The intra-competition among tumor subpopulations is a promising target to modify and control the outgrowth of the resistant subpopulation. Adaptive therapy lives up to this principle well, but the gain of tumors with an aggressive resistant subpopulation is not superior to maximum tolerated dose therapy (MTD). How to integrate these two therapies to maximize the outcome? According to the model and system reachability, the ‘restore index’ is proposed to evaluate the timing of the transition from the treatment cycle of adaptive therapy to high-frequency administration, and to juggle the benefits of intra-competition and killing of the sensitive subpopulation. Based on the simulation and animal experiment, the effectiveness of this method in treating tumors with an aggressive resistant subpopulation has been confirmed.Adaptive therapy exploits the self-organization of tumor cells to delay the outgrowth of resistant subpopulations successfully. When the tumor has aggressive resistant subpopulations, the outcome of adaptive therapy was not superior to maximum tolerated dose therapy (MTD). To explore methods to improve the adaptive therapy’s performance of this case, the tumor system was constructed by osimertinib-sensitive and resistant cell lines and illustrated by the Lotka-Volterra model in this study. Restore index proposed to assess the system reachability can predict the duration of each treatment cycle. Then the threshold of the restore index was estimated to evaluate the timing of interrupting the treatment cycle and switching to high-frequency administration. The introduced reachability-based adaptive therapy and classic adaptive therapy were compared through simulation and animal experiments. The results suggested that reachability-based adaptive therapy showed advantages when the tumor has an aggressive resistant subpopulation. This study provides a feasible method for evaluating whether to continue the adaptive therapy treatment cycle or switch to high-frequency administration. This method improves the gain of adaptive therapy by taking into account the benefits of tumor intra-competition and the tumor control of killing sensitive subpopulation. Adaptive therapy (classic adaptive therapy) ,3,4,5,6 The classic adaptive therapy regimen consists of repeated treatment cycles, the duration of the treatment cycle generally includes a period of administration and withdrawal ,3,5. TheHence, it is meaningless to maintain intra-competition in the later period of classic adaptive therapy. At this time, killing a large number of sensitive cells like MTD may increase the outcome of classic adaptive therapy. To maximize the outcome of competitive stress and killing of the sensitive subpopulation, the timing to bridge these two therapies should be chosen wisely. Therefore, the control degree of competitive stress on resistant subpopulations should be quantitatively evaluated. In this way, the timing of treatment switching from the treatment cycle of classic adaptive therapy to high-frequency administration of MTD can be explored effectively. Then system reachability would be introduced to assess the degree of control. In this tumor system, system reachability mainly relies on the proportion of sensitive subpopulation, because it is easily regulated by drugs, while the resistant subpopulation can only be indirectly regulated by sensitive subpopulation. Therefore, an indicator of reachability should be constructed that is associated with the two participants and can predict the duration of each treatment cycle. Based on the purpose of integrating classic adaptive therapy and MTD, the threshold of the reachability indicator would be explored in the study to evaluate the timing of treatment switching, and the feasibility of this reachability-based adaptive therapy would also be verified.In this research, the fitness cost for the resistant subpopulation of tumor system was avoided, because this property does not always exist in resistant subpopulations, unless the resistant mechanism is mediated by energy-consuming methods, such as multidrug resistance transporters ,9. Then Human lung adenocarcinoma cells (H1975 and A549) were purchased from the National Collection of Authenticated Cell Cultures  and confirmed to be mycoplasma-free by Gmyc-PCR kit . All cell lines were cultured in RPMI-1640 (Gibco (Thermo Fisher Scientific), Waltham, MA, USA, cat# 21870076) containing 10% fetal bovine serum  and 1% Penicillin-Streptomycin (5000 U/mL) .In terms of drug sensitivity, H1975 is sensitive to osimertinib, and A549 is resistant to osimertinib. In addition, H1975 contains EGFR T790M mutation, A549 contains KRAS G12S mutation. H1975/GFP (green fluorescent protein) cells, expressing GFP to track cells, were transfected by eGFP-puromycin resistant lentivirus  and selected with 0.5 mg/L puromycin to maintain the expression of label proteins. A549/RFP (mCherry fluorescent protein)/luc (firefly luciferase) cells, expressing RFP and firefly luciferase, were transfected by RFP-Luciferase-puromycin resistant lentivirus  and selected with 1.6 mg/L puromycin  to maintain the expression of label proteins.The nutrition restriction model was used to demonstrate one aspect of the competition between subpopulations in vitro. To obtain the growth curve, 24-well plates containing 1:1 mixed H1975/GFP and A549/RFP/luc, the corresponding single cell line as control groups were detected under high-content imaging analysis system Opera Phenix  from 24 h after cell planting. The number of cells was calculated by the Image Analysis and Evaluation module of high-content analysis software Harmony  every 12 h . As for Animal experiments were carried out in compliance with the Chinese law on animal welfare. All animal care and experimental procedures were approved by the animal ethics committee of the Pharmaceutical Animal Experiment Center of China Pharmaceutical University. 5 to 7-week-old female BALB/c nude mice  were allowed to acclimatize (in groups of 3–4 mice per cage) for at least 7 days prior to the experiment and were maintained on a 12-h light:12-h dark cycle with free access to standard rodent chow and water.6 cells per mouse) into the left forelimb flank of mice. Firstly, mice were inoculated with H1975 or A549 to fit the tumor growth curve. Then, mice inoculated with H1975 or A549 would be given osimertinib 20 mg/kg/day to fit the parameters of the drug effect. After that, H1975 and A549 were mixed at a ratio of 9:1 and then inoculated to get the competition parameters. The content of H1975 and A549 in the mixed tumor was obtained by In vivo imaging every 3–4 days. Tumor volume was measured by caliper two to three times a week, and calculated according to the formula In this experiment, mice were inoculated with different cells to obtain the data for fitting. The cell suspension was inoculated subcutaneously (~5 × 106 cells per mouse), the mixed ratio of H1975 and A549 was 9:1 before inoculation. Because the sensitive subpopulation should occupy a high proportion at the beginning of therapy, then several rounds of treatment cycles can be carried out. When tumor volume reached 220–300 mm3, mice were randomly grouped according to tumor volume and weight. Tumor volume was measured by caliper two to three times a week and the A549 content in the mixed tumor was collected sparsely. As for the content of H1975, because the duration of in vivo imaging of H1975 for each mouse was more than half an hour, for animal welfare considerations and to ensure the physical condition of the animal, the content of H1975 was not collected at this stage.To compare various therapy protocols, mice were inoculated subcutaneously with mixed tumor cells  was used to evaluate the content of each subpopulation of the mixed