and hence control the formation of interlocking M12L8 nanocages in rare M12L8 TPB-ZnI2 poly-[n]-catenane both as crystalline (1) and amorphous (a1) phases. Conversely, slow self-assembly  leads to a statistical mixture including a new coordination polymer TPB-ZnI2 (2)  and poly-[n]-catenane 1  is crucial for the structure–function correlation in M12L8 TPB isostructural poly-[n]-catenanes. Additionally, solid-state synthesis by neat grinding yields amorphous M12L8 poly-[n]-catenane (a1′) but not coordination polymers in a selective manner in a short time (15 min). The dynamic behavior of the M12L8 poly-[n]-catenane chains demonstrated by the amorphous-to-crystalline (a1′ → 1) transformation upon the uptake of ortho-, meta- and para-xylenes and other aromatic molecules shows the potential of M12L8 poly-[n]-catenanes to be used in functional applications . Finally, combining structural single crystal X-ray diffraction (SC-XRD) data and DFT calculations specific for the solid-state, the role of the guest molecules in the stability of the 1D chains of M12L8 nanocages has been studied. Various energy interactions, such as interaction energies (E), lattice energies (E*), host–guest energies (Ehost-guest) and guest-guest energies (Eguest-guest), were analysed considering 1 without solvent and including ordered nitrobenzene. DFT analysis considering guest molecules has been performed for the first time in the structural analysis of M12L8 poly-[n]-catenanes, providing important structural information regarding the dynamic behavior and guest inclusion/exchange properties in systems that do not have continuous channels.Herein, we show that in a supramolecular system with more than 20 building blocks forming large icosahedral instant synthesis27 at room temperature conditions in the preparation of mechanically interlocked large M12L8 cages is uncommon. The instant synthesis method allows selective synthesis using kinetic control, with which it is possible to obtain the poly-[n]-catenane of interlocked M12L8 nanocages as a homogeneous phase . Although we have demonstrated instant synthesis using TPB and ZnCl2 or ZnBr2, using ZnI2 has not been reported. Changing one of the building blocks in the self-assembling components  can have a drastic effect on the final products. For instance, it can lead to different and unexpected structures, particularly in self-assembling systems where the building blocks are more than twenty components, as it is in the poly-[n]-catenanes self-assembled of M12L8 icosahedral nanocages including solvent.Using the extremely fast crystallization method, 2 into the vigorous stirring light-yellow solution of TPB in nitrobenzene instantaneously formed a suspension that was stirred for 5 min  with a very large bump covering the 10–30 range in 2θ/° Bragg diffraction angles and with the absence of sharp Bragg reflections  solution in a round bottom flask and stirred for 12 h (overnight experiment). Then, the suspension was filtered, and the solid was analysed by powder XRD. The diffractogram showed that the amorphous phase a1 transformed to a crystalline material, as sharp Bragg reflections were observed  using the instant synthesis method, as observed by the repetition of three experiments. This behavior is different from that observed using ZnCl2 and ZnBr2, which formed crystalline M12L8 poly-[n]-catenane under the same experimental conditions.Using TG analysis, we quantified the included guest molecules, which amount to 1.3 guest molecules in the asymmetric unit (considering only toluene) (13.8% weight loss) and 7.8 guests per ene Fig. . The TG Instant synthesis was also carried out using chlorobenzene and toluene as templating solvents following the same protocol as in nitrobenzene synthesis . Clearln]-catenane can be formed using TPB and ZnI2 using slow crystallization experiments has been carried out by preparing single crystals by self-assembling TPB and ZnI2. Pristine single crystals of TPB-ZnI2 poly-[n]-catenane were obtained by layering diffusion of Zn(II) to a solution of TPB in nitrobenzene. In a crystallization tube, as a bottom layer, TPB was dissolved in a nitrobenzene/MeOH mixture, and then as a middle layer, methanol was added until a clear biphasic solution was formed  Å; b = 38.6805(7) Å; c = 16.0202(3) Å; α = β = 90° and γ = 120°; V = 20,757.9(8) Å3. Crystallographic analysis revealed the formula [(ZnI2)12(TPB)8]6(C6H5NO2) (1) with two ZnI2, one TPB ligand and one-third of a second TPB ligand in the asymmetric unit  was solved in the trigonal space group 00 K was ral Fig. .Figure 3M12L8 cage framework is formed of twelve Zn(II) metal centers at the vertices of the icosahedron with a tetrahedral geometry with three Zn-N bonds  and four Zn-I bonds ranging from 2.523 to 2.563 Å. The icosahedron is defined as “opened icosahedrons”20 due to the large windows formed by the absence of 12 of the 20 TPB ligands forming the faces (triangles) of the icosahedron. This large window allows good interpenetration of M12L8 cages and thus mechanical bond formation, resulting in 1D chains of interlocked icosahedral cages. The 1D chains extend along the crystallographic c-axis and pack in the crystalline state in such a way that the interactions among the rods are through weak C–H···I–Zn electrostatic interactions.The mechanical bond among the cages takes place because of the efficient π-stacking between the benzenic core of the TPB ligand (d = 3.868 Å) of adjacent cages. The good benzene-benzene interactions among TPB ligands are one of the key factors contributing to the formation under kinetic control18 of the poly-[n]-catenanes of interlocked M12L8 nanocages through mechanical bonding. This aspect is favourable from the enthalpic point of view. It is known that switching from benzene to triazine core in the exotridentate ligand -1,3,5-triazine (TPT)), the outcome of the self-assembly is a completely different structure that does not form the M12L8 interlocked cages but a 3D coordination polymer with doubly interpenetrated  networks29.The M12L8 cages does not lead to a porous structure but an array of isolated cages with large internal volumes of ca. 2600 Å3 , similar to the other reported isostructural structures21. Interestingly, the interchain interactions for the zinc iodide catenane have an important role in the structure stabilization. In fact, terminal ligand I in the Zn metal interacts with the aromatic rings of TPB via C–H···I interactions with distances of 3.169 Å and 3.145 Å from adjacent chains.The packing of the 1D chains of interlocked  Å3 Fig. . The volM12L8 cages can be resolved with reasonably small atomic thermal parameters. One of the four pyridine rings in the asymmetric unit is disordered over two positions, which is important to explain the guest exchange reactions despite not having continuous channels  for the nitrobenzene guest and TPB host, as shown in Fig. 321.The nitrobenzene guest interacts with the 2 group. The C–H···O distance is 2.556 Å, which, from an enthalpic point of view, contributes to the stabilization of the M12L8 cage , but in all cases, different crystal structures were identified, as evidenced by the different unit cells.To gauge more structural information on the self-assembly of nts Fig. . The solM12L8 poly-[n]-catenane, which was confirmed by the unit cell parameters as block-like crystals, and (ii) a new colourless crystalline phase (2) easily distinguishable due to its thin-plate habit. Crystals of 2  are stable at room temperature for several hours; therefore, due to their thermal stability, SC-XRD was recorded at room temperature (300 K). The thin plates  Å, b = 14.4843(2) Å, c = 18.2201(2) Å, β = 100.8630(10), and V = 6247.57(15) Å3, crystallizing in the monoclinic system in the C2/c space group  tes Fig. a have laup Table .Figure 6TPB ligand and one and a half ZnI2, as one of the Zn atoms sits at a special position on a twofold axis. There is also one chlorobenzene guest molecule 1(ZnI2)1.5]n·(C6H5Cl) (2). The minimum metal–ligand circuit is formed by four TPB ligands and four Zn(II) metal centers expanding along the c-axis, which leads to infinite 1D channels, including chlorobenzene. The smallest TPB-ZnI2 circuits form pockets of rectangular shape with windows of ca. 10 Å × 15 Å dimensions 1.5]n·CH5Cl (2).c-axis  C–H···I interactions (d = 3.045 Å). The included guest does not form a particular interaction with the host structure but guest-guest interactions through π–π electrostatic contacts with distances among the centroids of each chlorobenzene of 3.743 Å 30 corresponding to 30% of the total unit cell volume  coexists with a coordination polymer (thin plates) with unit cell parameters similar to those of the crystal including chlorobenzene: a = 29.721(4) Å, b = 13.3437(15) Å, c = 17.2043(15) Å, β = 99.804(17), V = 6723 Å3. Details of this structure along with other coordination polymers will be reported elsewhere.Importantly, if toluene is used as an aromatic templating solvent instead of chlorotoluene, a mixture of crystals is also obtained where the 2 or any of the other coordination polymers observed using instant synthesis were unsuccessful. The M12L8 poly-[n]-catenane as crystalline or amorphous phases were always obtained instead of coordination polymers. This clearly demonstrates that instant synthesis crystallization is an effective method to prepare 1D M12L8 poly-[n]-catenane in a selective manner, as it does not allow the error-checking process31. The error-checking mechanism is possible due to the labile nature of the coordination bond. During slow crystallization (stratification method), the formation of alternative structures such as the abovementioned coordination polymers or other potential structures can take place because the coordination bond can be broken and reformed until the most thermodynamically stable structure is self-assembled . It is important to see that crystal