Chloride penetration resistance is one of the most important performance measures for the evaluation of the durability of concrete under a chloride environment. Due to differences in theory and experimental conditions, the durability index (chloride diffusion coefficient) obtained from laboratory accelerated migration tests cannot reflect the real process of chloride ingress into concrete in the natural environment. The difference in test methods must be considered and the transfer parameter  Chlorine ion transmission in concrete and induced steel corrosion are the main causes leading to the damage of coastal reinforced concrete structures. The durability and lifetime of the concrete structures in chloride environments depend on the anti-chloride ion permeability of concrete; therefore, the diffusion coefficient of chloride ions is one of the most important indicators in measuring the capability of concrete resisting chloride erosion. According to the test principles and test conditions, two types of diffusion for chloride in concrete can be categorized. One is the natural diffusion coefficient, which reflects the long-term transmission of chloride into concrete and can be directly used for durability design and service life prediction for marine concrete structures. Extensive research has been carried out on the natural diffusion coefficients ,2,3,4,5.The second is the chloride diffusion coefficient obtained from the accelerated test, including the electrical non-steady-state migration coefficients, the apparent diffusion coefficient of the salt ponding test, and a variety of steady and non-steady-state chloride migration coefficients proposed by Wang and Xu . Those dIn summary, existing comparative studies of diffusion coefficients focused on differences between a variety of coefficients obtained from laboratory tests with standard methods, and, generally, the steady-state diffusion coefficients were specified as the input parameter to the durability life prediction of concrete, lacking in consideration for the disparity between laboratory rapid test results and the long-term performance of concrete in the natural environment. Due to the difference in temperature and humidity, salinity, chloride ion adsorption, and the saturation state of concrete, the chloride diffusion (or migration) coefficients obtained by laboratory tests can hardly reflect the chloride ingress process of concrete in natural marine situations. Wang and Fu  deemed tIn 2002, we started to place concrete test specimens at the Exposure Test Station in Zhanjiang in southern China. The natural chlorine diffusion coefficients at different exposure durations were measured, and the relationship between the natural chloride diffusion coefficient and the exposure duration in the actual marine environments was established. We also compared the natural chloride diffusion coefficients of specific exposure duration with the apparent diffusion coefficients, which were obtained by applying an external electric field to drive the chloride ions in the concretes of the same compositions. By analyzing the diffusion coefficients from different test methods, we obtained direct conversion factors to rapidly characterize the long-term performance of concrete durability by the accelerated electromigration method.The tested binder materials include Portland cement (P·II.42.5), fly ash (FAII), and ground blast furnace slag . Due to the long interval between the two tests, two different binders were used. The concrete aggregates include crushed granite with 5~25 mm continuous gradation and river sand grains with a fineness modulus of 2.60. FDN-5 high-efficiency water-reducing agent, with its main components being the condensation polymer of β-naphthalene sulfonic acid and formaldehyde, is used to reduce water by > 20%.Concrete specimens were cut into cubes of 100mm to investigate the chloride ingress of each mix proportion over a specified period. Concrete was designed using Portland cement, fly ash, and GGBS, mostly with water-to-binder ratios (W/B) of 0.35 . CylindrThe specimens used were concrete rods with a diameter of 100 mm and a height of 50 mm. The concrete specimens were placed on the plastic support in the catholyte reservoir, which was filled with about 12 L of 10% NaCl solution. The sleeve was filled above the specimen with 300 mL anolyte solution (0.3 M NaOH). The test device is shown in Cubic concrete specimens (100 mm × 100 mm × 100 mm) were first removed from molds one day after being cast and were then cured in fresh water at 20 °C for 28 days. As shown in The concrete was broken into particles smaller than 4.75 mm, and the particles with diameters between 2.36 mm and 4.75 mm were screened for the mercury injection test. The PASCAL140 and 240 automatic mercury injectors manufactured by Thermoelectric Finnegan Italy were used.Due to the continued hydration of the binder, concrete permeability decreases over time. From the 10-year exposure test results in South China, the relationship between time and the chloride diffusion coefficients of concrete in the actual marine environment can be obtained, as shown in tD is the chloride diffusion coefficient at time t, refD is the chloride diffusion coefficient at time reft, and m is the age factor of the chloride diffusion coefficient.Takewaka and Mastumoto  proposedm of the diffusion coefficient can be derived for concrete with different cementitious materials, and the water-to-binder ratio is 0.35 in South China coastal areas of the marine environment  of concrete at 28 days and 56 days, which corresponds to long-term resistance to chloride permeability for concrete in marine environments in South China.According to the curvilinear relationship in RD) was measured through an RCM experiment after curing in standard conditions for 28 days and 56 days. All the diffusion coefficients obtained from exposure and RCM are shown in With the development of testing technologies, the non-steady-state electro-migration method (RCM method) becomes the main method to measure the chloride ion resistance of the concrete in China. The Hong Kong–Zhuhai–Macau Bridge and many other large projects applied this method as the standard method for the quality control of concrete durability ,27. To cAccording to De and RD have a similar value for concrete without an active admixture at the same age, meaning that the results of the non-steady-state electric acceleration test based on the Nernst–Planck equation can reflect the short-term durability indicators and long-term performance when not considering the influence of an active admixture.The coefficients De is smaller than RD. Since the accelerated tests hardly reflect the secondary hydration effect on durability improvement, the diffusion coefficients RD of 56 days are closer to the natural diffusion coefficients compared to the 28-day test results.Due to the secondary hydration of the active admixture of fly ash and GGBS, the concrete structure tends to be denser over time, and the concrete durability improves. Therefore, the natural diffusion coefficient The pore structure characteristics of concrete are the most important factor affecting the chloride ion transport in concrete. appD(t) is the effectual diffusion coefficient at time t used for life prediction, RD(reft) is the apparent diffusion coefficient at time reft measured by the RCM test, kt is the transfer parameter, ke is the environmental variable, t is the time when the diffusion coefficient becomes stable , and m is the age factor.The effective chloride diffusion coefficients used to predict the service life of reinforced and non-cracked concrete structures exposed to chloride is an indicator of the long-term durability of concrete. The steady diffusion coefficients after long-term decrescence can be calculated by using the following equation based on Equation (1):t0) is plotted together with the exposure test results, and a regression line (dotted line in m in Equation (2) is obtained through regression analysis, where the transfer parameter kt is 1. The disadvantage of this approach is the introduction of an accelerated index for the electric field to describe the long-term migration of chloride ions, resulting in a deviation from the attenuation theory. In this paper, natural diffusion coefficients at t0 are calculated according to exposure regression curves firstly , without considering the external influencing factors such as tide, temperature and so on . In somem can be shown in kt can be obtained by a coefficients analysis between natural diffusion and rapid tests, according to Therefore, the age factor RD and De at the age of 28 days and 56 days are shown in kt is approximately 0.9. After using activated admixtures, the value is related to the age, type, and dosage of the admixture. For fly ash concrete, kt is in the range of 0.25 to 0.31 (28 d) or 0.32 to 0.43 (56 d). For GGBS concrete, kt is in the range of 0.26 to 0.48 (28 d) or 0.30 to 0.35 (56 d).The relationships between RD of the same age greater than the De. In addition, the RCM method is based on electric acceleration, and the selection of the initial current of the applied electric field is affected by the pore structure. Referring to De derived from the long-term natural diffusion coefficient fully reflects the adsorption effect of FA and GGBS on chloride ions. Therefore, the kt of FA concrete or GGBS concrete is much smaller than that of Portland cement, and, according to the comparison of pore structure parameters and the analysis of the chloride ion adsorption effect, the values of the FA concrete and GGBS concrete are very close.The chloride diffusion coefficient is concentration-dependent, while the RCM method uses a 10% chloride concentration solution, which is much higher than the chloride concentration in natural seawater , resulting in a kt might be only adequate for coefficient conversion and durability design for similar environmental conditions in the South China coastal area. For other environmental conditions, such as cold areas, the transfer parameter kt should be reanalyzed by considering the influence of temperature differences and frozen thaw and be amended by the test results in areas exposed to cold.The discussion above is based on the comparison of RCM results with the exposure diffusion coefficients in the South China coastal environment; therefore, the value of (1)m of the chloride diffusion coefficient is calculated based on the 10 years of data from marine exposure tests in South China.The age factor (2)With the extension of age, the pore diameter distribution of the concrete shifts to the direction of small size. Moreover, using FA or GGBS refines the pore diameter of concrete. At 12-years-old, the gel hole of concrete with FA or GGBS is more than 50% of the total pores.(3)Chloride diffusion coefficients measured by the electric field accelerated method (RCM) are significantly greater than the natural diffusion coefficients at the same age, being calculated from the long-term exposure test results in South China. Since short-time accelerated test results do not reflect the effect of secondary hydration caused by active admixtures on concrete pore structure and chloride ion adsorption improvement, there is a difference between the two diffusion coefficients, which are more obvious for concrete using FA and GGBS.(4)kt is related to the engineering environment conditions, test method, test age, and binder system. When the concrete structures are situated in a high-temperature and high-humidity environment, which is similar to South China, the transfer parameter kt can take the values of 0.9 for Portland cement concrete, 0.25~0.31 (28 d), and 0.32~0.43 (56 d) for FA concrete, and 0.26~0.48 (28 d) and 0.30~0.35 (56 d) for GGBS concrete.The durability rapid test results should be converted to an effective chloride diffusion coefficient in the service lifetime prediction of reinforced concrete structures. The transfer parameter