tumor through in vivo imaging, and the data was processed using Living Image software . Serially diluted H1975/GFP or A549/RFP/luc cells were cultured in a 96-well black plate with a transparent bottom. This was used to build a quantification database of bioluminescence and fluorescence intensity with cell numbers. And for luminescence imaging, D-Luciferin potassium salt  should be given to mice 12 min in advance by intraperitoneal injection at a dose of 150 mg/kg body weight. After that, fluorescence imaging tomography or diffuse luminescence imaging tomography was acquired to reconstruct a fluorescent or luminescence source in 3D space and calculate the absolute intensity of that source at depth [When tumor volume was about 200 mmat depth , then weLotka-Volterra model was used to describe the competition of subpopulations . The tumequation , vector H and A represents the content of H1975 and A549, The detailed model of normal growth condition was as following equations ,3, the mThe above model-related parameters were estimated by Monolix 2019R2 . The fitting results of parameters contained typical value and inter-individual variability. The typical value was the mean of the population parameter and the inter-individual variability was the variance of the parameter distribution. The optimization method of parameters was the maximization of the likelihood function to obtain the appropriate values by stochastic approximation expectation-maximization algorithm . The modic plots ,17.Before the start of the first treatment cycle of classic adaptive therapy and reachability-based adaptive therapy, the observations of tumor volume and single point of A549 content were collected. Based on this data, the individual estimates were obtained from individual empirical Bayes estimates assessed by fitting to the population model.In reachability-based adaptive therapy, there was an indicator of system reachability for evaluating whether to interrupt the treatment cycle. The process of finding the threshold of this interruption point was divided into two steps, the first step was to find the range of the interruption point, and the second step was to find the appropriate value.The first step was to get the treatment plans of reachability-based adaptive therapy that were not inferior to the classic adaptive therapy through simulation. The regimen of reference classic adaptive therapy was shown in the following Treatment protocols. The tumor composition at the beginning of the simulation was the average value of the mixed tumor samples of the animal experiment of model fitting, and the biological related parameters were the typical value of parameters representing the characteristics of the experimental animals. In the simulation of reachability-based adaptive therapy, the start time of the first treatment cycle was set to different time points, and the treatment cycle was set to 3 times. In each treatment cycle, the duration of drug administration and withdrawal was set at different lengths of time. In each treatment cycle, we can skip the drug withdrawal part and continue to administer it to achieve the purpose of interrupting the treatment cycle in advance. The above simulation treatment plans with the same or better outcomes as the reference classic adaptive therapy were screened out to obtain the range of the threshold.Then, each integer in the above range was used as a threshold to simulate the outcomes of reachability-based adaptive therapy under different conditions. The different condition means the intrinsic growth rate of resistant subpopulation was defined as 50–150% relative to the typical value of H1975, carrying capacity was defined as 80–120% relative to the typical value of H1975, and competition parameter was defined as 0–200% relative to the typical value of H1975. Finally, the value that maximizes the performance improvement of tumors with aggressive resistant subpopulation and minimizes the similarity to classical adaptive therapy was selected as the threshold.Combined with the administration of each sample in the experiment, GUN Octave (version 6) simulated the changes of tumor volume and A549 content of each sample over time with individual parameters.3, the end event was set as tumor volume reaching 1000 mm3 [The starting time point of each therapy was calculated from the tumor volume reaching 220–300 mmControl: Normal saline with 3% Tween 80 was given after grouping.3.Positive control: Osimertinib was given 20 mg/kg/day when tumor volume was as close as possible to 1000 mmMTD: Osimertinib was given 20 mg/kg/day after grouping. The MTD is aimed at eradicating all cancer cells . Moreove3. Then the treatment cycle consisted of two days of osimertinib administration and drug withdrawal until the tumor volume was as close as possible to 1000 mm3. If the tumor volume does not decrease with two days of osimertinib administration, osimertinib would not be withdrawn.Classic adaptive therapy (AT1): To maximize the use of tumor intra-competition, the first treatment cycle of adaptive therapy started from the tumor volume as close as possible to 1000 mm3 with two days osimertinib administration, the treatment cycle would be interrupted and osimertinib would not be withdrawn. As for the reason for 100 mm3, it was explained in the results.Reachability-based adaptive therapy (AT2): The start of the treatment cycle was the same as AT1. However, if the tumor volume reduction was less than 100 mmt-test every time point by SPSS and visualized by ggplot2. And survival analysis of different treatments was used pairwise comparisons between group levels with corrections by the survminer package. The acceptable level of significance set at p < 0.05; p-values were shown in figures as *: p < 0.05, **: p < 0.01 and ***: p < 0.005, ****: p < 0.0001, N.S.: not significant.In all figures, the data presented were representative of at least 3 independent experiments. Data were expressed as the mean ± SEM and statistically analyzed using the R (3.6.3) and SPSS (version 21.0). The significance analysis of growth curve among different groups used multivariate analysis of variance followed by a post-hoc Bonferroni test, drug effect, and effect of the conditioned medium used analysis of The competition between subpopulations mostly occurs under resource-constrained conditions, such as space and nutrition constraints ,20,21. HThe difference between the growth curves of the H1975 and A549 normal single (NS) group and dilution single (DS) group indicated that H1975 was more susceptible to nutrition restriction than A549, and A549 was more susceptible to space restriction. The influence of space restriction was mainly reflected in the end part of the growth curves of H1975_NS and A549_NS. When the two cell lines were mixed at a ratio of 1:1 under a normal culture medium, H1975 still maintained the advantage of obtaining space resources. C showed 3, the composition of each tumor subpopulation of mixed tumor-bearing nude mice was evaluated, and the subpopulation volume was converted from the number of cells according to In vitro experiments confirmed that these two cell lines compete for space and nutrition when resources are limited. And tumor cell lacks space and nutrition in vivo exactly ,23,24,25The established model was shown in Equations ,3,4. TheThe model described the volume dynamics of H1975 and A549 in single and mixed conditions well. The ic plots ,17. As sTo present an overview of each therapy for tumors that mix H1975 and A549, we simulated the subpopulations variation over time in MTD, AT1, and AT2, as shown in . For AT2According to the model, a parameter ‘restore index’ was introduced for better integration the adaptive therapy and MTD. In this system, it is calculated as following