structures 1 and 2 are completely different because the self-assembly process follows different crystallization conditions . Another important aspect is that kinetic products tend to be dynamic and hence can perform guest exchange/inclusion reactions but can also undergo further transformations towards more stable structures upon external stimuli  following crystal-to-amorphous-to-crystal transformations33. Thus, the crystallization process has a direct effect on the final structure formed and is therefore very important to gain control. The ability to direct at will the product formed in a chemical reaction has a crucial role both in chemical synthesis and material sciences.Our attempts to obtain coordination polymer n]-catenanes is not common and even less common in systems such as M12L8 interlocked cages. While it has been demonstrated that it is possible to obtain TPB-ZnBr2 poly-[n]-catenane as an amorphous material, neither the TPB-ZnI2 nor TPB-ZnCl2 structures have yet been reported in the solid state following a solvent-free approach. Here, we report, for the first time, the synthesis of TPB-ZnI2 poly-[n]-catenane by means of neat grinding.The solid-state synthesis of poly--catenanes, and in particular in M12L8 poly-[n]-catenanes, has not yet been reported. Thus, an amorphous polycatenane synthesized by neat grinding was used to adsorb o-xylene, m-xylene and p-xylene molecules using heterogeneous solid–liquid reactions at room temperature.Because toluene can be included in the a1′ was immersed in 4 ml of o-xylene and 1 ml of methanol. The suspension was stirred overnight, filtered, and analysed by powder XRD analysis. As observed in the diffractograms, a1′ transformed from an amorphous to a crystalline phase whose diffractogram corresponds to that of the M12L8poly-[n]-catenanes . Thus, we retain that the use of xylenes as templating solvents in the instant synthesis is not ideal. The inclusion of xylenes in the M12L8 nanocages is much more efficient using amorphous phases a1 or a1′, and from an industrial point of view, amorphous poly-[n]-catenane obtained in the solid state is preferable.Instant synthesis was also attempted using 1, including nitrobenzene, allows DFT calculations to be carried out, giving insights into the structural stability and intrinsic local dynamic behavior of the TPB-ZnI2M12L8 poly-[n]-catenane system. Therefore, DFT calculations specific for solid crystalline states have been carried out considering the energy interactions among M12L8 cages, among the 1D chains, and host–guest energy interactions. DFT calculations were carried out at the PBE/DNP level 37. The strategy adopted here was shown to be good in several recent studies of crystalline systems such as molecules, polymers, and hybrid metal–organic materials44. The explicit van der Waals contribution, according to the approach proposed by Grimme, was determined46.The availability of the room temperature structure of system to be adopted in DFT calculations. First, the “interaction energies” (E) among interlocked and noninterlocked M12L8 nanocages, which can give us a view of the stabilities of the 1D ribbons and of individual  M12L8 cages, are considered. The second aspect is the “lattice energy” (E*), which considers the energy of a single M12L8 cage or a 1D chain of interlocked cages immersed within the crystalline lattice (1). The third point considers the interaction energy among the M12L8 host and the guest and guest-guest interactions, referred to hereafter as Ehost-guest and Eguest-guest, respectively.It is important to define the model E can be analysed considering the interaction energies of two close interacting dimers: two interlocked M12L8 cages or two first neighbour cages that are not mechanically linked but that are both stable. The interaction energy into 1 considering the interlocked M12L8 cages is ca. 89 kcal/mol, which is almost 178 kcal/mol per 2 cages, and this interaction appears to be strongly affected by the aromatic ring interactions (d = 3.868 Å) from the central TPB ring. The interaction energies among the noninterlocked M12L8 cages that interact via van der Waals interactions correspond to 47 kcal/mol, or 23.5 kcal/mol per cage.M12L8 cage immersed in the crystalline structure is very high: approximately 505 kcal/mol. In a similar way, if we consider the polycatenated chain along the c-axis  and calculate the energy required to remove a single infinite chain of M12L8 interlocked nanocages from the crystalline structure, the calculated E* is 398 kcal/mol, indicating that the model of an infinite 1D chain “immersed” in the structure is a good model explaining the high stability of the poly-[n]-catenane architecture.The average energy required to extract a single n]-catenane, we carried out the same sort of calculations but included the six crystallographic nitrobenzene guest molecules in the M12L8 cages. In this way, it is possible to compare the previously calculated energies against the empty model not including guest molecules. The interaction of the M12L8 cages with the solvent molecules, the Ehost-guest, is also important in this case. The Ehost-guest is approximately 46 kcal/mol for each solvent molecule, which is comparable to the energies observed among the noninterlocked cages interacting via electrostatic and van der Waals interactions . The Eguest-guest interactions  values for a single cage and a single chain are higher and the interaction energies (E) for interlocked cages are also higher  among chains for structure 1, as observed by DFT. This has a direct effect on the guest exchange/inclusion properties and the dynamic behavior of the 1D chains of interlocked cages, which might help to explain the guest inclusion/exchange observed in these systems formed of large, interlocked cages.From the DFT calculations, it has been seen that almost all the interactions increase in the presence of guest molecules. This confirms that when the solvent is included in the M12L8 cages considering two main directions, one perpendicular to the chain propagation (c-axis) and the second parallel to the chain extension , has a low energy cost. While a 1 Å displacement orthogonal to the 1D rod direction has an energy penalty of 10 kcal/mol, in the sliding direction, the energy penalty is 0.2 kcal/mol per 2 Å translation. These calculations were carried out considering a TPB-ZnBr2 model system of two M12L8 cages belonging to different chains 20. The DFT results reported herein go in that direction, as the van der Waals interactions among neighboring chains are much weaker when the solvent is included in the DFT calculations.It has been reported that the relative displacement among 1D chains of interlocked n]-catenanes formed by the interlocking of large icosahedral M12L8 nanocages using TPB and ZnI2 as building blocks has been reported for the first time using three different crystallization methods. Fast crystallization by means of one-pot instant synthesis using nitrobenzene as the templating solvent gives amorphous poly-[n]-catenane phases. Compared to the other experiments using the same solvents where TPB is self-assembled with ZnCl2 or ZnBr2 using instant synthesis that yields crystalline poly-[n]-catenane, the TPB-ZnI2 system using nitrobenzene as the templating solvent yields an amorphous phase (a1). Importantly, fast crystallization takes place under kinetic control, homogeneously producing M12L8 poly-[n]-catenane. Instead, the slow crystallization by layering solution allows the formation of large single crystals of interlocked M12L8 poly-[n]-catenane including nitrobenzene (1), whose structure has been determined at room temperature conditions allowing unambiguous resolution of the guest nitrobenzene. In contrast to kinetic control, under thermodynamic conditions, the products are not obtained selectively but as mixtures. Slow crystallization under thermodynamic control using different templating solvents yields a mixture of the M12L8 poly-[n]-catenane and 1D coordination polymers (2), including aromatic guest molecules that yield 1D channels. The M12L8 poly-[n]-catenanes have also been synthesized in the solid-state in the absence of solvent as an amorphous phase by neat grinding. In the presence of aromatic guest molecules, the amorphous phase a1′ becomes crystalline following an amorphous-to-crystalline transformation by using suitable guests such as o-DCB, toluene, o-xylene, m-xylene or p-xylene. Solid-state DFT calculations were carried out to analyze the stability of M12L8 poly-[n]-catenane 1 with and without nitrobenzene guests. Considering that the MOC cavity is considerably larger than the guest molecule, the host–guest interactions are highly specific, allowing its binding within the cage. The DFT analysis allowed us to rationalize the importance of the guest effect in the stabilization of the 1D chains of interlocked cages. The lattice energies of interlocked and noninterlocked M12L8 nanocages as well as host–guest and guest-guest energy interactions were calculated. The results suggest that while interactions within the 1D chains are enhanced by the presence of guests, the electrostatic interactions among the chains are less intense. The lower local van der Waals interchain interactions observed in 1 add additional proof to the dynamic behavior described in isostructural M12L8 poly-[n]-catenanes allowing 1D chain displacement and guest exchange properties in structures that do not contain channels.The self-assembly of poly-[n]-catenane formed in the solid-state shows good potential for functional applications of this class of mechanically interlocked materials in areas such as molecular separation of xylene isomers but can also find applications for including other guests such as drugs for drug delivery purposes.The guest uptake ability by the amorphous phase of the poly-[Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.