The D1 and D2 are core proteins of the photosystem II (PSII) in cyanobacteria, algae and plants and influence the proper function of oxygenic photosynthesis (OP) in all oxyphototrophs on Earth. The frequent occurrence of psbA and psbD in cyanophages raises the question whether these genes coevolved with hosts during the early stages of cyanophage and cyanobacterial evolution, or whether they are direct descendants of genes adopted from the genomes of cyanobacterial hosts. The phylogeny of D1/D2 proteins encoded in the genomes of selected cyanophages and oxyphototrophs was reconstructed. In addition, common ancestral sequences of D1 and D2 proteins were predicted for cyanophages and oxyphototrophs. Based on this, the reconstruction of the 3D structures of D1 and D2 proteins was performed. In addition, the ratio of non-synonymous to synonymous (dN/dS) nucleotide substitutions in the coding sequences (CDSs) of psbA and psbD was determined. The results of the predicted spatial structures of the D1 and D2 proteins and purifying selection for the CDSs of psbA and psbD suggest that they belong to the ancient proteins, which may have formed the primordial PSII. It cannot be ruled out that they involved in water oxidation in cyanobacteria-like organisms at early stages of the evolution of life on Earth and coevolved with ancient cyanophages. The data are also discussed in the context of the origin of viruses.Cyanophages are viruses that infect cyanobacteria. An interesting feature of many of them is the presence of  To date, the genomes of more than 70 cyanophages, mainly living in marine environments, have been identified and sequenced3. Interestingly, the vast majority of described cyanophages mainly infect cyanobacteria from the two genera Prochlorococcus and Synechococcus, which in turn are the dominant oxyphototrophs on Earth, as they inhabit all oceans7. Cyanophages are among most abundant ‘semi-life’ forms that play a crucial role in global biogeochemical cycles of carbon, oxygen and nitrogen9.Cyanophages, or bacteriophages (phages) that infect cyanobacteria, were first described in the 1990spsbA for protein D1 and psbD for protein D211. The first photosynthetic gene identified in cyanophages was psbA13. 80% of the cyanophage genomes sequenced so far have psbA, and 42% have both psbA and psbD genes3. The function of the cyanophage genes psbA and psbD is not yet fully understood, but it is likely that they play a key role in infection under changing light conditions that cause photoinhibition of host D1/D2 proteins14. Light-induced photoinhibition causes a more rapid degradation of D1/D2 than the completion of the cyanophage developmental cycle. Thus, providing copies of psbA (D1) and psbD (D2) during viral infection allows the infected cell to be photosynthetically active for as long as is required for full viral particle replication17.A particularly interesting feature of cyanophages is the presence of genes in their genome that encode proteins involved in the photosynthetic activity of cyanobacteria. The best known in this context are the genes coding the PSII core proteins that are most abundant in cyanophages, namely psbA and psbD in cyanophages raises the question of whether these genes evolved in co-evolution with hosts in the early stages of cyanophage and cyanobacterial evolution or whether they are direct descendants of genes that cyanophages later adopted from the genomes of cyanobacterial hosts. The answer to this question relates to at least two interrelated issues. First, it concerns the origin of viruses in general and cyanophages in particular. Second, it sheds light on the evolution of oxygenic photosynthesis (OP) too, since cyanobacteria-like organisms are generally considered to be the first oxyphototrophs on Earth20. According to the most widely held view, the ancestor of cyanobacteria did not possess photosynthetic genes encoding proteins involved in O2 production. OP probably arose about 2.3–2.6 billion years ago during the so-called Great Oxidation Event (GOE) through lateral gene transfer and the fusion of two photosynthetic systems derived from primordial, non-oxygen evolving bacteria23. Some recent phylogenetic analyses suggest that OP may have arisen at very early stages of the evolution of life on Earth26. Therefore, the molecular evolution of proteins at the coding sequence (CDS) level for psbA and psbD was also studied here in the context of nucleotide substitutions in codons. Most nucleotide substitutions occur at the 3rd position in the codon, less frequently at the 1st and 2nd positions27. A nucleotide substitution in a codon can be synonymous and we usually find it at the 3rd position. Generally, synonymous substitutions do not lead to a change in the encoded amino acid in the protein. As a result, the protein retains its biological function. In other words, such a change is neutral and invisible to natural selection. Non-synonymous nucleotide substitutions at the codon lead to a change in the amino acid in the protein. The altered protein can in turn affect the fitness of an organism. Determining the number of non-synonymous (dN) and synonymous (dS) nucleotide substitutions therefore allows us to estimate what type of selection we are dealing with within the CDS under study29. By comparing the dN/dS ratio, we were thus able to determine which type of selection acts on the CDS for psbA and psbD in cyanophages and selected oxyphototrophs  and/or D2 protein (psbD) in the genome. In the group of cyanophages examined, no case was found in which only psbD was present; when psbD was present, it was only together with psbA : D170, E189, H332, E333, D342, and A34433, https://www.rcsb.org/3d-view/6W1P, Supplementary Information Notes 34. Although the predicted model protein is shorter than the template, namely only shifted by 37 amino acid residues, there is an identical set and pattern of amino acids required for the functioning of OEC: D144, E163, H305, E306, D315 and A317 , which is 10 amino acids shorter at the N-terminus than the full D1 sequence . We obtained a much more similar, almost identical 3D model of the predicted, probably ancestral D2   Fig. ”. The rents Fig. . These r25. The occurrence of early branched cyanobacterial D1 and D2 in clades dominated by cyanophage sequences also suggests an early and continuous gene exchange between cyanophages and their hosts  in their genome10. It is likely that psbD was later lost in some cyanophages. The results also show that purifying selection CDSs of psbA and psbD, early branching in the trees for D1/D2 proteins, and separate clades with dominance of cyanophage proteins indicate that the psbA and psbD genes for D1 and D2, respectively, must have evolved very early in both cyanophages and cyanobacteria. It cannot be excluded that the genes psbA and psbD had a common ancestor prior appearance of cyanobacteria-like organisms. Moreover, the reconstructed spatial structures for the predicted hypothetical ancestral D1 and D2 proteins suggest that they may have been functionally similar enough to the modern forms. The ability to bind the Mn4O5Ca cluster may even have been present in the case of the ancient form of the D1 protein. The common ancestor of the psbA/psbD genes and the corresponding D1 and D2 protein products proposed here is made plausible by previous data that the D1/D2 heterodimer may have preceded the homodimeric form D025. The predicted structure of the hypothetical D0 protein suggests that it was more similar to the existing D1 than to the D2 sequences, and contained almost the same amino acids as D1, which are responsible for binding the Mn4O5Ca cluster. The only predicted mutation was at position 170, where D replaces E (D170E)25. Another important point related to the evolution of OP is also the structural similarity of the Ca-binding site between the homodimeric type I reaction centre (RCI) of green sulphur bacteria and heliobacteria and the Mn-binding site with the D/E residues in PSII37. Taken together, all these common structural features and the data based on molecular phylogeny provide further evidence for an early evolution of PSII and OEC.The D1/D2 proteins are homologues formed by duplication of the original gene, and this duplication must have occurred long before the appearance of cyanobacteriapsbA and psbD in cyanobacteria and cyanophages, one can ask whether it can not only be a rough model for the evolution of cyanobacteria and cyanophages, but whether it can also give an indication of how the evolution of viruses has proceeded in general. In the case of cyanophages and D1/D2 proteins, the results obtained indicate that the primordiality hypothesis of viruses in relation to their cellular hosts can be considered41. Our phylogenetic data showed that both the cyanophage and cyanobacterial D1/D2 proteins are deeply related, suggesting that the cyanophage and cyanobacterial ancestral sequences of psbA and psbD arose at least at the same