equation . The numThe range of restore index to interrupt the treatment was determined through the first step of estimation the appropriate threshold of restore index for reachability-based adaptive therapy. The plans of AT2 that achieved the same or better outcomes than AT1 were screened out. The restore index changes with therapy time of the above plans were depicted in the left subplot of C. The or3 after 2-days of osimertinib administration, the treatment would transition to the high-frequency administration.According to the second step (see methods part), each integer in 6–23 was used as the threshold of restore index to interrupt the treatment cycle for testing. Finally, the threshold was set to 15, and its selection was based on maximizing the improvement of the poor outcome AT1 case and choosing a larger threshold to minimize the similarity between AT2 and AT1 D. When AMice inoculated with H1975 and A549 mixed in a ratio of 9:1 were randomly assigned to AT1, AT2, MTD, Control, and Positive control groups. The growth data before grouping was used to calculate the individual parameters, which was applied to the simulation of tumor volume and A549 content versus therapy time. These simulation results could further verify the model. For AT1 and AT2 groups, three samples from each group were presented in A. The bl3, and A549 was set to 20 mm3, and the result was shown in The purpose of reachability-based adaptive therapy was to improve the outcome of AT1 when the tumor has an aggressive resistant subpopulation. To further clarify the applicable fields of AT2, we simulated the outcomes of MTD, AT1, and AT2 in the cases of resistant subpopulation A549 with different values of intrinsic growth rate, competition parameter, and carrying capacity. The change percentage of each parameter was relative to the typical value of the corresponding parameter, and the parameters of sensitive subpopulation H1975 were fixed at the typical value. In addition, the initial volume of H1975 was set to 180 mmIt has been studied those factors such as the initial fraction of resistant subpopulations, the proximity of the tumor to carrying capacity, the intrinsic growth rate of a resistant subpopulation, and the rate of cellular turnover affect the time gained of adaptive therapy compared to MTD . These fIn this research, we introduced a new method that considers the benefits of the treatment cycle and high-frequency administration. To find the switching point of the above two treatments, the restore index that takes into account the response of sensitive subpopulation to the drug and the expansion speed of resistant subpopulation is proposed to measure the ability of the drug to regulate the state of the tumor system at the end of each treatment cycle. This is an approximation of the system reachability of this non-linear tumor system. It is found that the restore index can predict the outcome of each treatment cycle through the simulation. Thus, the restore index can evaluate whether to continue the treatment cycle or switch to high-frequency administration.3 in the animal experiment of AT2. In addition, when tumors have more than two subpopulations, they can still be divided into sensitive and resistant subpopulations in terms of drug sensitivity. At this time, the reachability-based adaptive therapy can be further explored for its applicable fields.The switching point plays an important role in the outcome of reachability-based AT. At the switching point, if the treatment cycle continues, there are not enough sensitive cells that can be killed during the high-frequency administration period, then the benefits of MTD cannot be realized. However, if the treatment cycle is interrupted prematurely, the intra-competition cannot be fully utilized, resulting in reduced outcome of reachability-based AT. Therefore, an appropriate switching point can maximize the benefits of both, and the restore index is an effective tool to find this switching point. In addition, the correlation between restore index and the tumor response to the drug was found through the simulation. This correlation converts the calculation of the potential restore index during the therapy into a direct observation of treatment response. Thus, the treatment would transition to the high-frequency administration when tumor volume response of 2-days osimertinib administration was less than 100 mmWhen we confirmed the in vivo competition of H1975 and A549, mice inoculated with mixed tumors were given osimertinib for 7 days. Based on the experimental results, the comparison between the observed volume of H1975 and the simulated volume without A549 protection was shown in Reachability-based adaptive therapy could improve the performance when the tumor has an aggressive resistant subpopulation; this therapy maximizes the outcome of intra-competition and the killing of sensitive subpopulations by the restore index.


T, Cu, Zn, CN−, and SCN− were 94.14, 94.53, 98.14, 98.55, and 93.13%, respectively. The main reaction in anode was the electroly oxidation of CN− and SCN−, while the electrolytic deposition of Cu, Zn, and other metal ions in the cathode surface. There were not only adsorption and electric adsorption of various ions, but also an electrolytic deposition reaction of Cu, Zn, and other metal ions on the surface of the activated carbon particle electrode. During the electrochemical reaction, the concentration of hydrogen ions near the anode increases locally, which produces the precipitation of CuSCN, Cu2Fe(CN)6, and Zn2Fe(CN)6, etc. in the solution, which are helpful for the removal of cyanide and heavy metal ions in cyanide wastewater.The electrochemical treatment of wastewater is widely used for cleaning due to its efficiency. In this paper, two-dimensional (2D) and three-dimensional (3D) electrochemical systems were used to treat cyanide wastewater. The effect of the applied voltage and the material of the main electrode on the removal of various ions and the characteristics of chemical reactions were mainly studied. The results show that the applied voltage was the key effect of the electrochemical treatment process. The removal of ions from the wastewater at 2 V is mainly due to the effect of electro adsorption and enrichment precipitation, while at 4 V, it is mainly due to anodization and cathodic deposition. The treatment effect of the 3D electrode system was significantly better than the 2D system. The 3D electrode system by used granular activated carbon as the particle electrode, with the carbon filled stainless mesh (CM) and coal based electrode (CB) as the main electrode, the treatment effect were better than main electrode of stainless steel mesh (M). The 3D system with CB as the main electrode had an applied voltage of 4 V, a treatment time of 5 h, plate spacing of 10 mm, and the dosage of activated carbon particles was 2 g. The removal rates of CN Cyanide wastewater treatment has been the subject of research due to the difficulty of treating its complex composition, which contains a large number of free cyanide and copper, zinc, iron metal cyanide complex ions. Three-dimensional (3D) electrode electrochemical system treatment techniques are used to remove organic material and heavy metal ions from industrial wastewater of chemical, metallurgical, and material industries  coal-based electrode (CB) and stainless steel mesh (M) electrode. Commercial activated carbon particles were used as the particle electrode. The electrochemical treatment of cyanide wastewater by 2D and 3D systems and the effects of electrode materials and applied voltage on the removal law of various ions in cyanide wastewater and its mechanism was studied.The 304 stainless steel mesh (M) used in the experiment was from a steel product