Depression is a severe and widespread psychiatric disease that often accompanies epilepsy. Antidepressant treatment of depression comorbid with epilepsy is a major concern due to the risk of seizure aggravation. SAMe, a universal methyl donor for DNA methylation and the synthesis of brain monoamines, is known to have high antidepressant activity. This study aimed to find out whether L-methionine (L-MET), a precursor of SAMe, can have antidepressant and/or anxiolytic effects in the WAG/Rij rat model of depression comorbid with absence epilepsy. The results indicate that L-MET reduces the level of anxiety and depression in WAG/Rij rats and suppresses associated epileptic seizures, in contrast to conventional antidepressant imipramine, which aggravates absence seizures. The antidepressant effect of L-MET was comparable with that of the conventional antidepressants imipramine and fluoxetine. However, the antidepressant profile of L-MET was more similar to imipramine than to fluoxetine. Taken together, our findings suggest that L-MET could serve as a promising new antidepressant drug with anxiolytic properties for the treatment of depression comorbid with absence epilepsy. Increases in the level of monoamines and their metabolites—DA, DOPAC, HVA, NA, and MHPG—in several brain structures, is suggested to be a neurochemical mechanism of the beneficial phenotypic effect of L-MET. Depression is a severe chronic disease that affects more than 350 million people worldwide, making it one of the most common psychiatric disorders . DepressThe Wistar Albino Glaxo/Rijswijk (WAG/Rij) rat strain is a well-validated genetic model of childhood absence epilepsy with mild depression-like (dysthymia) comorbidity ,11,12,13Spike-wave discharges (SWDs), the main hallmark of absence epilepsy, are expressed age-dependently both in WAG/Rij rats  and GAERAlthough a pathologic phenotype in WAG/Rij rats is genetically determined, previous data have shown that the absence epilepsy and depression-like comorbidity can be altered by early postnatal environmental impacts , indicating that epigenetic mechanisms might be involved ,20.Even though genetic factors play an important role in the pathogenesis of depression , epigeneThe potential role of the insufficiency of methyl-group donors in the pathogenesis of depression was confirmed by the effectiveness of L-methyl folate in the treatment of depressive disorders in humans . It was S-adenosyl-L-methionine (SAMe) is a universal donor of methyl groups necessary for DNA methylation and the synthesis of the neurotransmitters DA, noradrenaline (NA), serotonin (5-HT), the deficiency of which in the brain leads to depressive disorders. A reduced SAMe content was found in depressed humans . In placThis study aimed to test the assumption that L-MET can have an antidepressant effect in the WAG/Rij rat model of depression comorbid with absence epilepsy.Does L-MET exhibit an antidepressant and/or anxiolytic activity in tests relevant to the assessment of anxiety and depression?Is the antidepressant effect of L-MET comparable with that of conventional antidepressant drugs, such as the tricyclic antidepressant imipramine and the selective serotonin reuptake inhibitor fluoxetine?What is the effect of L-MET on the associated absence epilepsy, and is it different from the effect of the reference antidepressant imipramine?Are the effects of L-MET on depression-like behavior and absence seizures related to alterations in the brain monoamine systems?To achieve the aim, several questions were asked:p < 0.05), the number of the squares crossed  = 7.46, p < 0.05), and the number of center entries  = 16.19, p < 0.001). In L-MET-treated WAG/Rij rats, the latency to leave the center was lower, but the number of crossed squares and center entries was greater compared with their vehicle-treated counterparts. L-MET caused a tendency toward a smaller number of boli  = 5.83,  of boli .p = 0.05) and the number of risk assessments  = 6.13, p < 0.05). In L-MET-treated WAG/Rij rats, the time in the light was greater than and the number of risk assessments was less than the vehicle-treated controls. The number of transitions between compartments tended to be lower  = 4.32, be lower .p < 0.05), the number of hangings from the open arms  = 7.64, p < 0.05), and the number of boli  = 5.51, p < 0.05). In L-MET-treated WAG/Rij rats, the time in open arms and the number of hangings from the open arms were greater, but the number of boli was lower, compared with the vehicle-treated group. The number of risk assessments and rearings in the closed arms tended to be less than that of the vehicle-treated control group  = 4.81, ol group .p < 0.05), the duration of the first episode of active swimming  = 6.51, p < 0.05), and the number of dives  = 15.70, p < 0.01). L-MET did not significantly affect the duration of swimming  = 1.76, p = 0.21). L-MET treatment reduced the immobility time, increased the duration of the first episode of active swimming (“climbing”), and did not significantly affect the duration of swimming, indicating an antidepressant-like effect  = 4.63, e effect . The conp < 0.001). A significant decrease in the number of SWDs was observed only in the “sensitive” subgroup of WAG/Rij rats and only on the 14th day of L-MET administration . L-MET decreased the number of seizures in a subgroup of WAG/Rij rats with an initially smaller number of seizures (59.33 ± 9.88), called “sensitive”, but did not change the number of discharges in a subgroup of WAG/Rij rats with an initially larger number of seizures (99.5 ± 15.54), called “insensitive”. Two-way ANOVA with repeated measures showed a significant effect of the initial number of seizures on the effect of L-MET on the number of SWDs  = 21.24, stration A. L-MET stration C or on tstration B and lonstration D mean duOn the 14th day of L-MET treatment, a decrease in the number of well-developed mature SWDs registered before treatment A was accAnalysis of SWDs with a Fast Fourier Transform procedure revealed that on the 14th day of L-MET treatment, the averaged spectral power at the fundamental frequency and the first and second harmonics was significantly lower compared with the baseline (before treatment) and the 7th day of treatment. Even though on the 7th day after the cancellation of L-MET, the number of epileptic discharges was restored to the initial level, the averaged spectral power of discharges remained lower than the initial one and did not significantly differ from that on the 14th day of treatment .Treatment with L-MET did not change the background EEG , indicatp < 0.05) and “treatment option”  = 18.3, p < 0.001) as well as the interaction of these factors  = 16.66, p < 0.001) on the mean duration of the SWDs. Acute administration of imipramine reduced the mean duration of absence seizures compared with acute and chronic vehicle administration, while chronic treatment did not significantly affect this measure (p = 0.21) and “treatment option”  = 3.8, p = 0.06) as well as the interaction of these factors  = 3.66, p = 0.07) on the number of seizures. The effect of acute and chronic imipramine administration on the number of SWDs was the opposite: acute treatment reduced the number of seizures, while chronic administration increased the measure of absence seizure severity (The effect of imipramine on the number of SWDs in WAG/Rij rats depended on the treatment option (acute or chronic). The two-way ANOVA revealed a significant effect of the factors “drug”  = 5.22,  measure B. The twseverity A.p < 0.001) and asymmetry index  = 25.19, p < 0.001) of epileptic discharges. L-MET treatment reduced the amplitude and asymmetry index of SWDs compared with vehicle-treated and imipramine-treated WAG/Rij rats. Although imipramine treatment also reduced the amplitude of epileptic discharges, the effect of L-MET was greater than that of imipramine. Unlike L-MET, imipramine did not significantly change the asymmetry index of the SWDs  = 33.86, the SWDs .The increase in the number of SWDs caused by 14-day treatment with imipramine indicates its negative effect on absence seizures. Even though the mean duration of SWDs after chronic imipramine treatment did not significantly change compared with vehicle administration B, the inThe duration of individual discharges after imipramine administration could reach 100 s or even more, while the mean duration of the discharges after vehicle administration did not exceed 20 s. Thus, the negative effect of imipramine treatment was also manifested in the form of a fusion of individual discharges into one very long discharge, resembling absence status epilepticus. To determine whether imipramine can have a pro-epileptic effect on absence seizures, the effect of a two-fold higher dose of imipramine (30 mg/kg) was studied. On the 4th day of imipramine injection at a higher dose, atypical  epileptic discharges appeared on the EEG, accompanied by convulsive twitching of the head .Atypical  epileptic discharges had an amplitude (about 3000 µV) several times higher than the amplitude of typical SWDs , as well as poorly expressed asymmetry. In addition, they were characterized by the disorganization of their time–frequency dynamics and a substantial change in the power spectra, as evidenced by the wavelet B and FouTo determine whether L-MET can have a pro-epileptic effect like imipramine, the effect of two-fold, four-fold, and six-fold higher doses of the drug  on the number of SWDs was compared with the effect of L-MET at a dose of 50 mg/kg. Saline served as the control drug. Different doses of L-MET were tested in the same animals. The different doses of drugs were administered at intervals of at least 7 days . The resBiochemical data have shown that chronic administration of L-MET causes an increase in the level of monoamines and their metabolites in all studied brain structures in WAG/Rij rats . Of notep < 0.05). Changes in other monoamines and their metabolite levels were insignificant.In the prefrontal cortex, chronic injections of L-MET induced an increase in the content of NA  = 8.49, p < 0.05) and, at the trend level, the content of DA (p(U) = 0.09). L-MET also caused a tendency to increase the level of the NA metabolite MHPG  = 3.02, p = 0.1) and the ratio of 5-HIAA to 5-HT  = 3.0, p = 0.1; p(U) = 0.028). 