time. This also suggests close co-evolution of psbA and psbD between hosts and their cyanophages. Therefore, the data seem to fit, at least in part, the 'virus early' hypothesis42. However, a certain problem with this virus/cyanophage origin scenario is the question of the origin of the proteins that build their capsids. Viral capsid proteins have no obvious homologues among the proteins of modern cells44. An analysis of cyanophage capsid proteins is beyond the scope of this study. Nevertheless, following new comparative analyses of capsid proteins, it has been hypothesised that protoviruses, the first replicons or mobile genetic elements, originated with the first cellular forms, i.e. the last universal common ancestor (LUCA), but that the capsid proteins were recruited from later primordial cellular forms44. All analysed here cyanophages belong to the realm Duplodnaviria that possess double-stranded DNA (dsDNA) . Comparative analyses of viruses and their hosts suggest that Duplodnaviria-like dsDNA viruses may have evolved from a population of replicators in the pre-LUCA era, which later gave rise to dsDNA viruses and cellular forms39.As far as the origin of viruses is concerned, in connection with the phylogeny of psbA and psbD within them. Purifying selection for CDSs of psbA/psbD has already been described for the major groups of oxyphototrophs26. This fact, together with the conserved 3D structure of D1/D2 proteins predicted here, suggests that the ability to bind Mn4O5Ca clusters and to oxidise water may have arisen very early.At this point, a hypothetical scenario about the origin of viruses and cyanophages in particular is linked to the advent of OP. Cyanophages must have co-evolved with their hosts, as evidenced by the relatively abundant presence of psbA and psbD in the studied groups of oxyphototrophs and cyanophages suggest that D1/D2 are ancient proteins that may have formed the primordial PSII that could oxidise water at very early stages of the evolution of life on Earth.In summary, the D1/D2 proteins and the genomes of the cyanophages that code for them arose in very early stages of the evolution of cyanobacteria and must have been in close relationship with the cyanophages (co-evolution). The in silico reconstructed 3D structures of the D1 and D2 proteins and the purifying selection estimated for the CDSs of www.ncbi.nlm.nih.gov) and UniProt (uniprot.org) databases  and the consensus sequences were determined  and D2 (6dhe.1.D) for T. elongatus were used. The accession numbers, i.e.: 6w1p and 6dhe correspond to the 3D protein structures of PSII deposited in the Protein Data Bank (PDB) (https://www.rcsb.org). The selection was based on the fact that the 3D structures of the D1 and D2 proteins were determined by X-ray diffraction with a resolution of 2.26 and 2.05 Å, respectively49. The templates and the obtained models were pairwise alignment by EMBOSS needle software , UniProt (www.uniprot.org), Kyoto Encyclopedia of Genes and Genomes , and European Nucleotide Archive  databases for details see Supplementary Information Notes N) and synonymous (dS) nucleotide substitutions was determined in the analysed CDSs. The ratio of dN to dS (dN/dS) was calculated to estimate the type of selection. According to the dN/dS method, dN/dS = 1 means neutral evolution, dN/dS < 1 indicates purifying (negative) selection, and dN/dS > 1 means positive selection29. The sequences were aligned using the default parameters of ClustalW implemented in MEGA645. The dN and dS values were calculated using the software DnaSP v. 6.12.0350, then dN/dS values were determined.The molecular phylogenetic analysis was based on CDSs for Supplementary Information.


Furthermore, an unconfined compressive strength (UCS) test was performed on the backfill with the addition of ITs. Compared to the control group (without ITs), the UCS of backfill with 20% ITs increased from 1.08 MPa to 1.33 MPa, indicating that the addition of solid waste could be beneficial to the strength development of the backfill by mitigating the interference of phosphate with the hydration process. The backfill cured for 28 d was selected for the toxic leaching test, and the phosphate concentration in the leachates was always below 0.02 mg/L, indicating that ITs can effectively immobilize phosphate in backfill for a long time.Compared with the post-treatment of pollutants, such as the removal of phosphate from wastewater, it is more important to develop effective emission control strategies to reduce phosphate pollution. Phosphogypsum (PG) is a typical solid waste byproduct of phosphate production and contains high amounts of residual phosphate. In order to control the phosphate emissions during the recycling of PG aggregates for cemented backfill, another solid waste product—iron tailings (ITs)—was added during the preparation of backfill slurry. The results showed that the ITs effectively accelerated the phosphate removal in cemented PG backfill, enabling the quick reduction in the phosphate concentration to the discharge standard (<0.5 mg/L) within 15 min. This means that the emissions of phosphate to bleeding water were effectively controlled. The adsorption experiment showed that phosphate was adsorbed by the ITs, and the adsorption data fitted well with the Langmuir adsorption model  Secondary pollution should be fully considered in the recycling of solid waste . PhosphoAt present, there are several methods for removing phosphate, such as chemical precipitation, biological treatment, adsorption, and membrane separation ,16,17,18Iron tailings (ITs) are a kind of solid waste with a low content of valuable elements produced during the exploration of iron resources. It can be used for the production of road materials, building brick, trace-element fertilizers and concrete ,29,30,31However, most of the previous studies used ITs for the removal of phosphate from sewage, and there has been no research on the use of ITs for controlling phosphate pollution in cemented PG backfills. In fact, compared to the treatment of phosphate after the drainage of bleeding water, it is more meaningful to control phosphate emissions from backfill slurry. Therefore, this study aimed to study the feasibility of using ITs for the immobilization of phosphate in cemented PG backfills. With the addition of various amounts of ITs during the preparation of the backfill slurry, the adsorption rate and the adsorption mechanism were studied. In addition, the effects of ITs on the performance of backfills were tested by measuring the pH, initial setting time and final setting time, and unconfined compressive strength (UCS) of the backfill. Finally, toxic leaching tests were performed on the backfill after curing for 28 d to evaluate the long-term effects of ITs on the immobilization of phosphate.The ITs used in the present experiments were collected from a mine in Yunnan, China, and washed thrice with deionized water to remove the adhered dust and soluble substances. After washing, they were oven-dried for 24 h at 105 ℃. The PG and binder were collected from a phosphate fertilizer company in Guiyang, China. The PG was a moist gray–black powder with a pH of 1.9. The binder was composed of yellow phosphorous slag, fly ash and cement clinker in 6:1:3 ratio . The parThe diffraction patterns of the ITs and PG were characterized via X-ray diffraction (XRD) using a D8 X-ray automatic diffractometer  with a resolution of 0.02°/s in the angular domain 5–80°, and the results are shown in A scanning electron microscope equipped with an energy-dispersive X-ray spectroscope (SEM-EDS) was used to characterize the ITs and backfill. The microstructure and surface elemental content of the materials were analyzed using a TESCAN MIRA4 scanning electron microscope . 2(AlSi3O)(OH)2), Fe2O3 and Ca(MgFe)(CO3)2;. The XRF result shows that ITs , aluminum (Al3+), calcium (Ca2+), and other metal ions contained in ITs have an affinity for phosphate [According to previous studies, the reaction of functional groups on the surface of substances—caused by van der Waals forces—is the reason for the adsorption of the molecules . Therefothat ITs  containehosphate . TherefoTo investigate the change in phosphate concentration in the cemented backfill, the backfill slurry was prepared with the mixing proportions shown in 2PO4 at different concentrations  were added to the flask. At room temperature, the conical flask was placed in an oscillating device and vibrated for 24 h at 150 rpm HCl or NaOH was used to maintain the pH of the solution at 7. Then, the mixture was filtered through a 0.45 µm microporous membrane, and the filtrate was used to measure the phosphate concentration. The data were fitted to the Langmuir and Freundlich isotherm models to analyze the adsorption equilibrium parameters and adsorption behavior.In order to explore the phosphate adsorption process of ITs, adsorption isotherm experiments were carried out. First, 3 g of ITs was weighed and added to a 100 mL conical flask, and then 50 mL standard solutions of KH2PO4 solution with a phosphate concentration of 20 mg/L were added to a 250 mL conical flask. The pH of the mixed solution was maintained at 7 with HCl or NaOH. The conical flask was placed in an oscillating device at room temperature and shaken at 150 rpm. The supernatant of the sample was filtered after 1, 2, 4, 8, 12, 24, and 30 h. Then, the filtrate’s phosphate concentration was measured. The data were fitted to the pseudo-second-order model to determine the phosphate removal kinetics model.To study the adsorption kinetics, 3 g of ITs and 150 mL of KHIn order to explore the influence of ITs on the strength development of the cemented PG backfill, backfill with various amounts of ITs was prepared with the mixing proportions shown in In order to evaluate the impact of adding ITs on the mechanical properties of the backfill, UCS tests were performed for backfill samples cured for 7 d, 14 d, and 28 d. According to the JGJ/T 70-2009 standard , the tesCemented PG backfill is transported to underground stopes where it is in long-term contact with groundwater . Therefo43−-P), which was determined via the ammonium molybdate tetrahydrate spectrophotometry method. First, 100 µL of 10% ascorbic acid solution was added to 5 mL of the sample. After the mixture was shaken well, 200 μL of H8MoN2O4 was added in solution. After standing for 15 min, the solution was put into a Shimadzu UV1800 spectrophotometer  and measured at the wavelength of 700 nm. The pH of the backfill slurry was measured using a Mettler Toledo FE28 pH meter .The phosphate concentration measured in this study was the concentration of soluble orthophosphate  were 1.63 mg/L and 0.70 mg/L, respectively, which meant that the phosphate concentration in the bleeding water exceeded the discharge standard (<0.50 mg/L) . HoweverBased on the above results, during the preparation of the backfill slurry, adding sufficient ITs could quickly and effectively solidify the phosphate, resulting in the phosphate concentration in the bleeding water being below the discharge limit. This means that the post-treatment of wastewater could be avoided, indicating that adsorption by ITs in backfill preparation is an effective emission control strategy for phosphate pollution. This provides a new direction for the removal of phosphate from cemented PG backfill. Furthermore, this method achieves the disposal of waste using other waste, representing a reproducible approach for the large-scale recycling of solid waste.