factory in Jiangsu Province. The filler and the particle electrode were commercial coconut-shell activated carbon. The low rank pulverized coal and liquefaction residue was from a coal chemical factory in Yulin, Shaanxi Province. The cyanide wastewater was from a gold smelter in Henan Province, and its main composition is shown in Fifty milliliter cyanide wastewater was accurately measured taken into the electrolytic cell. The electrode plates were, respectively, connected with the positive and negative electrodes of the DC power supply. The plates were connected with wires plugged into the solution in parallel with 10 mm intervals. The experiment under different applied voltages, for a treatment time of 5 h, and the dosage of particle activated carbon in the 3D system was 2 g. At the end of the experiment, the solution was treated by liquid-solid separation, and several ions concentrations were regularly analyzed. The precipitates and plates were repeatedly washed with distilled water until pH 7 was reached, and then dried for analysis and characterization. The connection diagram of the experimental device was shown in Yao et al. . In the T and CN− in wastewater were analyzed by chemical titration, the concentration of Cu and Zn were analyzed by AA-1800H Atomic Absorption Spectrometry, and the thiocyanate ion was determined by 723PC Spectrophotometer.The surface morphology of the electrode material and the composition of the loading material were analyzed by JMS-6390Lv Scanning Electron Microscope (with energy-dispersive IE300X). The composition of the precipitations was analyzed by X-ray Diffractometer (D8 ADVANCE A25). The concentration of CNT, Cu, Zn, CN−, SCN− at 4 V were all about 20% higher than those under 2 V. In addition, the treatment effect of the CM electrode was better than the CB electrode and M electrode, but the overall treatment effect was not good. When the main electrode was M, the removal rate of ions in 2 V was generally low and the highest removal rate was only 28.14% (CN−). The removal rate of all ions was greatly improved at 4 V, but only maintained levels at about 35%. This may be due to the small contact area between the electrode and wastewater. When CM and CB were used as the main electrodes, the removal rates of each ion in the 2D system reached more than 62% under 4 V conditions, and the removal rates were significantly improved. When the main electrode was CM, the removal rates of CNT, Cu, Zn, CN−, and SCN− were 69.11, 62.17, 65.45, 74.43, and 64.14%, respectively.Treatment cyanide wastewater used the main electrodes of M, CM, and CB, respectively, under 2 and 4 V conditions by the 2D system. The removal rates of several main ions were shown in T, Cu, Zn, CN−, and SCN− were 94.14, 94.53, 98.14, 98.55, and 93.13%, respectively.Treatment cyanide wastewater in the 3D system used various main electrodes under the conditions of 2 and 4 V. The removal rate of several main ions is shown in Compared with the 3D system, the 2D system had a low treatment efficiency and was similar to the reaction process of the main plate with the 3D system. Therefore, the SEM-EDS analysis of the main plates and particle electrodes in this paper focused on the 3D system.+ and resulted in the hydrolysis of OH− near the anode. This was caused by the precipitate reaction of H+ with complex ions and SCN− in the solution, mainly including CuSCN, CuCN, Zn(CN)2, Cu2Fe(CN)6, Zn2Fe(CN)6, Cu3Fe(CN)6·4H2O, Zn(OH)2, and Cu(OH)2. The removal of ions in wastewater at 2 V was thus caused by the interaction of electric adsorption and anodic precipitation.When the voltage was 2 V, gas was produced at the anode. Oxygen evolution reaction occurs at the anode due to the electrode terminal voltage reaching the decomposition voltage of water. White precipitate appears near the anode, which was due to the high concentration of H− → CNO− → CO2 + N2, and SCN− mainly occurred and there was a small amount of hydrolysis side effects.− at 2 V takes place, which then adsorbs on the cathode.− and Fe ion generated complex which was deposited on the cathode at 4 V. However, in the spectrum of the CB electrode, the higher Cu content was mainly based on electrodeposition, while the Cu content in CM was relatively low. On the one hand, only a small part of activated carbon in the cathode was used for detection and analysis, on the other hand, this depends on the resistance and the conduction efficiency of CM.2Fe(CN)6, Zn(OH)2, and CuCN. The difference is that the precipitate contains a large amount of Zn2Fe(CN)6 when the electrode was CM. This is because the iron in the stainless steel mesh would be dissolved in the solution and combine with the cyanide ion to form a large number of iron cyanide complexes, and precipitate with zinc in the solution to generate a large amount of Zn2Fe(CN)6.To study the reaction mechanism furtherly, the precipitates in the solution under different electrode systems and voltages were analyzed by XRD. The XRD analysis results of the precipitates at 2 V are shown in 2Fe(CN)6 in the precipitates of the CM system was high. This is mainly caused by the Fe dissolved in the solution. The precipitation of the CM system was much larger than that of the CB electrode, which proves that under the condition of 4 V, due to the influence of the CM material's resistance and conduction efficiency, the actual voltage in the solution system may not reach the deposition voltage of Cu and Zn. It could also be because only a small amount of metal separates, so the precipitation reaction of Cu and Zn mainly occurs in the solution. However, the electrodeposition reaction of metal mainly occurs in the CB electrode system, the quantity of precipitate was smaller.The XRD of the precipitates at 4 V is shown in According to the research results above, which used CM and CB as an anode and cathode, and granular activated carbon (GAC) as particle electrode, when cyanide wastewater was treated by the 3D system under different voltage conditions, the ions in the solution interacted by adsorption, directional migration, enrichment precipitation, and electrolytic deposition. Under the action of different voltages, the reactions in the solution were different. The main mechanism is shown in 2 in the anode, and caused a large amount of H+ to gather near the anode, and complex precipitation reaction 6, Zn2Fe(CN)6, and so on. The iron in the CM electrode dissolution also leads to the formation of Zn2Fe(CN)6 with the iron cyanide complex and zinc ion in the solution. The reaction equations were as follows:At a voltage of 2 V, the actual voltage in the system exceeds the decomposition voltage of water, which produced O− and SCN− were converted into CNO− firstly, and then CO2 and N2 were mainly generated. At the same time, water decomposition reaction also exists in the anode and cathode [formulas ]. The reaction of the particle electrode is shown in the figure, under the action of the electric field the oxidation-reduction reaction occurs at both ends of the particle electrode  The results of the present study show that the 3D electrode system with activated carbon filled stainless steel mesh (CM) and coal-based electrode (CB) are better than stainless steel mesh (M) as the main electrode. The CB electrode is especially suitable for cyanide wastewater treatment. The main mechanism of the system of 2 V is electro adsorption and enrichment precipitation. At 4 V, the system mainly uses electrodeposition to remove all ions in wastewater, and the treatment efficiency of electrodeposition was higher than that of electro adsorption.T, CN−, and SCN− in wastewater were 94.53, 98.14, 94.14, 98.55, and 93.13%, respectively. The surface of the CB anode has an oxygen evolution reaction and electrochemical oxidation reaction of CN−, SCN−, and there is electrolytic deposition of Cu, Zn, and other metal ions on the cathode surface. There was adsorption and electro adsorption of various ions on the surface of the activated carbon particle electrode, and the electrolytic deposition reaction of Cu, Zn, and other metal ions at the same time.