5-HIAA is the primary product of the enzymatic degradation of serotonin by monoamine oxidase A (MAO-A). The 5-HIAA-to-5-HT ratio is an indication of the serotonin turnover rate in the brain and serves as an estimated index for assessing the rate of release of 5-HT into the synapse.In the nucleus accumbens, L-MET treatment significantly increased the content of the DA metabolite DOPAC  = 4.8, p < 0.001), the HVA/DA index  = 4.61, p < 0.05) and, at a tendency level, the content of DA  = 4.0, p = 0.06) and its catabolic product DOPAC  = 4.10, p = 0.06).In the striatum, L-MET treatment significantly elevated the level of HVA, the end product of DA degradation  = 22.15, p < 0.01); the metabolic product of NA, MHPG,  = 28.36, p < 0.001); and, at the level of a tendency, the content of DA  = 4.07 p = 0.06) and the 5-HIAA/5-HT index  = 3.00, p = 0.1).In the hypothalamus, L-MET significantly increased the content of NA  = 14.18, p = 0.1), DOPAC  = 3.37 p = 0.08), and 3-methoxytyramine (MT)  = 3.00 p = 0.1).In the hippocampus, L-MET caused a tendency to increase the levels of DA  = 3.00, The present study describes for the first time the anxiolytic and antidepressant effects of L-MET treatment in the WAG/Rij rat model of depression comorbid with absence epilepsy. The results indicate that L-MET not only reduces the level of anxiety and depression in WAG/Rij rats but also suppresses associated epileptic seizures, in contrast to conventional antidepressants, especially tricyclic and tetracyclic, many of which can decrease the seizure threshold and aggravate epileptic seizures ,43,44,45The anxiolytic effect of L-MET is evidenced by a reduction in anxiety measures in three well-validated tests for the assessment of anxiety-related features of behavior: the open-field, light–dark choice, and elevated plus-maze tests .In the open-field test, L-MET caused a significant increase in the number of crossed squares, center entries, and unassisted (unsupported) rearing (the measures of exploratory motivation) and a tendency to decrease the number of fecal boli (a measure of fear/anxiety provoked by a new unfamiliar open space). Interestingly, L-MET treatment did not affect assisted (supported) rearing. The open-field test is a conflict test based on opposing motivations: to explore a new environment (exploratory motivation) and to avoid a central (more “dangerous”) area due to fear/anxiety. A classic exploratory behavior recorded in the open field is rearing behavior. However, previous studies have shown that supported rearing  and unsupported rearing  are two different behavioral traits in the open-field test ,48. SuppIn the light–dark choice test, the anxiolytic effect of L-MET is evidenced by an increase in the time spent in the lit compartment and a decrease in the number of risk assessments, an ethologically relevant measure of anxiety ,50. In LIn the elevated plus-maze, L-MET induced an increase in the time spent in open arms, the number of head dips from the open arms, and a decrease in the number of boli. Observing stress-related anxiety in rodents typically relies on species-specific  behaviors such as increased risk assessment, the reduction of exploration, seeking shelter, escape, and defecation . TherefoIn the forced swimming test, L-MET treatment reduced the immobility time, increased the duration of the first episode of active swimming (climbing), and did not significantly affect the duration of swimming compared with saline treatment, indicating an antidepressant-like effect. Of note, the antidepressant profile of L-MET (decreases in immobility) was comparable with that of classical antidepressants such as imipramine and fluoxetine. However, the antidepressant-like effect of L-MET in the forced swimming test was more similar to the effect of the tricyclic antidepressant imipramine than that of the selective serotonin reuptake inhibitor fluoxetine. Thus, L-MET, like imipramine, increased the duration of climbing and did not affect the duration of swimming, while fluoxetine increased the duration of swimming and did not affect the duration of climbing. It should be noted that the effect of L-MET on anxiety levels in WAG/Rij rats also has a greater similarity with the effect of imipramine than of fluoxetine. L-MET, like imipramine , showed One of the most important characteristics of the beneficial action of L-MET in the WAG/Rij rat model of depression and absence epilepsy comorbidity is the combination of anxiolytic and antidepressant effects with an overwhelming effect on absence seizures. This seems to indicate that L-MET affects the common mechanisms underlying absence epilepsy and its neuropsychiatric comorbidities. Previous behavioral, electrophysiological, and pharmacological studies have demonstrated functional deficits in the brain DAergic system responsible for depression-like behavior in WAG/Rij rats ,14,18,52L-MET also increased the NA content in the prefrontal cortex and the content of NA and its metabolite, MHPG, in the hypothalamus. There is convincing evidence that the brain’s NAergic system is the intrinsic anti-epileptic mechanism. NA regulated the excitability of cortical neurons , adjustiL-MET is the immediate precursor of SAMe and the end product of the one-carbon cycle . SAMe isOne important question is why the smallest dose of L-MET was the most effective in suppressing absence seizures. First, apparently, because it is this dose of L-MET that increases the SAMe content in the brain but not in the blood. These data indicate that L-MET causes an increase in the local synthesis of SAMe in the brain, rather than an increase in the synthesis of SAMe peripherally, with subsequent transport of SAMe from the periphery to the brain . We agreIn the present study, it has been also demonstrated that one of the mechanisms of the beneficial phenotypic effects of L-MET is an increase in the level of monoamines and their metabolites, mainly DA, DOPAC, HVA, NA, and MHPG, in several brain structures. Reduced monoaminergic tone, especially mesolimbic DAergic insufficiency, has been earlier reported to be associated with depression-like pathology in WAG/Rij rats ,18.In our previous work, a maternal diet enriched with methyl-group donors and cofactors of the one-carbon cycle  during the perinatal period enhanced the DAergic tone of the brain mesolimbic system , increasMales of inbred WAG/Rij rats were used as experimental subjects. WAG/Rij rats were born and raised at the Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences and represented approximately the 72nd generation from parents originally obtained from Radboud University Nijmegen . All rats were kept under a 12/12 h light–dark cycle (lights on at 8.00 a.m.). Animals were housed in standard plastic cages in groups of 3–4 animals per cage. Food and tap water were available ad libitum. Experiments were performed according to the European Union Directive 2010/63/EU on the protection of animals used for scientific purposes. Animal care and use were in accordance with the institutional policies and guidelines. The experimental protocol was approved by the Ethical Committee of the IHNA (protocol № 5 of 2 December 2020). All efforts were made to minimize the number of animals used in experiments and their suffering from experimental procedures.L-MET  was injected intraperitoneally (i.p.) at a dose of 50 mg/kg for 14 days. The 50 mg/kg dose was chosen because it has been shown that it is at this dose that L-MET causes an increase in the SAMe level in the brain. Other doses (25 mg/kg and 100 mg/kg) were less efficient . In addiAdult 7-month-old WAG/Rij rats (vehicle-treated and L-MET-treated) were randomly divided into 2 groups: one of them was subjected to EEG registration and behavioral testing (n = 48) and the other (n = 16) to measuring the level of brain monoamines and their metabolites. Animals intended for biochemical studies were not subjected to behavioral testing to prevent any possible effects of testing on the brain monoamines and their metabolite levels.Stereotactic surgery was performed under chloral hydrate anesthesia . Rats were equipped with bilateral epidural electrodes (stainless steel screws) over the frontal  and occipital  cortex. The electrodes were implanted into small round holes (0.8 mm in diameter) in the skull and fixed with dental acrylic. EEG registration for 3 h per day (from 16.00–19.00) was conducted in freely moving animals using the wireless 8-channel biopotential measurement system “BR8V1” based on Texas Instruments ADS1298 Analog Front-End. The EEG was recorded monopolarly, and a reference electrode was placed over the cerebellum. Animals were placed in Plexiglas recording cages (15 cm × 30 cm × 26 cm) and habituated to the experimental situation for 1 h before the beginning of the recording session. SWDs were detected in the EEG as repetitive trains of sharp asymmetric large-amplitude spikes and slow waves lasting ≥1 s with amplitude at least two-fold higher than the baseline EEG signal. Calculation of SWDs was performed by a “blinded” unbiased expert. The severity of absence epilepsy was assessed by the number and mean duration of the SWDs. The mean duration of SWDs was calculated as the ratio of the total duration to the number of SWDs. The number and mean duration of the SWDs were calculated at baseline, on the 7th and 14th days of L-MET injections, and on the 7th day after L-MET cancellation. The time–frequency dynamics of the SWDs were analyzed using the complex wavelet Morlet. The frequency spectra of the EEG with SWDs were computed using the Welch method , and then the spectra were averaged.Of note, the 2 s time window supplies a frequency resolution of 0.5 Hz. The SWDs’ time–frequency dynamics and spectral power were compared between 2 groups of WAG/Rij rats (drug-treated and vehicle-treated). The averaged power spectra of the background EEG during quiet wakefulness (fragments lasting 8–10 s and separated from SWDs at least by 2 s) were compared between the experimental and control groups of animals. In addition to the spectral power density, the SWDs’ amplitude (μV) and asymmetry index (the ratio of the negative component to the sum of negative and positive components of the SWDs in %) were calculated. Custom-made software was used for the semiautomatic measurements of the SWD amplitude and asymmetry index. The amplitudes of all spikes in a whole SWD were computed and then averaged.L-MET-treated and vehicle-treated WAG/Rij rats were compared for differences in behavioral tests relevant to anxiety and depression. The level of anxiety was determined in the light–dark choice, open-field, and elevated plus-maze tests. The forced swimming test assessed depression-like behavior in WAG/Rij rats. Behavioral tests were carried out in compliance with the recommendations developed for testing epileptic animals .The apparatus consisted of two compartments with openings between them. The large (36 cm × 18 cm) compartment was light (100 lx) and the small (18 cm × 17 cm) one was dark (<5 lx). Each rat was placed in the light compartment facing away from the opening, and the following behavioral reactions were measured for 5 min: latency of entering the dark compartment, the time spent in each compartment, the number of transitions between compartments, and the number of risk assessments (aborted attempts to enter into the light compartment). The shorter the time spent in the light compartment, the lower the number of transitions between compartments, and the greater the number of risk assessments , the higher the level of anxiety in the test and vice versa ,46.The apparatus was a circular arena, 96 cm in diameter with a 40 cm wall, divided into 16 sectors and 3 circles. The central circle with a diameter of 32 cm was considered the center of the field. Animals were placed in the center of the field, and the following variables were recorded for 5 min: latency to leave the center, the number of crossed squares, the number of rearings (assisted and unassisted), center square entries, grooming reactions, and fecal boli (defecation). The open field was cleaned after testing each rat with 10% ethanol and wiped thoroughly to remove the odor. The test room had a dim illumination (40 W) for decreasing the aversiveness of the test. The lower exploratory activity in the open field, especially in the central area, is commonly ascribed to a higher level of anxiety and vice versa .The elevated plus-maze is a standard and widely used test to assess anxiety-like behavior in rodents. Anxiety-like behavior is characterized by an increased avoidance of open arms compared with closed arms. The elevated plus-maze used in our study was constructed of dark gray polyvinyl chloride . It consisted of two opposing open arms without side walls , two opposing enclosed arms  with side walls (height 38 cm), and a central square (10 cm × 10 cm). Two lights illuminated the maze . The maze was elevated 73 cm above the floor. The test duration was 3 min. Rats were brought to the test room 15 min before the start of the test (habituation period). Each rat was placed in the center of an elevated plus-maze