- due to the hydration reactions of the binder [2PO4− and HPO42− [e binder . When the binder . Anotherd HPO42− . When thd HPO42− . As showtQ is the amount of phosphate adsorbed in time t (mg/g), eQ is the equilibrium adsorption amount (mg/g), and K2 is the adsorption equilibrium rate constant of the pseudo-second-order equation (g(mg/h)).The adsorption kinetic model is usually used to estimate the adsorption rate, which is an important feature to judge the adsorption performance of an adsorbent. Pseudo-first-order and pseudo-second-order kinetic models have been widely used to describe adsorption systems ,47. AccoThe curve of the adsorption rate of phosphate by ITs was evaluated, and the results are shown in R2) of the pseudo-second-order kinetic equation is 0.99, indicating that the phosphate adsorption process of ITs is consistent with the pseudo-second-order kinetic model. This illustrates that the adsorption process is chemical adsorption and that there is a chemical bond between the ITs’ surface and the phosphate [The fitting of the pseudo-second-order model of ITs is represented in hosphate , and theeQ is the adsorption capacity of the adsorbent at adsorption equilibrium (mg/kg), mq is the adsorption capacity when the adsorbent reaches saturation (mg/kg), LK is the Langmuir equilibrium constant (L/mg), and eC is the concentration of the adsorbed ion when the adsorption in the solution reaches equilibrium (mg/L). In addition, from the Langmuir equation, a separation factor (LR) can reflect the feasibility of the adsorption process. This can be expressed as follows:C0 is the initial P concentration (mg/L). The correlation coefficient LR of the Langmuir equation is equal to 1, indicating that the adsorption is linear; when the LR value is less than 1, it means that it is favorable for adsorption, while LR > 1 indicates that it is not conducive to adsorption [The adsorption isotherm describes the adsorption processes at the solid–liquid interfaces and can be studied to calculate the adsorption capacity and explain the adsorption mechanism ,54. In tsorption .FK is the Freundlich constant (mg/kg) and 1/n is the adsorption strength parameter.The linear form of the Freundlich isotherm model can be expressed as follows :(4)logqeR2 = 0.985) is higher than the Freundlich correlation coefficient (R2 = 0.898), indicating that the Langmuir isotherm model is more suitable for the adsorption process of ITs. The adsorption of phosphate on Its mainly takes place in a single, uniform layer [LR was between 0 and 1, indicating that it is conducive to adsorption. According to the curve-fitting results, the equilibrium adsorption capacity of the ITs is 163.93 mg/kg, which is also close to the results of the adsorption kinetics.The fitting results of the experimental data based on the two adsorption models are shown in rm layer . In addi2O3 in ITs and includes ligand exchange, precipitation, and electrostatic adherence [2O3, Al3+, Ca2+, and other components in ITs may also promote the removal of phosphate through adsorption or precipitation to some extent [The adsorption isotherm and kinetics studies show that the adsorption process of phosphate on ITs is mainly monolayer chemical adsorption. Previous studies have shown that the adsorption process of phosphate on ITs is mainly attributable to the Fedherence . In addie extent . These ce extent . CombineThe changes in phosphate removal efficiency and the contribution of ITs were calculated, as shown in The development of the early strength has an important relationship with the setting time of the backfill. The initial and final setting times (IST and FST) of the backfill with various proportions of added ITs are shown in The strength of the backfill plays a decisive role in maintaining the stability of mines ,62. The The 28 d UCS of cemented PG backfill was also enhanced by ITs. As shown in 2+ and OH− derived from the hydration of the binder. When the ITs were introduced into the cemented PG backfill system, the phosphate in the PG aggregate was quickly and firmly adsorbed onto the ITs by ligand exchange, precipitation, and electrostatic adherence. Without the interference of phosphate, more Ca2+ and OH− could participate in the hydration on reaction and produce more hydration products to fill the gaps between aggregates, which benefited the strength development of the backfill. Macroscopically, the slurry was easier to coagulate, and the UCS of the backfill was effectively improved, as discussed above.In order to determine the distribution of phosphate in the backfill, EDS mapping was performed on the backfill without ITs and with 20% ITs, as shown in In order to explore the stability of phosphate immobilized by ITs, the backfill cured for 28 d was selected for toxicity leaching tests. With various proportions of ITs in the cemented PG backfill, the phosphate concentration in the leachates was always lower than 0.02 mg/L—far below the discharge standard (0.5 mg/L). This shows that the immobilization of phosphate in the backfill is not disturbed after adding ITs. Therefore, adding ITs to the PG aggregate can solidify the phosphate in the backfill for a long time, which is of great significance for environmental protection.After the PG aggregate was treated with the ITs as proposed in this research, the phosphate could be effectively immobilized within the ITs, rendering the aggregate harmless and useful. The stable existence of phosphate in the backfill also indicates that the impurities could be well immobilized during the preparation of the backfill, reducing the amount of phosphate released into the bleeding water and the leachates. This greatly reduces the costs of subsequent wastewater treatment. Furthermore, the immobilization of phosphate can enhance the strength performance of the backfill, with substantial significance for the recycling of solid waste.(1)The addition of ITs to the cemented PG backfill slurry effectively accelerated the removal of phosphate. When the proportion of ITs was 20% or more, the phosphate concentration in the bleeding water could drop to below the discharge standard (0.5 mg/L) within 15 min. The phosphate concentration in the toxic leaching solution of all test blocks was lower than 0.02 mg/L—far lower than the national standard (0.5 mg/L)—indicating that the addition of ITs would not affect the long-term environmental behavior of the backfill.(2)The linear correlation coefficient in the pseudo-second-order kinetic model was 0.99, indicating that the adsorption of phosphate by ITs takes place through a chemical adsorption process, and there is a strong bond between molecules. ITs are more consistent with Langmuir isotherm model, indicating that the predominant adsorption mode is monolayer adsorption. During the preparation of the backfill, the actual decrease in phosphate attributed to ITs was close to the theoretical values.(3)The addition of ITs positively affected the UCS of the backfill. With the addition of 20% ITs, the IFT and FST of the backfill were reduced by 35% and 33.9%, respectively, and the 28 d UCS was improved by 23.1%, compared to the control group. Furthermore, it is recommended to study the transportable properties of IT-based cemented PG backfill in the future.(4)Due to their low cost and good phosphate adsorption performance, ITs have the potential to be used in the preparation of cemented PG backfill to control phosphate pollution, providing new insights into the economical and efficient recycling of solid waste.In this study, ITs were used as additives to adsorb phosphate in the process of producing cemented PG backfill. The phosphate immobilization mechanism and the effect of the addition of ITs on the strength of the backfill were investigated. The following conclusions can be drawn:


To do so, a simple one-compartment model was used to describe the transport of a permeant across a membrane (skin). This model provides relatively simple expressions for the amount of permeant in the skin, the cumulative amount of permeant that crosses the skin, and the flux of permeant, for both the infinite and the finite dose regimens. Transdermal administration of the formulated NT gel to rats resulted in plasma levels of approximately 150 ng/mL between 8 and 30 h post-administration. These levels were higher than the minimum concentration of 40 ng/mL recommended for smoking cessation therapy and slightly higher than the upper limit of the therapeutic range for the treatment of depression in humans. The one-compartment model used to describe transport across the skin was connected to a two-compartment pharmacokinetic model used to predict NT plasma concentrations in rats using the outk determined in vitro and the values of other pharmacokinetic parameters obtained in vivo. The predicted concentrations were close to the observed plasma levels and the time profiles were similar for both types of data. These results show the usefulness of the outk parameter determined in vitro to predict plasma concentrations of drugs administered percutaneously.The aims of this study were to evaluate the feasibility of a nortriptyline (NT) formulation for transdermal administration and to assess the usefulness of an estimated kinetic parameter ( The transdermal route of drug administration has received a great deal of attention from researchers and drug manufacturing companies in recent years . TransdeRQ), can be calculated considering the