(2) The treatment efficiency of the 3D system is higher than that of the 2D system because of the addition of the particle electrode the reaction efficiency and removal rate of ions in wastewater improve greatly. When the applied voltage of the 3D system is 4 V, with a treatment time of 5 h, an electrode spacing of 10 mm, and the dosage of activated carbon particles is 2 g, the removal rates of Cu, Zn, CN−, SCN− and metal cyanide complex ions will occur under the action of the electric field, generating precipitation of CuSCN, Cu2Fe(CN)6, Zn2Fe(CN)6, which is the main reason for the removal of cyanide and heavy metals ions from cyanide wastewater.(3) In the process of cyanide wastewater treatment by 3D electrode based on CB electrode, due to the concentration of hydrogen ion increase in the local area near the anode caused by oxygen evolution reaction, the precipitation reaction between CNThe original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author.YS: conceptualization, methodology, writing—reviewing, and editing. SL: data curation, writing—original draft, software, and formal analysis. Both authors: contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A performance evaluation criterion (PEC) is defined to characterize the performance based on both friction factor and heat transfer. It was found that increasing the number of semi-twisted tapes increases the number of swirl flow streams and leads to an enhancement in the local Nusselt number as well as the friction factor. The average Nusselt number increases from 15.13 to 28.42 and the average friction factor enhances from 0.022 to 0.052 by increasing the number of the semi-twisted tapes from 0 to 4 for the Reynolds number of 1000 for the base fluid. By using four semi-twisted tapes, the average Nusselt number increases from 12.5 to 28.5, while the friction factor reduces from 0.155 to 0.052 when the Reynolds number increases from 250 to 1000 for the base fluid. For the Reynolds number of 1000, the increase in nanofluid concentration from 0 to 3% improves the average Nusselt number and friction factor by 6.41% and 2.29%, respectively. The highest PEC is equal to 1.66 and belongs to the Reynolds number of 750 using four semi-twisted tape inserts with 3% nanoparticles. This work offers instructions to model an advanced design of twisted tape integrated with tubes using multiple semi-twisted tapes, which helps to provide a higher amount of energy demand for solar applications.The hydrothermal performance of multiple semi-twisted tape inserts inside a heat exchanger pipe is numerically examined in three-dimensions. This study aims to find the optimum case for having the highest heat transfer enhancement with the lowest friction factor using nanofluid (Al The increasing energy demand will require improving the energy efficiency of heat transfer applications ,2. Due tInserts have been attracted lots of attention, though not regarding their effect on the characteristics of the employing fluid ,14. TherLim et al.  investigThere are limited studies in the literature incorporating a higher number of twisted tape inserts in a tube in the forms of separated and joint twisted tapes . DalkiliNanofluid has been recently widely employed in various heat transfer applications to improve the thermophysical properties of the working fluid toward higher heat transfer performance ,45,46,472O3/water nanofluid is used to improve the properties of the working fluid toward a higher heat transfer efficiency using a two-phase mixture model. Both the Nusselt number and friction factor are studied to provide a comparative study on the advantages of heat transfer enhancement and the disadvantages of pressure drop penalty. The effect of the Reynolds number is also investigated. This study points out some hydrothermal aspects, such as the tangential and radial velocities of the nanofluid, which has been rarely discussed in the literature. It should also be mentioned that, in this study, the use of one semi-twisted tape is introduced for the first time. This study provides guidelines to design an improved configuration of twisted tape inserted tubes using multiple semi-twisted tapes. The more efficient usage of twisted tape inserted tubes could help to provide a higher amount of energy transmission suitable for application in parabolic trough solar collectors.According to the presented literature review, there are limited studies on the use of multiple twisted tape inserts in a tube, especially in the presence of nanofluid. Furthermore, there is a limited comparative investigation on the performance of multiple semi-twisted tape inserts in a tube ,57,58. TThe schematic of the proposed system under investigation is presented in 2O3-water nanofluid enters the inner and outer tubes at a temperature of 300 K, considering four different Reynolds numbers. Alumina (Al2O3) is among the more common and cheaper nanoparticles utilized in different commercial and experimental applications and has been well reported by a number of researchers. The alumina/water nanofluid shows strong enhancement on the thermophysical properties of the base fluid [2O3 nanoparticles have other advantages, such as chemical and thermal stability and excellent dispersion properties in the base fluid [se fluid . Moreovese fluid .2O3-water nanofluid, neglecting the effects of radiation and viscosity losses. The two-phase mixture model [2O3 nanoparticles dispersed in water. The governing equations are defined as follows [Continuity equation:The present work will investigate a 3D steady-state laminar flow of incompressible Alre model  is emplo follows :ContinuiMomentum equation:ned from : (3)V→drEnergy equation:mV and mρ are given as:In Equation (5), the subscription k is related to the kth phase of the mixture. given as :(7)V→pf=The equation developed by Manninen  and ShilTherefore, the drift velocity is given as:2O3 nanofluid i.e., density [The properties of water-Al density ,65, heat density , effecti density , and eff density  are give2O3 nanofluid with three values of volume concentrations is used in the present study, as listed in A water-Al2. The twisted tape rotates at three different angular velocities of Constant temperature and velocity are used at the tube inlet, while zero relative gauge pressure is used at the outlet for the fluid. The channel wall is exposed to a uniform heat flux of 5000 W/mThe hydrothermal parameters investigated, along with the performance evaluation criterion (PEC), are defined as follows ,62:(16)DANSYS FLUENT software (version 18.0) is employed to resolve the conventional governed equations using a coupled algorithm to solve the velocity–pressure coupling, and a second-order upwind scheme is used to discretize the convection terms. The convergence criteria are set to 10-6 for both, whereas it is 10-11 for the energy equation.An overview of the computational grid is illustrated in To ensure the non-dependence behavior of the results from the computational grid, the average Nusselt number for various numbers of elements was examined. To obtain a reliable result, the experimental results of Qi et al.  for the The numerical simulations are conducted to evaluate the thermo-hydraulic performance of laminar swirl flow through the circular tube equipped with multiple semi-twisted tapes. The importance of the number of twisted tapes, Re numbers, and nanofluid concentrations are evaluated in this research.The reason for