facing a closed arm, and the following behavioral measures were recorded: the number of entries into and time (s) spent on each arm and central square, the number of head dips from the open arms, the number of rearings in the open and closed arms, the number of risk assessments (aborted attempts to enter into open arms), the number of transitions between open and closed arms, the number of grooming episodes, and the number of fecal boli. The maze was cleaned with 10% ethanol between trials to minimize scent trails. The shorter the time spent in open arms and the central square, the greater the number of risk assessments, and the higher anxiety and vice versa ,50,74,75The forced swimming test is a standard test to measure depression-like behavior in experimental animals. This test is widely used to assess the antidepressant potential of various pharmacological drugs. In this study, the forced swimming test for the assessments of stress coping, “behavioral despair” or depression-like behavior was modified from the test originally described by Porsolt . Only a 4 containing 3,4-dihydroxy benzylamine (0.5 nmol/mL) as an internal standard and centrifuged at 10,000× g for 10 min at 4 °C. The supernatant (20 µL) was removed and analyzed by HPLC to determine the concentration of noradrenaline (NA), 4-hydroxy-3-methoxyphenylglycol (MHPG), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine or serotonin (5-HT), 5-hydroxyindolacetic acid (5-HIAA). Chromatograph LC-304 T  with analytical column ReproSil-Pur   was used. The mobile phase for HPLC analysis was 0.1 M citrate-phosphate buffer containing 1.1 M octane sulfonic acid, 0.1 mM ethylenediaminetetraacetic acid (EDTA), and 9% solution of acetonitrile (pH = 3.0). Measurements were made using the electrochemical detector LC-4B  on a glass–carbonic electrode (+0.85 V) against the reference electrode Ag/AgCl. The turnover of monoamines was expressed as the ratio of tissue concentrations of the primary acidic metabolite  to the parent amine . Samples from all animals (vehicle-treated and drug-treated) were processed in parallel on the same day for each brain structure. We used a solution that contained 3,4-dihydroxy benzylamine (DHBA) , NA , 3-methoxy-4-hydroxyphenylglycol (MHPG) , DA , 3,4-dihydroxyphenylacetic acid (DOPAC) , homovanillic acid (HVA) , 3-methoxytyramine (3-MT) , 5-hydroxytryptamine (5-HT) , and 5-hydroxy indole acetic acid (5-HIAA)  as a standard at a concentration of 500 pmol/mL. The “internal standard” method was used to determine the levels of monoamines and their metabolites in brain structures as the ratio of the peak areas in the standard mixture and sample (nmol/g wet tissue). Samples were registered using the hardware–software complex Multichrom 1.5 . All the reagents used for the analysis were of high purity.Vehicle-treated and L-MET-treated WAG/Rij rats were decapitated with a guillotine for laboratory rodents. Then, the brain structures  were removed on ice, frozen in liquid nitrogen, and weighed. The samples were stored in liquid nitrogen and later assayed for levels of monoamines and their metabolites using high-pressure liquid chromatography (HPLC) with electrochemical detection. The details of the method used have been described earlier ,18. BrieData were analyzed using the program “STATISTICA Release 7”. A two-way analysis of variance (ANOVA) with repeated measures and one-way ANOVA with the Newman—Keuls post hoc test or a non-parametric equivalent, the Kruskal—Wallis H test (one-way ANOVA by ranks), and the Mann—Whitney U test with Bonferroni corrections were used when appropriate.The results of the present study provide the first evidence that L-MET treatment causes anxiolytic and antidepressant effects, which are associated with absence seizure suppression. The correction of the pathologic phenotype in WAG/Rij rats was accompanied by an increase in the content of monoamines and their metabolites, the insufficiency of which, as previously shown, is associated with pathology in this model.Behavioral, EEG, and biochemical studies indicate the therapeutic efficacy profile of L-MET in the WAG/Rij rat model. The antidepressant-like effect of L-MET is closer to that of the tricyclic antidepressant imipramine than that of the selective serotonin reuptake inhibitor fluoxetine. L-MET appears to be an antidepressant with anxiolytic properties, similar to imipramine. However, in contrast to imipramine, L-MET does not aggravate epileptic seizures in WAG/Rij rats. It is assumed that neurochemical changes in brain structures caused by L-MET underlie its therapeutic effect. However, the exact mechanisms by which L-MET exert its favorable complex effects on anxiety, depression, and associated absence epilepsy await full elucidation.L-MET is the immediate precursor of SAMe, which is a major donor of methyl groups not only for the synthesis of monoamines in the brain but also for DNA methylation and epigenetic modifications of gene expression. Emerging evidence suggests an important role of epigenetic mechanisms in the pathogenesis of depression and epilepsy as well as in the mechanisms of antidepressant and anti-epileptic effects of drugs ,79,80,81


Foodborne diseases caused by foodborne pathogens pose risks to food safety. Effective detection and efficient inactivation of pathogenic bacteria has always been a research hotspot in the field of food safety. Complicating these goals, bacteria can be induced to adopt a viable but non-culturable (VBNC) state under adverse external environmental stresses. When in the VBNC state, pathogens cannot form visible colonies during traditional culture but remain metabolically active and toxic. The resulting false negative results in growth-related assays can jeopardize food safety. This review summarizes the latest research on VBNC foodborne pathogens, including induction conditions, detection methods, mechanism of VBNC formation, and possible control strategies. It is hoped that this review can provide ideas and methods for future research on VBNC foodborne pathogenic bacteria. Campylobacter being the cause of more than 60% of confirmed cases. Other common foodborne pathogens include Salmonella and Yersinia . WheStaphylococcus aureus, Escherichia coli O157:H7, Salmonella, and others [More than 100 kinds of bacteria can enter the VBNC state. These include many foodborne pathogenic bacteria, such as d others . The bacVibrio cholerae and E. coli present in marine and estuarine environments [Initially, the VBNC state was observed in ronments . Since tS. aureus revealed that colonies enter the VBNC state within a short period of time [E. coli to enter the VBNC state [ of time . These b of time  have beeNC state .relA and spoT genes upon entry into the VBNC state leads to the accumulation of (p)ppGpp, resulting in the enhancement of stress resistance [The stringent response is a key survival mechanism often used by bacteria in response to adverse environmental conditions , which isistance .rpoS, spoT, relA, ppx, ppk, and lon) as well as the antitoxin proteases Lon and ClpP are upregulated in bacteria after induction of the VBNC state upon exposure to an adverse environment  were performed to study the effect of oxidative stress resistance on bacterial survival under aerobic conditions. The three mutants displayed reduced viability and morphological changes exclusive to VBNC bacteria. When antioxidant treatment was restored, the number of colonies entering the VBNC was relatively reduced [Increased oxidative damage and oxidative stress are observed upon entry of bacteria into the VBNC state, as evidenced by an increase in the production of reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide, and hydroxyl radicals . For insnduction . In addibacteria . Bacteribacteria ,48. Expe reduced . The finE. coli O157:H7 in the normal and VBNC states. The authors identified 97 genes and 56 proteins that were significantly altered upon entry into the VBNC state, which mainly involved genes and proteins related to membrane transport, metabolism, DNA replication, and cell division, and which led the cells to the VBNC state of low metabolic activity [V. cholerae revealed changes in the expression of 1420 genes, with upregulation of genes related to biofilm formation and stress response, and downregulation of genes related to cell division and ribosome activity [E. coli was in the VBNC state, including stress response-induced genes, three genes encoding toxic proteins , and others [The mechanism of bacterial entry into VBNC involves complex processes which cannot be separated from the regulation of genes. RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods have been used to compare transcriptional and protein expression differences in activity . Similaractivity . A totald others . Taken tV. cholerae, where 19 proteins related to carbohydrate metabolism, phosphate utilization, and stress response appeared to be highly expressed when bacteria recovered from the dormant state. Bacteria can become persistent cells, which is defined as a dormant, non-dividing, and metabolically inactive state. Indeed, bacteria in the VBNC could be considered as a deeper level of dormancy. Since persistent and VBNC cells are very similar in their dormancy and tolerance of high antibiotic concentrations, the increasingly accepted view is that persistent and VBNC cells might represent different stages of the same dormancy process . During S. aureus to enter the VBNC state, where the oxidative stress generated by NTP triggered an oxidative stress response in the bacterium, which consumed part of the cellular ATP and made other energy-dependent physiological activities  less energetic  or iodonitrotetrazolium chloride (INT) staining, substrate uptake determined by direct viable counting (DVC), membrane permeability (SYTO 9/propidium iodide (PI) staining), nucleic acid detection , and RNA expression (RT-qPCR) . The folE. coli in the VBNC state after UV treatment using CTC flow cytometry. The number of E. coli entering the VBNC state was proportional to the germicidal power in the UV range of 0–30 mJ/cm2. This is a relatively simple detection method. However, the toxicity of CTC can lead to an underestimation of the level of active cells, and stained bacteria grow at the same rate as untreated bacteria only at low concentrations of the dye [The redox test method is based on the fact that VBNC bacteria retain low respiratory activity and can reduce a specific dye to a fluorescent product. When the dye enters the cell, it can be reduced by respiratory chain enzymes of VBNC bacteria to produce a red fluorescent precipitant. The fluorescence can be observed and quantified by fluorescence microscopy or flow cytometry. The typically used dyes include CTC or INT ,58,59. FListeria monocytogenes [V. cholerae [S. typhi [L. monocytogenes and observed the normal morphology of dead cells versus the elongated morphology of live cells by fluorescence microscopy. The proportion of live cells was determined by random counting of multiple microscopic fields [V. cholerae were measured by the conventional culture method versus ciprofloxacin by fluorescence microscopy. Only 15.11% of the samples were positive for V. cholerae in the conventional culture method, whereas 40.70% of the samples were positive for V. cholerae using the DVC live microscopy assay, showing the accuracy of the measurement by DVC microscopy [The DVC method was proposed in 1979. Nalidixic acid is a DNA helicase inhibitor. When nalidixic acid was added to Gram-negative (G−) bacteria samples, viable bacteria were inhibited from dividing with the morphology changed to become significantly longer and thicker. When the samples were further stained with