measured concentration, the volume of fluid in the receptor chamber, and the exposed surface of the skin.In vitro studies measure the diffusion of the chemical of interest, or permeant, into and across the skin. To carry out these studies, Franz diffusion cells with two chambers separated by a piece of skin are usually employed. The formulation containing the permeant is introduced into the donor chamber and the concentration of the permeant in the fluid that fills the receptor chamber is measured at different sampling times. For each sampling time, the cumulative amount of permeant per unit area diffused across the skin, or amount at the receptor chamber per unit area  that show a curved profile when plotted vs. time  obtained at that pseudo-steady state, Equation (1) can be simplified as follows:In the above equations, ssJ):t-axis intercept is the lagT value  obtained by linear regression according to Equation (4) and the constant concentration of the permeant in the donor chamber.In this equation, the slope represents the steady-state flux , while ssC) will be reached.RQ vs. time derived from Fick’s second law of diffusion is much more complex than Equation (1) [maxJ) at time JmaxT (maxC) at some time (CmaxT) after the administration  , a maximme TJmax E is achistration F. ssJ, pK, and lagT, with a relatively simple interpretation, and the possibility of predicting a ssC value using the following equation: S is the surface of the skin exposed to the formulation and CL is the plasma clearance of the permeant. However, the in vivo scenario is often closer to a finite dose than an infinite dose regimen, with plasma concentration curves similar to that in The infinite dose regimen is employed more often in in vitro studies than the finite dose regimen because of its less complex mathematical treatment, its obtention of parameters, such as In addition to the diffusion models, compartmental pharmacokinetic models and physiologically based pharmacokinetic (PBPK) models have been used to describe the in vivo percutaneous absorption of different substances ,13,14.Nortriptyline (NT) is a tricyclic antidepressant drug that belongs to the class of non-selective monoamine uptake inhibitors. It is widely used in the treatment of unipolar depression  and therThe oral administration of NT is associated with some disadvantages, such as low oral bioavailability (between 30 and 50%) and side effects associated with plasma level fluctuations ,23. HencThe objectives of this study were to evaluate the feasibility of an NT formulation for transdermal administration and to assess the usefulness of the in vitro infinite dose technique to predict in vivo plasma levels of NT in rats percutaneously administered with the aforementioned formulation. To enable this, the experimental data obtained in vitro were analysed using a compartmental model to obtain the value of a kinetic constant for the diffusion of the drug from the skin to the receptor chamber of the diffusion cell. The value of this constant was combined with the pharmacokinetic parameters of NT obtained in rats to predict plasma concentrations, which were compared with those obtained after the in vivo administration of the formulation.A simple one-compartment model can be used to describe the transport of a permeant across a membrane (skin) that separates the donor from the receptor chambers in a diffusion cell .In this model, the concentration of the permeant is assumed to be homogeneous inside the compartment (membrane or skin), the entrance of permeant into the compartment from the donor is described by means of zero-order kinetics (constant-rate input) in the case of an infinite dose experiment A or firsMQ is the amount of permeant in the membrane per unit area, R0 is the constant rate per unit area at which the permeant enters the membrane, and outK is the first-order rate constant characterizing the permeant output from the membrane to the receptor chamber.The differential equation describing the change in the amount of permeant in the membrane with time when the permeant enters into the membrane at a constant rate is as follows:An equation describing the time course of the amount of permeant in the membrane can be obtain by integrating Equation (7) :(8)QM=R0R0 × t), the amount of permeant in the membrane (obtained with Equation (8)), and the cumulative amount in the receptor (RQ = R0t − MQ) provides the following equation:A mass balance considering the cumulative amount of permeant that enters the membrane  obtained in an infinite dose experiment in order to estimate the values of R0 and outk.Equation (9), which is simpler than Equation (1), can be fitted to the data  obtained at the pseudo-steady state, Equation (9) can be simplified as a straight line:R0 and the t-axis intercept is the value of 1/outk  in order to estimate the value of pK as the ratio of R0 to DC. Additionally, the inverse of outk can be interpreted in a similar way as lagT in Equations (1)–(4). lagT is related to the time to reach a certain fraction of steady state. In this way, the flux (J) after 2.7 times the lagT value has been estimated in about 97.5% of ssJ [J as a function of time according to the proposed model (When considering only the data (f 1/kout B. In theate flux C and caned model A:(11)J=RssJ is 3.7 times the 1/outk value.This equation can be used to deduce that the time required to obtain 97.5% of ink is the first-order rate constant for permeant input into the membrane. By integrating these equations, the following equations describing the time course of DQ and MQ can be obtained [Q0 is the dose, or initial amount, of permeant per unit area in the donor chamber. A mass balance considering the initial amount of permeant and the amounts at the donor chamber, membrane, and receptor chamber (RQ = Q0 − DQ − MQ) yields:First-order kinetics are considered in the model for both the input of permeant into the membrane and its output to the receptor chamber D. The seobtained :(14)QD=QThe cumulative curve described by Equation (16) is shown in RQ, t) or  in order to obtain the values of ink and outk.Equations (16) and (17) can be fitted to the experimental data (maxJ:maxJ can be obtained by replacing t in Equation (17) with the jmaxT value obtained with Equation (18).By making the first derivative of Equation (17) equal to zero, the following equation can be obtained for the time to achieve outk represents the same constant, which means that its value can be obtained with an infinite dose experiment and used later for simulations considering a finite dose administration, as described below.In both kinds of experiments—infinite and finite dose—® 80 (T80), oleic acid (OA), propylene glycol (PG), and 9-fluorenylmethyl chloroformate (Fmoc-Cl) where purchased from Sigma-Aldrich . All other reagents were of analytical or HPLC grade.Nortriptyline hydrochloride (NT), hydroxypropyl methyl cellulose (HPMC), tweenMale Wistar rats (280–310 g) were used in this study, and the experiments were performed in accordance with the authors’ institutional rules for the use of animals in research. All animals were housed under standard conditions and had ad libitum access to water and a standard laboratory diet. Protocols for the in vivo studies using rats were approved by the Ethics Committee for Experimentation and Animal Welfare , approval code A1352991914316 (3 May 2013).The composition of the NT gel was (m/m): water (34%), PG (30%), ethanol (25%), NT (5%), OA (2%), T80 (2%), and HPMC (2%). For the gel preparation, HPMC was dispersed in water with the help of a stirrer for 12 h at room temperature. Then, a solution containing the corresponding amounts of PG, ethanol, OA, and T80 was added to the aqueous dispersion of HPMC and, finally, NT was added to that mixture, which was continuously stirred for one additional hour.2. NT gel (0.4 mL) was uniformly spread on the skin surface, the donor chamber was covered with parafilm®, and the cells were immersed in a water bath of 37 °C. To maintain infinite dose conditions, the gel was removed from the donor chamber and replaced with fresh gel of the same composition 9 h after starting the experiment. The receptor fluid was withdrawn (200 µL) and refilled with fresh medium at 3, 5, 7, 9, 24, 26, 28, and 30 h. The collected samples were analysed by HPLC [RQ values. Briefly, chromatographic separation was performed on a Waters Spherisorb ODS2 (4.6 × 250 mm) column at room temperature. The mobile phase consisted of a mixture of acetonitrile/water , delivered at a flow rate of 1 mL/min. Samples were derivatised with Fmoc-Cl and the fluorescence detector was set at excitation and emission wavelengths of 260 nm and 310 nm, respectively.Rat full-thickness skin was obtained from previously sacrificed rats. Their hair was cut with an electric trimmer and pieces of skin (about 25 mm diameter and 1 mm thick) were mounted between the halves of vertical Franz-type diffusion cells, with the stratum corneum facing the donor chamber containing the gel with the NT. The receptor solution being in contact with the dermal side was composed of phosphate buffer saline pH 7.4 (9 mL). The receptor solution was continuously stirred with a small magnetic bar. The area available for diffusion was 1.45 cm by HPLC  to deterRQ, t) using the NONMEM program  to obtain the values of the parameters R0 and outk.Equation (9) was fitted to the experimental data (2 (exposure area) was fixed to their skin with cyanoacrylate adhesive [v/v). The obtained NT plasma concentrations were plotted against the sampling times and the area under this curve extrapolated to time infinity (AUC) was determined using the trapezoidal rule [appCL) was calculated as the quotient between the administered NT dose and the AUC value.To study the in vivo skin permeation of NT, five rats were cannulated in the jugular vein , the haiadhesive . NT gel adhesive . At the dal rule . The appoutk constant obtained in the in vitro skin permeation experiments, and a ink value estimated using Equation (12), the administered dose, and the amount of NT remaining in the gel determined in the in vivo skin permeation experiments. The pharmacokinetic parameters of NT in the rats administered intravenously were obtained by fitting, using the NONMEM, a two-compartment model to the plasma concentrations obtained in a previous study [The plasma concentrations of NT were simulated in rats percutaneously dosed with the formulated NT gel using the pharmacokinetic