the behavior of Nu is connected to the flow behavior under different circumstances. The analysis of radial and tangential velocity provides details about the role of multiple semi-twisted tape addition on the intensity of secondary flow. As seen in To assess the thermal performance of multiple semi-twisted tapes at different Re numbers, the friction coefficient and average Nu number as a function of Re number is depicted in As a case in point, The effect of the number of semi-twisted tapes and Re number is investigated on the thermo-hydraulic performance of laminar swirl flow through the circular tube. This section is devoted to the importance of nanofluid addition in the considered configurations. As shown in It should be noted that because the variation of PEC is not linear, as shown in 2O3/water) flow in a 3D circular tube integrated with multiple semi-twisted tapes were investigated numerically. The range of nanoparticle volume fractions of 0, 1, 2, and 3%; the range of Reynolds numbers of 250, 500, 750 and 1000; and the number of the semi-twisted tapes of 0, 1, 2, 3, 4 were examined comprehensively. The computational results conclude that increasing the twisted tape number enhance both heat transfer and pressure drop; however, its effect on the heat transfer enhancement is more pronounced. The following key outcomes were achieved:The higher average friction factor is reached when a higher number of semi-twisted tapes are applied in the tube. The average Nusselt number, x, increases from 15 to 28.5 and the average friction factor enhances from 0.155 to 0.052 by increasing the number of the semi-twisted tapes from 0 to 4 for the Reynolds number of 1000 for the base fluid.Increasing the Reynolds number enhances heat transfer performance while it reduces the friction factor. By using 4 semi-twisted tapes, the average Nusselt number increases from 12.5 to 28.5, while the friction factor reduces from 0.155 to 0.052 when the Reynolds number increases from 250 to 1000 for the base fluid.Increasing the nanoparticle concentration results in a higher Nusselt number and friction factor. For this case at the Reynolds number of 1000, the increase in nanofluid concentration from 0 to 3% improves the average Nusselt number and friction factor by 13.24% and 3%, respectively.The highest PEC is 1.66 and belongs to the Reynolds number of 750 for the system using four semi-twisted tape inserts where the volume fraction of nanoparticles is 3%.Heat transfer improvement and friction factor characteristics of laminar nanofluid (Al


This work is about the study of the correlation of pore formation in welded joints of Al–MG–LI alloy with zirconium additives with the state of the base metal, thermal vacuum treatment, and welding technologies MIG and EBW. Metallographic analysis has been carried out, the phase composition of the alloy and weld metal has been investigated, and thermal cycles of welding have been calculated, allowing to estimate the residence time of metal in the alloying zone and weld metal in the liquid state. The nature of the allocation of strengthening fine-dispersed phases in the welded joints of the alloy has been determined. The regularity and character of pore formation in welded joints depending on the applicable thermal vacuum treatment (TVT) and welding technology have been revealed. It was established that TVT with subsequent hardening and aging has no effect on the phase composition of the alloy. However, this type of treatment contributes to the formation of a more homogeneous and uniform nature of the separation of fine-dispersed strengthening phases. It was revealed that the MIG technology  is characterized by a large length of the fusion zone, the high residence time of metal in the fusion zone and weld metal in the liquid state, and the formation of pores. Phase formation in the temperature range of the beginning and end of the alloy crystallization occurs not only in the weld at the final stage of crystallization but also in the fusion zone, which may induce pore formation, whereas EBW welding shows the opposite trend and no pores. It was found that EBW technology prevents pore formation and makes it possible to obtain welded joints of 1420 Al alloys of the required quality. Al–MG–LI alloys with zirconium additives are used in the aerospace industry and other areas of production due to their unique properties. For every 1% of lithium, the density of the alloy decreases by 3%, while Young’s modulus increases by 5%. The modulus of elasticity of aluminum, magnesium, and lithium are 7100, 4300, and 500 kg/mm3, and it is the lightest among aluminum alloys [Al–MG–LI alloys, despite the extremely low corresponding lithium characteristic, have a significantly higher modulus of elasticity, consequently, these alloys have an exceptionally high modulus of elasticity ,7. Due tm alloys .Both the most efficient and promising technological process for the production of monolithic components of various structures are welding. The weldability of 1420 aluminum alloy has several features associated with the physical, chemical, and technological properties of the material ,13,14,152O3·nH2O) and other impurities interact with the liquid metal of the weld pool and release hydrogen gas bubbles into the molten metal. Aluminum, which is characterized by high chemical activity, easily interacts with air oxygen and forms an oxide film [The moisture adsorbed on the porous oxide film in the form of variable aluminum hydroxide  recommended, as one of the solutions to prevent porosity in welds of Al–MG–LI alloys in thin welded constructions in the aviation industry, the use of the thermal vacuum treatment of material before welding. Thermal vacuum treatment is applied to change the composition of joints and reduce the concentration of hydrogen in the base metal, which will improve the quality of the initial material, reduce the amount of released hydrogen, and as a consequence, reduce pore formation.Hydrogen is the main cause of porosity in welding aluminum alloys. In the liquid state, aluminum dissolves hydrogen quite easily, which is due to the high rates of effective diffusion of hydrogen in liquid aluminum . The solThe pore-forming hydrogen can enter the weld pool not only from the metal that is welded but also from the welding materials and shielding gas . The rigElectron beam welding (EBW) is an alternative method for welding Al alloys of thin joints, which provides better weld quality. EBW is carried out by a highly concentrated energy source, in a vacuum chamber, which provides good degassing of the metal, and without the use of welding materials (welding wire and shielding gas).Although earlier studies of Al–MG–LI alloys welded joints have been conducted, the interaction between pore formation, thermal vacuum treatment, and welding technologies of Al–MG–LI alloys has not been investigated. Currently, a comparison between EBW and MIG welding of 1420 Al alloy is missing within the scientific literature. This study is aimed at substantiating the choice of welding technology in thin welded joints of Al–MG–LI alloys. The influence of base material, thermal vacuum treatment, and welding technologies on pore formation has been analyzed in welded joints of Al–MG–LI alloys.Two welding technologies were used to obtain experimental blanks with a thickness of 3 mm made of 1420 Al–MG–LI alloy with zirconium. Welding of thin plates of 1420 alloy with the different initial states was carried out by automatic argon arc welding (MIG) with an additive wire AlMg-6 and by electron beam welding (EBW). The average chemical composition of AMg-6 welding wire is shown in −3 mm Hg. Processing time is assigned depending on the thickness of the material. Cooling with furnace up to 80 °C, then in the