acridine orange, live bacteria could be distinguished by fluorescence microscopy based on the altered morphology of the viable bacteria. Nalidixic acid is less effective in inhibiting Gram-positive (G+) bacteria, so the division inhibitor used for the DVC test for G+ is often ciprofloxacin ,61. Thisytogenes , V. cholcholerae , and S. S. typhi . Besnardc fields . Similarcroscopy . This isBifidobacterium [E. coli entry into the VBNC state. The authors measured the percentage of live cells and demonstrated that elevated NaClO concentrations accelerated entry into the VBNC state [The use of fluorescent dyes to examine cell membrane permeability can also identify and quantify bacteria in the VBNC state. The most commonly used fluorescent dyes are SYTO 9 and PI. SYTO 9 is able to penetrate the cell membranes of both living and dead cells to bind DNA and RNA, resulting in green fluorescence. PI is only able to penetrate dead or damaged cells, resulting in red fluorescence . Differeacterium . Zhu et NC state . The SYTNC state ,64. In aNC state .S. typhimurium in lettuce. After incubating the samples with PMA dye, the invA gene was selected as the target gene for amplification. Typically, the final minimum detection limit for Salmonella in lettuce was 103 CFU/g. The detection limit could be reduced to 101 CFU/g when a 12 h bacterial enrichment was performed [The nucleic acid binding dyes ethidium monoazide (EMA) and PMA can enter dead cells with damaged cell membranes. Both dyes covalently cross-link to the dead cell DNA, thereby reducing or eliminating the amplification of DNA fragments in the subsequent PCR in dead cells, leaving live cells unaffected. The distinction permits a reasonably accurate estimation of the number of live cells. EMA is sometimes able to penetrate through live or intact bacteria, leading to false negative results, so the most commonly used nucleic acid amplification inhibitor is PMA . Liang eerformed . This tewzy gene of E. coli O157:H7 and agfA of S. enterica for LAMP and quantitatively determined VBNC bacteria in different agricultural products that included tomato and spinach. The detection limits for E. coli and S. enterica were 5.13 × 103 and 1.05 × 104 CFU/g, respectively. Peterse et al. developed a PMA-LAMP assay targeting the hipO gene of C. jejuni. The assay had a 100% targeting specificity for this bacterium and was successfully used for the quantification of VBNC C. jejuni in agricultural products that included chicken breast meat and romaine lettuce. The detection limits were as low as 102 CFU/g [LAMP is an automated cyclic DNA synthesis method that does not require complex thermal cycling apparatus and can amplify target DNA within 1 h at constant temperature (60–65 °C) using four to six specially designed primers and a strand-displacing DNA polymerase ,69. Liu 02 CFU/g . CompareSalmonella in milk samples was quantified based on RT-qPCR Salmonella invA mRNA. The method had a minimum measurement limit of 104 CFU/mL for Salmonella and sensitivity up to 101 CFU/mL after 12 h of bacterial enrichment [The large double helix structure can reduce the measurement sensitivity. Single-stranded mRNA is a more suitable target than DNA to assess cell viability by virtue of its small molecular weight, half-life of only a few minutes, and presence only in living cells. In the RT-qPCR measurement of cellular mRNA, the mRNA is reverse transcribed to cDNA, which is used as a template for PCR amplification. In one study, richment . The RNArichment .2O) actively participate in cellular metabolic activities, including the synthesis of fatty acids, proteins, and nucleic acids. The heavy hydrogen isotope deuterium (D) can replace H) in water molecules during fatty acid and protein synthesis to form carbon–deuterium (C–D) bonds. The integrated spectral intensity of C–D and C–H segments is detected using spectroscopy analysis to quantify the degree of substitution of H by D, and thus evaluate the metabolic activity of bacteria  aSalmonella. The bacteria retained their shape but released many membrane vesicles accompanied by transient overexpression of membrane vesicle protein PagC. This protein could be used as a biomarker to detect VBNC Salmonella cells by immunoblotting (V. cholerae revealed different trends of the up- and downregulation of different proteins, which might permit the detection of VBNC V. cholerae [The physiological response of the bacteria when they enter the VBNC state is altered. This is reflected in the different products produced by VBNC bacteria compared to their culturable counterparts. This difference can be exploited to detect VBNC bacteria. Jun et al.  chemicalblotting . The asscholerae . The manVBNC bacteria can recover when cultured under certain conditions. Particularly, the recovery of toxin-producing bacteria in foods can result in damage to the human body, necessitating the control of these bacteria . The comV. parahaemolyticus in a minimum salt medium. Interestingly, the upshift of the temperature to 37 °C instead of 25 °C significantly impaired the resuscitation [Several factors affect the resuscitation of bacteria from the VBNC state. These include temperature, humidity, oxygen concentration, acidity, and others. Wong et al. reported that a temperature upshift from 4 °C to 25 °C successfully recovered the cultivability of VBNC citation . The autL. monocytogenes and E. coli O157:H7 in process wash water. The authors reported that both bacteria were induced to enter the VBNC state after treatment with 2.5 mg/L chlorine. All L. monocytogenes cells were inactivated after treatment with 5 mg/L chlorine, but E. coli O157:H7 entered the VBNC state [S. aureus was induced to the VBNC state due to oxidative stress at a low temperature of 4 °C, followed by the addition of chloramphenicol. The recovery of the bacteria was completely lost because the antibiotic inhibited the synthesis of VBNC bacterial proteins [V. parahaemolyticus recovery experiment [Enterococcus faecalis. Benzathine and piperacillin can bind to penicillin-binding proteins that are required for the final stage of peptidoglycan synthesis in bacteria, resulting in the loss of enzymatic activity. Gentamicin inhibits protein synthesis [Besides physical conditions, bactericides that include antibiotics and chlorine can block entry into the VBNC state and can effectively decrease the viability of the VBNC bacteria. Truchado et al. evaluated the effect of chlorine on NC state . The autNC state . The finproteins . The samperiment . Similarynthesis .The diversity of bacterial species, their different environments, and the development and refinements of genomics and proteomics analyses have provided a wealth of data on VBNC bacteria. Recent research has increasingly addressed VBNC foodborne bacteria, with the emergence of new sterilization technologies that include pulsed light and neutral electrolyzed water sterilization technology, the development of more accurate and diverse detection technologies/instruments for VBNC bacteria, and the deeper exploration of the mechanisms related to the induction and control of VBNC bacteria.Since the proposal of the VBNC bacteria state 40 years ago, research in a variety of scientific disciplines has enriched the understanding of VBNC bacteria. Future explorations and refinements will involve five aspects. First, new sterilization methods will mainly focus on the creation of environmental stresses that will act to sterilize samples. Since this may induce VBNC bacteria, conditions that induce the VBNC state and their mechanisms of action will be explored in-depth. Second, the effects of different food systems and different food processing conditions on the production of VBNC bacteria will be systematically explored to determine if there is a universal mechanism of food processing conditions that induce VBNC bacteria. Third, food safety researchers will continue to develop and refine accurate quantitative detection technologies for novel VBNC bacteria. Indeed, the detection methods discussed above  have the


The paper presents the results of tests on concrete with recycled aggregate impregnated with the use of citric acid. Impregnation was carried out in two stages, with a suspension of calcium hydroxide in water  or diluted water glass used as the second impregnant. The mechanical properties of the concrete were carried out: compressive strength, tensile strength and resistance to cyclic freezing. In addition, concrete durability parameters such as water absorption, sorptivity and torrent air permeability were investigated. The tests showed that this type of impregnation did not improve most of the parameters of concrete with impregnated recycled aggregate. The mechanical parameters after 28 days were significantly lower compared to the reference concrete, although after a longer curing period, these differences decreased significantly for some series. The durability parameters of the concrete with impregnated recycled aggregate also deteriorated compared to the reference concrete with the exception of air permeability. The results of the tests carried out indicate that impregnation using water glass in combination with citric acid gives the best results in most cases and that the order in which the impregnation solutions are applied is very important. Tests also showed that the effectiveness of impregnation is very much influenced by the value of the w/c ratio. Depleting natural resources of raw materials, combined with increasing environmental concerns, are creating growing pressure to reduce landfill and reuse waste. This trend also applies to the construction industry, which is a major consumer of raw materials and producer of waste from, among other things, the demolition of various types of structures ,2,3,4. WOne obstacle is the deterioration in concrete performance usually obtained when replacing natural aggregate (NA) with recycled concrete aggregate (RCA) in concrete of the same composition: lower strength, higher water absorption and thus lower resistance to environmental corrosion. The deterioration of the properties of concrete with recycled aggregates can be counteracted in various ways with two approaches being the most common. The first involves enriching the composition with supplementary cementitious materials ,6,7. ThiMethods for treating recycled concrete aggregate are manifold, and new ones are still being proposed. They can be divided into two groups ,16,17. TWhen reviewing the literature, it is easy to see that there are relatively few descriptions of studies in which RCA is impregnated in two stages, and virtually none in which one of the impregnants is citric acid. It is relatively rare in the literature to find results of studies of such durability characteristics of concrete with impregnated RCA as resistance to cyclic freezing or air permeability. Taking this into account, this paper fills the knowledge gap on the effect of two-stage impregnation of recycled concrete aggregate with the use of citric acid on the mechanical and durability properties of concrete, with particular emphasis on permeability tested by the Torrent method.The research presented in this article is a continuation of investigations presented in ,34. As iA total of 10 series of concrete with varying w/c values was made, 8 of which contained recycled concrete aggregate subjected to a two-stage impregnation procedure. One of the impregnants was a citric acid solution applied to the aggregate in all eight series.The citric acid used had a twofold function. The first was to remove some of the old mortar, and for this purpose it was used in the first impregnation step. Then, the goal of the second impregnation was to strengthen the mortar