parameters of NT in the rats administered intravenously, the value of the us study .A graphic representation of the pharmacokinetic model used for the simulation of NT plasma concentrations is shown in R0 (or ssJ) and outk were 229 ± 13 μg/cm2/h and 0.10 ± 0.01 h−1, respectively . Considering that the NT concentration in the donor chamber was 50,000 µg/mL, a value of pK = 0.0046 cm/h was obtained for the NT in rat skin when the formulated gel was applied.The pharmacokinetic parameters of NT obtained by fitting a two-compartment model to the plasma concentrations obtained after the intravenous administration of 0.5 mg are shown in ink value of 0.065 h−1 by means of Equation (14). Additionally, the appCL of the NT in these rats was 3126 mL/h; this value being higher than the CL value obtained in the rats administered intravenously  was 3% of the initial amount, which made it possible to estimate an in vivo venously . To simuoutk, an additional simulation was performed, which excluding this parameter in the model . This simulation provided an NT plasma profile far from the experimental concentrations , and a value of 4.7% was obtained. This predicted value was in good agreement with the value of 5.2% obtained experimentally.Equation (15) was used with the above values of The transport of permeants through the skin has traditionally been described by equations derived from Fick’s laws of diffusion. Although these equations are based on very sound concepts, their complexity makes them difficult to apply to the analysis of data obtained from in vitro experiments using infinite or finite dose regimens. A simple equation describing the experimental data can only be obtained if a steady state in the diffusion process is reached during an infinite dose experiment (Equation (4)).t with (t − t0), where t0 represents the time delay.In this work, a simple one-compartment model has been used to describe the transport of a permeant across a membrane (skin), which provides relatively simple expressions for the amount of permeant in the membrane, the cumulative amount that crosses the membrane, and the flux of permeant, for both the infinite and the finite dose regimens. A limitation of the model and derived equations is that no delay in the amount of permeant crossing the membrane is considered, i.e., as the permeant reaches the membrane, a given fraction leaves it. However, an initial delay can easily be incorporated into the equations by replacing P(o/w) in the −1 to 4 range, and a melting point of below 200 °C [P(o/w) is 3.9–4.7, and the meting point is 213–215 °C (pubchem). These values are within or slightly outside the ideal ranges, which suggests a good percutaneous transport of this molecule. The in vitro evaluation of the formulated NT gel using rat skin showed a measurable transport of NT, with quantifiable concentrations in the receptor chamber 3 h after the start of the experiments. Fitting Equation (9) to the corresponding cumulative amounts of NT allows for estimating the value of outk, in addition to the parameters usually determined in infinite dose experiments (ssJ and pK). In the proposed one-compartment model, the rate constant kout has the same meaning for both the infinite and finite dose regimens (ink and disposition parameters) to simulate the plasma concentrations of NT after the transdermal administration of a finite dose.Some ideal physicochemical properties proposed for transdermal drug delivery are: a molecular weight of below 400 g/mol, a log w 200 °C . In the regimens , and, coThe transdermal administration of the formulated NT gel to rats resulted in plasma levels of approximately 150 ng/mL between 8 and 30 h post-administration. These levels are higher than the minimum concentration of 40 ng/mL recommended for smoking cessation therapy  and sligCL) than distribution  processes. Therefore, the values of the distribution parameters determined in rats from the previous study, in which intravenous administration was applied, were used in the simulations, but the CL value was replaced by the appCl value obtained from the rats of this study. In order to use the model shown schematically in ink is required, but unlike outk, its value cannot be estimated from the results obtained in vitro using an infinite dose regimen. However, this value can be easily estimated in vitro by means of Equation (14), if the amount of permeant remaining in the percutaneous formulation is determined at some time after the start of administration. Finally, a good agreement was obtained between the simulated and observed plasma concentrations of NT (outk was not included in the model.The apparent plasma clearance of NT in rats administered percutaneously (3126 mL/h) was higher than the plasma clearance determined in rats administered intravenously in a previous study (1580 mL/h). Although part of the discrepancy between both values could be attributed to metabolic degradation of NT in the skin, this difference is probably due to the fact that both studies were performed at different times, separated by several years, and reflects the fact that different populations of rats were used. In addition, the plasma levels of NT obtained with intravenous administration were higher than those obtained with transdermal administration, which could cause some degree of saturation in the elimination of NT and, consequently, a decrease in plasma clearance. The differences between rat populations are expected to have a greater impact on elimination  and (13) or the integrated Equation (16), which describe drug entry into systemic circulation, can be combined with the differential equations of a typical two-compartment model that describes the drug disposition. Nevertheless, further studies with other molecules are needed to assess the general validity of the described approach.This work shows the potential usefulness of the


It is important to take care of a properly balanced amino acid composition in the diet in order to inhibit or delay the occurrence of processes and changes related to the destruction of nervous tissue. Therefore, an attempt was made in this manuscript to evaluate the effect of different ratios of the key amino acids arginine and methionine, relative to lysine, in relation to two turkey feeding standards. The amino acid guidelines formulated by British United Turkeys (BUT) suggest higher levels of lysine (Lys) in turkey diets than those recommended by the National Research Council (NRC). In order to assess the impact of such supplementation, we analyzed the level of indicators informing the presence or degree of advancement of neurodegenerative processes in the nervous tissue , indicative of the destruction of neurons). The level of low-density lipoprotein receptor-related protein 1, or LRP-1, which facilitates the breakdown of toxic amyloid-β, was also assessed. In addition, the effect of different doses of these amino acids on neurodegenerative changes in DNA, especially the degree of methylation of histone proteins resulting from covalent modifications was compared between lysine and arginine residues.We postulated that the use of optimal levels and proportions of Arg and Met relative to a low or high concentration of Lys in diets for meat turkeys would reduce the occurrence of metabolic disturbances in the nervous tissue that can lead to neurodegenerative changes. The aim of the study was to determine the effect of various proportions of Lys, Arg, and Met in diets for turkeys, with a low content of Lys in accordance with NRC (Experiment 1) recommendations, and in diets with high Lys levels that are close to the recommendations of breeding companies (Experiment 2) on selected indicators of potential neurodegenerative effects in the brain and liver of turkeys. The Experiment 1 and Experiment 2 was conducted using 864 day-old turkey chicks randomly assigned to six groups, in eight replicates (6 groups × 18 birds × 8 replicates). A full description of the methodology can be found in previously published papers using the same experimental design. Indicators informing about the presence or advancement of neurodegenerative processes in the nervous tissue were determined in the brain and liver . It was established that in the case of both a low  and a high  level of Lys in the diet of turkeys, the Arg level can be reduced to 90% of the Lys level and Met to 30% of the Lys level, because this does not cause neurodegenerative changes in turkeys. Unfavorable neurodegenerative changes may appear if the Arg level is increased from 100 to 110% of the Lys level recommended by the NRC. However, due to the lack of such a relationship when Arg is increased from 100 to 110% of the Lys level recommended by BUT, at this stage of research no definitive conclusions can be drawn regarding the risk of neurodegenerative changes caused by increasing Arg in the diet of turkeys. A diet with an appropriate amino acid profile plays a key role in exploiting the genetic potential of fast-growing turkeys . The maiAccording to Toue et al.  and KolaAccording to Rajagopal et al. , Arg stiLysine is another amino acid that is essential for the growing turkeys, and a Lys deficiency or defects associated with catabolism of this amino acid can result in neurodegenerative and other physiological changes . NutritiDiets for turkeys with a high Arg content (110% Lys) were found to be unfavorable, due to the risk of lipid oxidation, protein nitration, and adverse changes in the concentrations of hormones regulating metabolism, especially of sugars . Due to A further suggestion to explore the question of the effect of different levels and proportions of lysine, arginine, and methionine in diets for meat turkeys was the findings of other researchers, who evaluated the rearing efficiency, as well as biochemical and immune parameters of the blood, intestinal morphology, dressing percentage, and meat quality ,31,32. AWe found no reports in the world literature of research conducted in poultry in the context of neurodegenerative changes. Because Arg, Met, and Lys are essential amino acids for poultry and determine the biological value of fodder protein, it seems useful to observe such changes, especially when we attempt to improve the growth performance of turkeys by changing the proportions of these amino acids. We took an interest in potential neurodegenerative changes after an in-depth analysis of the results obtained by the co-authors of this study in their research on growth performance