air. (2) Followed by standard heat treatment T6. Thermal vacuum treatment of 1420 alloy workpieces was performed in the electric resistance chamber furnace of vacuum model ESCVE-16/16GM1 .Part of the samples before welding, after standard heat treatment T6, were subjected to thermal vacuum treatment (TVT) according to the following modes ,14,15,16Keller reagent  was used to reveal the microstructure on the sections. The research was conducted on an Altami CM0745-T optical stereomicroscope  and an Altami MET 1T inverted light microscope  with a magnification of up to 1000 times using Altami Studio 3.5 software . Hardness is measured by the Vickers method on microhardness PMT-3 at a load of 50 g. The microhardness was carried out along the welded joint along with the following directions: base metal, heat affected zone, welded seam, heat affected zone, base metal.Analysis of thermodynamic calculations of possible phases during equilibrium crystallization of the alloy (in coordinates of % phases from temperature) was made using the JMatPro the Materials Property Simulation Package the company Sente Software Ltd. . JmatPro is a simulation software which calculates a wide range of materials properties for alloys and is particularly aimed at multi-component alloys used in industrial practice. Using JmatPro allows calculations for: stable and metastable phase equilibria, solidification behavior and properties, mechanical properties, thermo-physical and physical properties, phase transformations, and chemical properties.The phase composition of the samples was studied using X-ray diffractometer XRD-7000. A monochromator was used to separate the Kβ component of the X-ray radiation during the analysis. Imaging conditions: scanning angle range 2 Theta from 30 to 90, X-ray tube—Cu, tube voltage 30 kV, tube current 30 mA, scanning speed 1 deg/min, scanning step 0.01 deg, slits DS 1 deg, SS 1 deg, RS 0.15 mm. Processing of X-ray images was performed using the software PANalytical X’Pert HighScore Plus Ver.2.1 . Determination of the phase composition of the analyzed samples was performed using the database “ICDD PDF-2+ 2012”.Calculated analysis of the Al–MG–LI alloy the phase composition after the standard heat treatment of the deformed metal and after TVT was carried out using the JMatPro program on the example of 1420 alloy. For the calculation, the average value of the main alloying elements was taken in accordance with OST 1-90048-90-90 .3Zr (AL3M_DO23)—formed at 815 °C in the liquid alloy; Mg2Si (MG2SI)—formed at 559 °C in the temperature range of the beginning and end of crystallization of the solid solution based on aluminum; Al2LiMg (TAU_ALLIMG)—begins to be released at 435 °C from the solid solution; Al3Mg2 (AL3MG2)—begins to be released at 190 °C from the solid solution.Al3Li (AL3M_L12) may be in the alloy at temperatures lower than 190 °C in addition to the stable phases  dissolve. After quenching, the alloy consists completely of solid solution-based aluminum, less than 1% of the non-dissolved phases Al3Zr (AL3M_DO23) and Mg2Si (MG2SI). As a result of artificial aging at 120 °C, the hardening stable phases Al2LiMg (TAU_ALLIMG) and Al3Mg2 (AL3MG2) and the metastable phase Al3Li (AL3M_L12) are released; the calculation of the release of this phase is shown in During the quenching process at 450 °C, the hardening phases Al2LiMg (TAU_ALLIMG), Al3Mg2 (AL3MG2), Al3Li (AL3M_L12).Thus, according to the calculated results, the main strengthening phases of 1420 alloy after T6 heat treatment will be AlCalculation of the thermal vacuum treatment simulation of the alloy showed that in the process of exposure at 450 °C for 6 h there is a significant alignment of chemical elements in the body of the grain . The homMetallographic analysis of the 1420 alloy metal (not previously subjected to TVT) showed a characteristic allocation of hardening phases of different shapes as shown in The metallographic studies of the alloy metal subjected to TVT were obtained as follows. In the alloy metal, there are both strengthening phases and a number of pores a,d. HoweThe calculations also showed that TVT with subsequent quenching and aging has no effect on the phase composition of the alloy. The calculations are confirmed by the results of qualitative X-ray phase analysis. Al, Cubic, spatial group Fm-3m (225) (№ 01-089-2837 in the database);2Mg, Cubic, spatial group Fd-3m (227) (№ 03-065-5657 in the database);Aluminum Lithium Magnesium AlLi8.9Li1.1, Cubic, spatial group Fm-3m (225) (№ 03-065-7533 in the database).Aluminum Lithium ALThe peaks in the diffractograms of the alloy are characteristic of the following compounds:2LiMg (TAU_ALLIMG), and the substance 03-065-7533 is close to the phase Al3Li (AL3M_L12). Other calculated phases with content less than 5% are practically not determined by the X-ray phase analysis.The chemical composition of the substance number 00-026-0042 is close to the calculated phase AlThe macrostructure investigation of welded joints was carried out on samples cut from the cross-section. The analysis of macrostructure studies showed that in the process of MIG welding pore formation is observed in the welded joints regardless of the initial state of the base metal. Pores are formed mainly at the boundary between the weld and the base metal. In the case of alloy welding after TVT, the pores are also present at the boundary between the layers . However2) is observed. Determination of the type of inclusions and pores was carried out using polarized light and temperature field illumination on an optical microscope. Volumetric and anisotropic phases such as intermetallic inclusions “glow” in the dark field, in polarized light they acquire a certain color and change their illumination. Silicate inclusions have a regular circular shape, in the light field concentric rings appear, and in the dark field and polarized light an optical cross effect is observed. Spherical pores in the volume of the material appear as dark spots, in the dark field and in polarized light a specific light figure is formed, the sharpness of which depends on the depth of occurrence [2)  .3Ti (AL3M_DO22), Al6Mn (AL6MN), Mg2Si (MG2SI), Al3Mg2 (AL3MG2). In this case, the two main phases (Al6Mn (AL6MN) and Al3Mg2 (AL3MG2)) are formed in the temperature range of the beginning and end of crystallization of the alloy. That is, these phases are formed not only in the weld at the final crystallization stage but also in the fusion zone. The formation of phases in the two-phase solid–liquid fusion zone can also initiate the formation of pores. The third hardening phase (Al3Mg2 (AL3MG2)) is formed at temperatures below 280 °C and is released inside the grain.Thermodynamic calculation of changes in the phase composition of the alloy as a function of temperature was made in the program JMatPro for the weld metal. The results are presented in Therefore, the use of TVT by MIG welding does not allow to improve the quality of welded joints to reduce pore formation.3Mg2 was released.EBW was carried out without additive material; thus, the metal chemical and phase composition will correspond to the base metal. The extent of the fusion zone in the heat-affected zone is considerably less in the welded joints made by the EBW than by the MIG welding. The fusion zone consists of 1–2 rows of fine equiaxed grains . The oveCalculation of the cooling time-temperature-transformational diagram was carried for 1420 alloy from 540 °C close to the temperature of the end of crystallization out to analyze the forming structure in the weld. According to the diagram shown in The microhardness has given the following results .As we can see from The hardness of the base material decreases more concerning the base metal when welded after