that had not been removed. The use of citric acid in the second impregnation step, on the other hand, was intended to have a synergistic effect with the first impregnation and lead to an improvement in the properties of the old mortar.Tests of selected mechanical and durability properties of concrete were used to assess the achievement of the intended effects. These effects have been only partially achieved, and the reasons for this are discussed later in this paper. Furthermore, although the results obtained are not very promising, the critical analysis of the research carried out in the paper provides grounds to conclude that with some adjustments to the impregnation procedure, the effectiveness of the presented methods of treatment of recycled concrete aggregate can be proven. It also gives guidance to the direction in which the modification of the presented procedures should go.Ten series of concrete were made for the study. Five of them were made assuming w/c = 0.45, and another five assuming w/c = 0.55. The concrete series with the same w/c value differed from each other only in the type of aggregate used and the amount of superplasticizer added to the mix. The aggregate was obtained by crushing the concrete in a laboratory jaw crusher. The concrete came from cubes left over from the strength test and was at least two years old at the time of crushing but not exceeding five years. The compressive strength of the crushed concrete varied and was approximately in the range of 20–40 MPa. The choice of such a raw material for the preparation of recycled concrete aggregate was dictated primarily by its availability. It was also significant that it was thus possible to guarantee a large diversity of source concrete in terms of strength, type of natural aggregate used, type of cement and age. The use of aggregate from the demolition of a particular structure would not have yielded the variety of raw material that positively influenced the versatility of the results obtained.The preparation of the aggregate involved several steps carried out in two stages. In the first, raw recycled aggregate was obtained, and this stage included pre-cutting of the concrete pieces, soaking (7–14 days), crushing, primary screening to separate oversize and sub-grains, crushing of oversize grains (>16 mm), screening by fractions (2–4 mm and 4–16 mm) and mixing of individual aggregate batches. In the second stage, the portion of aggregate to be used in specific series of concrete was twice impregnated and dried.Due to the amount of aggregate required, it was prepared in smaller portions. Because of this, as well as due to the random selection of the portions of material for subsequent operations, the aggregate obtained was homogenized to a large extent. Consequently, it could be considered that the initial variation in the parameters of the concrete serving as raw material would not affect the significant variation in the characteristics of the aggregate obtained.The second stage of preparation was double impregnation. Aggregates intended for a given batch of concrete were mixed with the impregnating solution for 30 min in a slow mixer—twice for 15 min each. There was a break between the first and second mixing, also lasting 15 min. Once the impregnation was complete, the excess impregnating solution, if any remained, was separated from the aggregate, and the aggregate itself was spread out in as thin a layer as possible to dry in the laboratory. After the first impregnation stage, the aggregate was left to dry for 4–5 days, after which a second impregnation was carried out. After the second stage, the aggregate, after drying for 3–4 days in a spread form, was transferred to open containers, where it was stored until it was used. The second impregnation stage took place at least 14 days before concreting. The flowchart of the impregnation process is presented in 2 to 2 parts water) and diluted water glass (1 part water glass to 2 parts water). The compositions of all impregnants were determined by weight. The impregnating solutions used and their order of application are reflected in the series designations. The first letter in the series name indicates the first impregnant used , the second letter indicates the second impregnant, and the number indicates the w/c value .Three types of impregnants were used for treatment. They were applied in different combinations and in different orders. These were as follows: 5% citric acid solution, which was used in each case, and interchangeably lime milk, which is a suspension of calcium hydroxide in water (1 part Ca(OH)In the two reference series, the aggregate was not impregnated, but in order to maintain similar conditions of preparation, it was washed with water and then dried. The concrete reference series were designated by the letters NI. These series used recycled concrete aggregate that was not impregnated. The compositions of all the series made are shown in In each series, 36 cubic specimens with a 100 mm edge and 2 cubic specimens with a 150 mm edge were prepared for testing. Due to the high content of unreacted citric acid in the aggregate, which is a compound that strongly slows down cement setting ,36,37, tCompressive strength (after 28 days and 36 weeks), tensile strength (after 28 days), water absorption, sorptivity and freeze–thaw resistance were tested on specimens prepared from each concrete mix.The compressive strength after 28 days was tested according to the procedure given in EN-12390-3  on 6 cubThe tensile splitting strength was tested according to the procedure described in EN-12390-6 [s). The weighed specimens were placed in a forced-air dryer at 110 ± 5 °C. After 14 days of drying, the specimens were removed and weighed, taking the mass thus determined as the mass of the dry sample (md). From these two mass values, the absorbability N was determined according to Formula (1):In order to determine the water absorbability, the 12 halves of the cubic specimens from each series remaining after the splitting tensile test were placed in water for a further 7 days. After this time, they were removed, surface-dried and weighed. The mass thus determined was taken as the mass of the specimen fully saturated with water  is a measure of the variability of the individual parameters for compressive strength after 28 days, tensile strength, water absorbability and sorptivity. In the case of concrete moisture content, the standard deviation of the results was calculated. As the compressive strength value of the concrete after 37 weeks was only tested on two specimens, the spread, i.e., the difference between the two values, was given in this case.ω—geometric mean of the multiples of the mean ωi calculated according to Formula (3):i—the value of a single measurement, Gx—the geometric mean of all measurements. In the case where the value of ωi satisfies the inequality (4), then the result to which the calculated value of ωi corresponds was considered an outlier, and the value of γ was recalculated with this result omitted.For the air permeability coefficient, it was decided to define its own measure of variability, which was defined as the average geometric deviation γ, and calculated according to Formula (2):The results of the freeze–thaw resistance test are shown in An analysis of the results obtained leads to the conclusion that the impregnation of the aggregate carried out did not improve the tested parameters of the concrete made. That is the most general conclusion, but a closer look at the obtained results of individual tests allows one to indicate cases in which there was, however, a slight improvement in the parameters. A closer analysis also makes it possible to indicate which of the solutions adopted have no prospects of success and which offer a chance of improving the properties of the concrete after some changes in the assumptions.The reason for the failure is undoubtedly citric acid—not, however, its use per se, but the assumptions made in doing so, most notably the amount of it remaining on the aggregate grains after the impregnation was completed. As written earlier, this compound was intended to play one of two roles in the mixtures being prepared. Either it was to remove some of the mortar from the recycled concrete aggregate, or it was to enhance the action of one of the other impregnations due to chemical reactions between both impregnations. It appears that the effectiveness of citric acid in both roles depended on which other impregnate it was used with. However, the effects of the impregnation were also influenced by the value of the w/c ratio of the concrete with the modified aggregate. The addition of citric acid did not work particularly well in combination with lime milk and also performed less well as a second impregnator. The combination with glasswater as a second impregnant yielded better results. In this combination, the order of application of the impregnating solutions was definitely more important for concrete with w/c = 0.55.As far as the impregnant combination is concerned, there are two reasons why citric acid and glasswater work better together. The first is the reaction products. When the acid reacts with glasswater, its product is a silicone gel . Its strThe second reason is the practically unlimited solubility of glasswater in water. This allows glasswater to be deposited in the pores of the aggregate in the first stage of impregnation in an amount that depends primarily on the concentration of the solution used and the available pore volume. At the same time, due to the high viscosity of glasswater itself, it is likely that the concentration dependence is not monotonic but has an extreme in its course. 2 that can be deposited in the pores of the aggregate per unit volume by introducing it as a solution. This is the reason why lime milk was used instead of a saturated solution of Ca(OH)2. The aim was to increase the amount of this compound deposited in the aggregate, although mainly on its surface.Lime milk, in contrast to glasswater, is very poorly soluble in water. Its solubility at 20 °C is 1.27–1.35 g/L, depending on particle size and dosage . This is2 with citric acid produces calcium citrate, whose solubility at 20 °C ranges from 3.91 mmol/L (hexahydrate) to 5.69 mmol/L (tetrahydrate) [2 deposited in the pores of the aggregate was so small that the citrate precipitate formed by the reaction with citric acid had little effect on sealing the pores.The reaction of Ca(OH)hydrate) , which t2 towards citric acid was also low with this sequence of the impregnation. This was unlike the case when citric acid was the first impregnating solution. In this case, the low solubility of Ca(OH)2 was not a hindrance, because as the calcium citrate crystallized, another portion of calcium hydroxide was dissolved in the water. This reaction continued as long as there were reagents available and water as the reaction environment. It can be assumed that this was still happening while the aggregate was still drying. Moreover, the neutralizing role of Ca(OH)The order of impregnation was also important when using water glass. It should be noted that during the second stage of impregnation, when there is an interaction between the two compounds contained in the impregnating solutions, the second impregnant is always in excess of the first. This is because there is only as much of the first as the aggregate has accepted. In this way, the compound contained in the second impregnating solution gives the aggregate its “character”, including determining its pH value. This has consequences during the setting and hardening of the concrete mixture made with the impregnated aggregate.A slightly different picture emerges