and the indicators of sugar metabolism, peptide nitration, lipid oxidation, and systemic antioxidant defenses ,3,22,23.Due to the fact that the nervous tissue plays a superior role in the functioning of the system, any interference leading to the improvement of its condition is justified, provided that the portion of amino acids used does not constitute a toxic dose, and we tried to avoid this problem by assessing the impact of individual proportions within the acceptable standards for poultry. We considered that, due to the fact that two different nutritional standards for poultry  that are commonly used in poultry farming give slightly different values for the nutritional recommendations of Arg, Met, and Lys, and that these amino acids limit the biological value of the dietary protein in poultry and may affect the metabolism of nervous tissue in a different way, it is important to balance them carefully and in our opinion there is a need for a thorough verification of this problem.We postulated that the use of optimal levels and proportions of Arg and Met relative to a low or high concentration of Lys in diets for meat turkeys would reduce the occurrence of metabolic disturbances in the nervous tissue that can lead to neurodegenerative changes. The aim of the study was to determine the effect of various proportions of Lys, Arg, and Met in diets for turkeys with a low content of Lys, in accordance with NRC  recommenThe experiment was conducted using 864 day-old turkey chicks randomly assigned to 6 groups, in 8 replicates (6 groups × 18 birds × 8 replicates). A full description of the methodology can be found in previously published papers using the same experimental design ,3,23,30.The experiment was conducted using 864 day-old turkey chicks, which were randomly assigned to 6 groups, in 8 replicates (6 groups × 18 birds × 8 replicates). A full description of the methodology can be found in previously published papers using the same experimental design ,24. DuriAt the end of the experiment the birds were weighed after an 8 h feed deprivation, and one bird from each replicate representing the group average BW was selected and euthanized after electrical stunning. Birds were then hung on a processing line and bled out for 3 min by a unilateral neck cut, severing the right carotid artery and jugular vein. The non-edible viscera, including the intestines, proventriculus, gall bladder, spleen, esophagus, and full crop, were manually excised after scalding at 61 °C for 60 s and defeathering in a rotary drum picker for 25 s. After removing the head, the brain and liver were collected for further analysis.The activity of acetylcholinesterase (AChE) was determined in homogenates of the brain tissue (Experiments 1 and 2) and liver tissue (Experiment 2) of turkeys using the Chicken Acetylcholinesterase ELISA Kit , and the amyloid-β level was determined using the Chicken Total β amyloid Protein (βAP) ELISA Kit . The levels of glycosylated acetylcholinesterase (GAChE) in the brain (Experiments 1 and 2) and liver (Experiment 2) of the turkeys were determined using the Chicken Glycosylated Acetylcholinesterase ELISA kit . Low-density lipoprotein receptor-related protein 1 (LRP1) in the homogenates of brain tissue (Experiments 1 and 2) and liver tissue (Experiment 2) were determined using the Chicken Low Density Lipoprotein Receptor Related Protein 1 ELISA kit . The level of phosphorylated Tau protein was determined using the Chicken Phosphorylated tau 231 (p Tau231) ELISA kit . The levels of epigenetic changes in the brain (Experiments 1 and 2) and liver (Experiment 2) were determined by analyzing global DNA methylation (methylome), using diagnostic kits from Sigma Aldrich.p < 0.05, and statistical calculations were performed using the GLM procedures of the STATISTICA software system ver. 12.0 .This experiment was performed in a completely randomized 3 × 2 factorial design, and the data (presented as the mean ± standard error of the mean) were subjected to a 2-way ANOVA to examine the effect of 3 levels of Arg  and 2 levels of Met (30 and 45%). The Shapiro–Wilk and Levene tests were applied to test the model assumptions of normality and homogeneity of variance. When a significant interaction effect was noted (F test), treatment means were separated using the post-hoc Tukey’s test. The significance level was set at p = 0.019), amyloid-β (p = 0.032), and Tau protein (p = 0.002) and a decrease in the level of LRP 1 (p = 0.035) in the brain of the turkeys. Decreasing Arg content to 90% of the Lys level caused a decrease in AChE (p = 0.019) in the brain. Increasing the Met content from 30 to 45% of the Lys level  [p = 0.05) was noted: when the intermediate Arg level (100% of the Lys level) was applied, increasing the Met content from 30 to 45% of the Lys level caused an increase in the content of Tau protein, which was not noted in the cases of the lowest and highest Arg levels used (90 and 110% of the Lys level) (Differences in Arg content relative to Lys  caused n p = 0.03 in the bs level) .p < 0.001, both) and the lowest percentage of DNA methylation (p < 0.001) in the brain (p = 0.047) in the brain. Increasing the Met content from 30 to 45% of the Lys level [p = 0.042), as the amyloid-β level in the brain decreased when the Met content was increased from 30 to 45% of the Lys level while maintaining the 1:1 Arg to Lys ratio, which was not observed in the case of the reduced Arg content (90% Lys) or increased Arg content (110% Lys). The two-way ANOVA showed an Arg × Met interaction for Tau protein in the brain (p = 0.035); in the case of the lowest Arg level (90% of the Lys level), increasing the Met content from 30 to 45% of the Lys level reduced the level of Tau protein, which was not observed in the case of the intermediate and highest Arg content  (p = 0.022) in the liver. The two-way ANOVA showed Arg × Met interactions for GAChE and amyloid-β in the liver . The interaction for GAChE resulted from the fact that, in the case of the highest Arg content (110% of the Lys level), increasing the Met content from 30 to 45% of the Lys level caused an increase in GAChE in the liver, which was not observed in the case of the intermediate and the lowest levels of Arg . In the case of amyloid-β, the interaction resulted from the fact that when the lowest Arg level was applied (110% of the Lys level), increasing the Met content from 30 to 45% of the Lys level caused a decrease in the level of amyloid-β in the liver, which was not observed in the case of the intermediate and lowest levels of Arg  (Turkeys that were fed diets with the highest Arg content (110%) relative to Lys  had the he brain . In compys level  had no ectively) . Differectively) . In compctively) .Due to the lack of similar studies in the world literature in animal models or data on human patients, it is difficult to compare our results on neurodegenerative changes in turkeys fed diets with different proportions of arginine and methionine relative to lysine. It is also difficult to explain the more specific mechanism of physiological reactions and changes in the biochemical parameters, due to the diametrically different effects in the two experiments, depending on the level of Arg.In Experiment 1, where the Lys content of turkey diets was based on NRC  guidelinAmong the pathological changes taking place in neurodegenerative diseases, in both humans and laboratory animals, the first symptoms are the excessive deposition of amyloid-β in the brain and neurofibrillary degeneration in the form of hyperphosphorylated Tau protein. According to Całyniuk et al.  Arg promAccording to Li et al. , amyloidThe decrease in the AChE level following the use of 90% Arg relative to Lys  may sugg2+ in the cell, stimulating calmodulin-dependent protein kinase II (CaMKII). Activation of CaMKII in turn stimulates the highly unfavorable phenomenon of hyperphosphorylation of Tau protein. These processes together can lead to oxidative stress, to which nervous tissue is particularly sensitive. This is because neurons use up large amounts of oxygen and have the highest mitochondrial activity of all cells of the body. An increasing amount of research indicates a relationship between oxidative stress of the nervous system and neurodegenerative diseases [2+/calmodulin, and its coenzyme is NADPH+ H+. The efficiency of this enzyme depends on an appropriate concentration of Ca2+ ions in the neuron, and excessive calcium inhibits the activity of nitric oxide synthase. Interestingly, research by Keith et al. [Pathological amyloid-β, produced by fragmentation of its precursor APP (amyloid precursor protein), which is a component of the cell membrane of neurons, is present in tissues in the form of insoluble clusters, and is toxic for nerve cells, called amyloid-β ,38. The diseases . In the diseases , which wdiseases ). The lidiseases ,50. In tdiseases , arginindiseases  showed th et al.  shows thh et al.  report th et al. . Accordi+ H+, which is used for the synthesis of fatty acids or cholesterol. A high concentration of cholesterol stimulates the production of amyloid-β from APP by activating β-secretase, which is much more efficient in a cholesterol-rich environment. It may be that a 110% proportion of Arg relative to Lys [According to Butterfield et al. , neurodee to Lys  is condue to Lys . As repoe to Lys  the neure to Lys  argue the to Lys . Accordie to Lys  argininee to Lys  urea proNeurodegenerative changes in liver cells are also associated with disturbances of mitochondrial function. On the one hand, toxic substances for neurons are produced, and on the other hand, processes that are associated with energy acquisition for neurons are disturbed . In our 2+ and a non-physiological elevation of the intracellular concentration of this ion in the nerve cell. This leads to a reduction in the rate of oxidative phosphorylation and the ATP level in the neuron [2+ and a non-physiological elevation of the intracellular concentration of this ion in the cell, which leads to a decrease in the rate of oxidative phosphorylation and the ATP level. Disruption of cellular respiration is conducive to the opening of mitochondrial mega channels, which can lead to the release of cytochrome C from the intermembrane space and activation of caspase 3. The disruption of cellular homeostasis leads to apoptotic cell death [2+ and Zn2+ ions. This results in oxidative stress and a decrease in ATP production in the neurons, which in combination with disturbances of Ca2+ metabolism induces a number of pathological changes leading to the death of neurons [Unfortunately, as the highest GAChE level was noted in the liver of turkeys from the treatment using 100% Arg and 45% Met  . AccordiAccording to Sharma , the decTo verify the hypotheses put forward in this paper, further research should include other useful indicators of neurodegenerative changes, especially levels of APP, crucial enzymes , calcium/calmodulin-dependent protein kinase type IV (CAMK4) and glucose 6 phosphate dehydrogenase), calcium, vitamins, especially B6 and B12, thyroid hormones, and biomarkers of inflammation and oxidative stress (cytokines and isoprostanes). Microscopic examination may prove useful as well (observation of plaques formed of Tau protein and clusters of amyloid β).It was established that in the case of both a low (NRC)  and a hiAmong the tested proportions of Lys and Arg in the diet of meat chickens, the safest variant is the higher level of lysine recommended by BUT  and an i