if the results obtained after 74 days are analyzed. The compressive strength after such a time was tested on the witness specimens from the cyclic freeze–thaw test. If the series with w/c = 0.45 are analyzed, in one case, the GC4 series, the obtained strength value of the concrete was higher than in the case of the reference series. The difference was very pronounced and certainly significant. The use of impregnated aggregate in concrete with w/c = 0.55 resulted in an increase in strength compared to the reference concrete in two cases: the CL5 and CG5 series. In all other cases, a decrease in compressive strength was recorded. Irrespective of the w/c value, concrete with aggregate impregnated first with citric acid and then with lime milk achieved quite similar strengths as did concrete with untreated aggregate, namely, once slightly higher and once lower. The reverse order of impregnation resulted in concrete with very low strength.The results of the test after 36 weeks, carried out on dried specimens with larger dimensions (150 mm × 150 mm × 150 mm), showed a slightly different relationship between the strength values obtained for the different series. Irrespective of the value of the w/c ratio, the highest strengths in these tests were obtained for the reference series. This was followed by series with aggregate impregnated first with citric acid and then with glasswater. Slightly lower strength values were obtained by series with aggregate that was impregnated with the same solutions but in reverse order. Concrete with aggregate impregnated first with citric acid and then with lime milk obtained significantly lower strengths than the previously mentioned series, but these were still acceptable values, which cannot be said about the results of the concrete in which the aggregate was impregnated first with lime milk and then with citric acid.The results of the tensile splitting strength test after 28 days and 74 days are presented in Of note is the very large increase in strength between 28 and 74 days for almost all series of concrete with impregnated aggregate. After 28 days, only two series with impregnated aggregate achieved a strength higher than 1 MPa, and in the case of one series, the strength was close to this value (0.98 MPa). All these series were impregnated with citric acid and water glass. However, these results still remained significantly lower than the tensile splitting strengths of the reference series, which exceeded 4 MPa (NI4) and 3 MPa (NI5), respectively.After 74 days, the tensile splitting strength developed in a much more balanced manner. While the reference series recorded only a slight increase (with a greater increase in the NI5 series), the increase in the series with impregnated aggregate was at least doubled. In the case of the CL4, CL5 and GC5 series, it was even much greater. In the group of concrete series with w/c = 0.45, the strength of the GC4 series even slightly exceeded the value obtained for the reference series. It is also worth noting the CL4 series, which, after 74 days, obtained a significantly higher strength value than the CG4 series, while after 28 days the situation was the opposite, with a difference of almost three times. Similarly, in the group of concrete series with w/c = 0.55, the greatest increase in strength was recorded by the series with aggregate impregnated with citric acid and lime milk (CL5), although the difference in strength values compared to the CG5 series was small. Within this group, the reference series achieved the highest strength.In contrast, irrespective of the w/c value and the test date, the series with aggregate impregnated with lime milk and citric acid obtained very low strengths. This was undoubtedly influenced by the order of application of the impregnation solutions. After the second impregnation step, a layer of citric acid-saturated old mortar remained on the aggregate grains, which had already been detached from the aggregate but not removed from it. This layer prevented good adhesion between the aggregate and the new mortar, a fact that the strength test mercilessly exposed.The results of the sorptivity test are shown in An analysis of the sorptivity results indicated that this parameter for concrete with impregnated aggregate had the opposite trend to its strength parameters. The w/c ratio had a certain influence on the formation of these trends. In the group of series with a lower value of this coefficient, the highest value of sorptivity was obtained for the LC4 series, and this value was at the same time higher than for the three series of concrete with a higher w/c ratio, including the concrete of the LC5 series, which was in the middle of the range in its group of series. If we exclude the two series that obtained extremely low results in the strength tests, then, after ranking the others according to increasing values of sorptivity, we obtained the same sequence in both groups of concrete series.Thus, the lowest sorptivity value was obtained for the reference series, followed by series impregnated first with citric acid and then with lime milk . Next in order are the series impregnated with citric acid and glasswater (CG4 and CG5). The GC4 and GC5 series close the ranks.The sequence was different for the results of the water absorbability test. The lowest water absorption was recorded for the reference series and the highest for the series in which the aggregate was first impregnated with lime milk and then with citric acid. The series in which citric acid was the first of the impregnants achieved similar water absorption values with the same w/c value. In contrast, the effect of w/c was marked for the series with aggregates impregnated first with glasswater and then with citric acid. At w/c = 0.45, this resulted in the second lowest absorption value, and at w/c = 0.55 in the second highest. In addition, the series with the higher w/c obtained a higher water absorption compared to the corresponding series with a lower value of this parameter.The difference in pore structure may explain the differences in the trends of the two parameters studied. In the study of sorptivity, capillary pores play a key role. Higher sorptivity with lower absorbability is a strong clue pointing to a higher proportion of capillary pores in the open pore structure. However, mercury intrusion porosimetry (MIP) studies would be required to verify this hypothesis.The results of the Torrent air permeability test are shown in Of the durability parameters analyzed so far, the air permeability of the concrete apparently showed that aggregate impregnation has the potential to improve at least some concrete parameters. Of the eight series of concrete with impregnated aggregate, four, i.e., half, obtained lower air permeability values in the tests than the corresponding reference series. These are the series in which citric acid and glasswater were used for impregnation. This is particularly evident in the case of the concrete series with w/c = 0.55. In this group of series, it was also more evident that the order of impregnation matters, as lower permeability was obtained in the study of concrete impregnated first with glasswater and then with citric acid. In the case of a series with a lower w/c value, the difference was small. However, attention should be paid to the moisture values of the concrete at the time of testing. For the GC4 series, it was significantly higher than for the CG4 series . Since the air permeability of the concrete increases as the moisture content of the material decreases , it can The relationship between moisture content and the permeability of concrete is twofold. On the one hand, as mentioned above, higher concrete moisture content results in lower permeability. This is due to the greater filling of the pores with water and the associated greater resistance that the material places on the flowing air. On the other hand, the rate of moisture loss through the concrete correlates positively with the permeability value. This correlation can be seen in Results of the freeze–thaw resistance test and the test of reduction of the tensile splitting strength after freeze–thaw cycles are presented in After 100 freeze–thaw cycles, only specimens from four series were suitable for strength testing. In addition to the two reference series, these were two concrete series with w/c = 0.45: a series with aggregate impregnated first with citric acid and then with lime milk, and a series with aggregate impregnated first with glasswater and then with citric acid. The smallest decrease in strength among the four series was recorded for the NI4 reference series, and only in this case was the decrease less than 20%. In the case of the series with impregnated aggregate, the decrease was twofold and fourfold.The decrease in splitting tensile strength after 100 freeze–thaw cycles was significantly greater. In the case of the NI4 reference series, the decrease was the smallest but still amounted to more than 50%. In contrast, the series with impregnated aggregate only reached just over 10% of the strength determined on the specimens not subjected to cyclic freeze–thawing tested at the same time. Despite the small number of examples (4 concrete series), it can be concluded that the splitting tensile strength of concrete is a parameter that is much more sensitive to the influence of cyclic freeze–thaw.The obtained results allowed us to formulate the following conclusions. Due to the strong retarding effect of citric acid on the setting and hardening of the cement, the negative effect of impregnation on the strength parameters of the concrete with impregnated aggregate was very evident after 28 days, while after 74 days, the strength values of the concrete decreased only slightly compared to the reference series. In three cases, the compressive strength at that time proved to be even higher.The sorptivity of concrete with impregnated aggregate was generally higher, but the increase of its value depended strongly on the value of the w/c ratio. For the higher w/c ratio, the increase in sorptivity was significantly greater.Water absorbability of concrete with the impregnated aggregate was higher, but citric acid combined with glasswater sealed the aggregate more efficiently than the other combination of impregnants.The air permeability of concrete made with aggregate impregnated with water glass and citric acid (in any sequence) was found to be lower than that of the reference concrete regardless of the value of the w/c ratio. Aggregate impregnation carried out with citric acid and lime milk resulted in an increase in air permeability of the concrete.The use of impregnated aggregate strongly negatively affected the resistance of the concrete for cycling freezing.The sequence of application of the impregnating solutions had a very strong influence on the results, with generally more favorable results being obtained when the citric acid solution was applied first. Glasswater performed better as the second impregnant, with the sequence of application having a smaller effect on the results obtained.To sum up, analyzing both the course of the impregnation process itself and the results obtained, it can be concluded that in order to reduce the negative effect of citric acid, it would be advisable to use it in a lower concentration or to introduce an additional operation of rinsing the aggregate after impregnation with the acid solution. Both of these modifications could be the subject of further research, as despite the generally poorer results obtained for concrete with impregnated aggregates, these treatments appear to have the potential to improve the performance of concrete with such aggregates.