Compaction of hot mix asphalt (HMA) requires high temperatures in the range of 125 to 145  High temperature is required for the construction of hot mix asphalt (HMA), which is typically 145 to 165 mixtures .The most widely used temperature reduction technology is the Warm Mix Asphalt (WMA), which reduces the production and compaction temperatures of HMA by 20–40 °C ,4. The t°C ,6. Organ°C ,8. Foami°C ,9. Numer°C ,14,15,16In previous studies, the authors have developed two different approaches for improving the compactability of asphalt mixtures, which are (1) the addition of Graphite Nanoplatelets (GNPs) ,18,19,20The objective of this study is to explore the effects of the two methods (adding GNP and optimizing aggregate packing) and the combination of them on reducing compaction temperatures. It is important to note that, although the GNP modification could also lead to a reduction of the mixing temperature, the focus of this study is only on the reduction of compaction temperature. Therefore, the mixing temperature is maintained at the traditional level in this study. The produced mixtures are therefore in between HMA and WMA, which can be classified as the hot-mixed warm-compacted (HMWC) mixtures, meaning that the mixtures are mixed at the same temperature as HMA but compacted at a lower temperature than HMA . SimilarIn addition, a new method is proposed to determine the compaction temperature. Most of previous methods assume that compaction is governed by the viscosity of asphalt binder and therefore determine the compaction temperature by the viscosity–temperature relationship of asphalt binder ,28. For In this study, a new method for determining the compaction temperature is proposed based on characterizing the compactability of mixtures by the gyratory compaction. The density at 30 gyrations  of GNPs to asphalt binder can result in 20% to 40% reduction in the compaction effort required for achieving the target air voids, 130% increase in the flexural strength of asphalt binders, and almost 100% increase in the fracture energy of asphalt mixtures. Meanwhile, other mechanical properties of binders and mixtures, e.g., rutting, stiffness, fatigue, and rheology, are not adversely affected by the GNP modification ,18.The physical mechanisms of GNPs on improving the compactability of asphalt mixtures have been investigated in the past years ,20. It wMicroscopically, it is believed that GNPs occupy the space between the asperities of the aggregates as shown in In a recent study on developing the Superpave 5 asphalt mix designs in Minnesota , a methodesign = 60) and was modified to a Superpave 5 mixture (5% air voids at Ndesign = 30) by this method. The mixture contains five aggregate sources and two reclaimed asphalt pavements (RAP). The aggregate gradations of the aggregate sources are plotted in One mixture from a previous study  is used mm, which represents a 1.08% increase compared to the original mix design. Therefore, the aggregate gradation of the Trial 2 (C/F = 1.33) was selected for the modified mix design.Trial blends were designed by adjusting the C/F ratio of the aggregates while keeping the binder content (5.6%) and the RAP contents (20% for R-1 and 10% for R-2) unchanged. The aggregate gradations of the Trial blends are shown in mm and 95.25 %Gmm, respectively. The performance test results are shown in Laboratory performance tests were conducted to compare the mechanical properties of the modified and the original mixtures. They are the Semi-Circular Bending (SCB) , Flow Nudesign = 60. The aggregate gradation of the Mix-C is shown in The traditional Superpave asphalt mixture mentioned in 2/g. The same GNPs have been used in previous studies [Based on the raw materials of the Mix-C, three modified mixtures were designed by (1) GNP modification, (2) optimizing aggregate packing, and (3) a combination of GNP modification and optimizing aggregate packing. The first modified mixture is denoted as Mix-GNP. It modified Mix-C by adding 6% GNPs by the weight of binder to the mixture. The GNPs used in this study are made of a synthetic graphite material with 99.66% carbon and 0.34% ash, characterized by an enhanced surface area equal to 250 m studies ,18,19,20The second modified mixture was designed by optimizing aggregate packing. Details of this approach is described in The third modified mixture was designed by combining the two former approaches. It contains 6% GNPs while it has the optimized aggregate gradation of the Mix-Agg. This modified mixture is denoted as Mix-GNP&Agg.Laboratory gyratory compaction tests (AASHTO T312)  were perThe gyratory compaction curves of the four mixtures at the two compaction temperatures  data of the project. The required field density level of Superpave 5 mixtures is 95 %Gmm, which is about 1–2% increase compared with the traditional Superpave mixtures [Another way to evaluate the compactability of mixtures is by the corresponding number of gyrations needed for achieving a certain density level. In this study, two density levels are of particular interest: (1) the average field density of the control mixture (Mix-C) and (2) the required field density level of the Superpave 5 mix design. The average field density of the Mix-C was 93.9 %Gmixtures ,49. Basemm), Mix-C needs 28 gyrations at the recommended compaction temperature . This result shows that, at 135 mm (requirement of the Superpave 5 mix design) with the regular field compaction effort. It is seen from A method is proposed for estimating the compaction temperature, which is based on the direct characterization of the compactability of asphalt mixtures by the gyratory compaction. A schematic diagram of this method is depicted in T and T and In this study, gyratory compaction tests were performed at two temperatures: 95 onlinear ,52,53,54tability ,51,54. HT and The linear approximations of the relationships between T and mm and 95 %Gmm, which is the average field density of the control mixture (Mix-C) and the required field density level of the Superpave 5 mix design, respectively. As shown in mm, the compaction temperature for Mix-C is interpolated as 128.3 mm, the compaction temperatures for Mix-GNP and Mix-Agg are interpolated as 124.2 Based on the obtained linear models between mm, the compaction temperatures are lower than 95 mm, Mix-C need a compaction temperature higher than 135 As shown in T and T-It is worthwhile to discuss the potential ways for improving the accuracy of compaction temperature estimation. One possible way is by increasing the number of experimental compaction temperatures, which would enable the characterization of the nonlinear relationship between In this paper, two approaches for improving the compactability of asphalt mixtures were introduced. One is the addition of Graphite Nanoplatelets (GNPs), and the other is the optimization of aggregate packing. An experimental study was performed to estimate the effects of the two approaches and their combination on reducing compaction temperatures. A method for determining compaction temperature was proposed based on the gyratory compaction. The main conclusions of this research are summarized as follows.1GNP modification, aggregate packing optimizing, and their combination can significantly improve the compactability of mixtures and thus reduce the compaction temperatures.2The combination of GNP modification and aggregate packing optimization has the most significant temperature reduction effect, followed by GNP modification, and aggregate packing optimization.3The three methods can reduce the compaction temperature of the original mixture by more than 30%, which is comparable to the temperature reduction effect of WMA.4The proposed method for compaction temperature estimation is based on the direct characterization of the compactability of asphalt mixtures. It can take into account many affecting factors which were not possible to consider with the traditional viscosity-based method, for example, the properties of aggregates and the lubricating effect of binder. Thus, the method can potentially be used in practice for estimating the compaction temperatures for asphalt mixtures.While the results presented in this proof-of-concept study are based on limited experimental data, it is reasonable to conclude that the proposed two methods (GNP modification and Aggregate packing optimization), and their combination, have good potential as practical methods for reducing the compaction temperatures of asphalt mixtures. It was also observed that the variability of the compactability of mixtures increased with the decrease in the compaction temperature, which implies that compaction temperature would influence the reliability of field compaction. Such an effect was not considered in the compaction temperature estimation in this study. It is recommended that future studies can consider the effect of compaction temperature on the reliability of field compaction. Moreover, it is seen from this study that one of the main difficulties for the compaction temperature estimation is the lack of knowledge on the relationship between the compaction temperature and the compactability of mixtures. To improve the accuracy of compaction temperature estimation, future research is suggested to explore the relationship between the compaction temperature and the compactability of mixtures, using a combination of experimental investigation and theoretical modeling.