TVT.V—welding speed, δ—thickness of the plate, τ—the times of the source, q—heat source power, η—coefficient of efficiency, c—heat conductivity, ρ—density of the metal, t0—operation time of the simulated heat source, depends on the concentration factor of the heat source.The formation of the welded joints structure, as well as the formation of pores directly depends on the temperature-time parameters of the welding process. To evaluate and compare the thermal cycles of arc and electron beam welding of thin welded joints of 1420 alloys, computational studies were carried out using a thermal model. A model of heating a semi-infinite plate by a normal-circular source, based on the differential equation of heat conduction in a moving coordinate system with a stationary source, and obtained by the analytical method using Green’s functions, was used for calculation:The instant cooling rate was defined as:dt = dx/V, where V is the welding speed), Equation (2) takes the following form:Given that in welding there is a relationship between the distance of the reference point from the heat source and time . The same instant cooling rate of the alloy zone metal and weld metal during crystallization at EBW is significantly higher than the cooling rate at MIG welding.2LiMg and Al3Mg2 and the metastable phase Al3Li are allocated.According to the results of the studies, the characteristics of the strengthening phases formation were revealed in the metal of the Al–MG–LI alloy with zirconium additives. In particular, it was found that in the metal without TVT there is an allocation of stable strengthening phases and one metastable phase. The nature of the allocation of strengthening phases in the metal with TVT and without TVT differs significantly. The content of chemical elements insignificantly changes in the released phases depending on temperature. In particular, after the standard heat treatment T6 the hardening stable phases Al3Li). The decomposition of the supersaturated solid solution in 1420 alloy occurs according to the scheme: α→ (Al3Li)→ S1(AL2MgLi). The phase (Al3Li) is an intermediate unstable phase that has a cubic Li2 lattice whose parameter is close to the lattice parameter of aluminum; thus, the precipitates (Al3Li) formed in the solid solution during cooling from the quenching temperature and during subsequent aging are completely coherent with the matrix and remain so in a wide range of aging temperatures. The second phase S1—the phase having a complex cubic lattice, is formed from—excretions or is isolated directly from the solid solution and is partially coherent with the matrix.From the literature data ,20,21, iAccordingly, thermodynamic calculations correlate with the literature data. In addition, the research shows that thermal vacuum treatment with subsequent heat treatment contributes to the alignment of the chemical elements along the grain body and fixation of homogeneous solid solution, which characterizes the uniform isolation of fine-dispersed strengthening phases, also confirmed by metallographic studies. The results of metallographic studies of welded joints showed not only the peculiarity of fine-dispersed phases allocation but also the nature of the formation and distribution of pores in the metal with TVT and without TVT. As known from ,13,16, pThe X-ray phase composition data correlate with the calculated results of thermodynamic analysis and show that TVT followed by quenching and aging has no effect on the phase composition of the alloy.In addition to the effect of hydrogen concentration in the base metal of aluminum alloys, both the ratio of alloying elements and the welding technology largely influence the quality of the weld, which determines the properties of the welded joint.6Mn and Al3Mg2) are formed in the temperature range of the beginning and end of crystallization of the alloy in the welds made by MIG. It was determined that the formation of these phases occurs not only in the weld at the final stage of crystallization but also in the fusion zone. The formation of phases in the two-phase solid–liquid fusion zone can also initiate the formation of pores. This fact can be correlated with the literature data, from which it follows that, when the crystallization front moves, gas accumulates in a narrow boundary layer of liquid metal. At a high saturation of the boundary layer of the liquid metal with gas, its concentration exceeds the standard solubility. During aluminum crystallization, the hydrogen available in it is completely fixed in the solid metal and then redistributed between the solid and liquid metal according to the laws of diffusion. In this case, the process controlling the transition of hydrogen to liquid becomes the diffusion of hydrogen in the solid phase [Research on the influence of TVT and welding technology on the formation of welded joints showed that pores form in MIG welds regardless of TVT. Pores were not detected in EBW welding. In addition, it was found that two main phases . Given the short residence time of the weld metal in the liquid state and high cooling rates at the time of crystallization at EBW, a homogeneous solid solution will be formed in the weld, which is confirmed by metallographic studies. The absence of pores in the fusion zone and weld metal at EBW is caused by processes of degassing of metal and a rigid thermal cycle of welding. At MIG welding, the formation of pores and large inclusions in the fusion zone are connected with the peculiarity of formation of strengthening phases in the liquid–solid state, its large extent, and a longer stay of metal in this state.Based on the results of the research on the interaction of base material, thermal vacuum treatment, and welding technology on pore formation in welded joints of Al–MG–LI alloys with zirconium additives, the following conclusions can be made.2LiMg, Al3Mg2, Al3Li is characteristic. In addition, calculations have shown that TVT with subsequent quenching and aging has no effect on the phase composition of the alloy, but the nature of the evolution of fine-dispersed strengthening phases becomes uniform.Thermodynamic calculations made it possible to determine the features of the formation of strengthening phases in the base metal with and without TVT. It was revealed that, in the metal after standard heat treatment without TVT, the formation of strengthening phases AlThe study of microstructure and calculations of thermal cycles of welded joints established that the MIG technology  is characterized by a large extent of the fusion zone, the high residence time of metal in the fusion zone, and weld metal in the liquid state, pore formation. As the weld pool cools, hydrogen atoms tend to be released due to a sharp decrease of solubility. However, hydrogen meets and combines with other hydrogen atoms, crystallization centers, and impurities in the metal recombine into molecules and form gas bubbles. These bubbles float as long as the ductility of the surrounding metal allows. Gas bubbles that do not resurface in time remain in the metal after it has crystallized, forming porosity, whereas in EBW welding, the opposite tendency is observed with the absence of pores.A more uniform distribution of hardness was revealed in the welded joints made by EBW. However, application of TVT before welding leads to a more uniform hardness of the weld metal, in both cases of welding.EBW technology prevents pore formation and makes it possible to produce welded joints of 1420 Al alloys of the required quality.


The purpose of this study was to analyze the light-dark variations in the concentrations of several neurotransmitters in the lumbar spinal cord of rats. Six groups of male Wistar rats were exposed to a 12 h light-12 h dark cycle for 70 days. At different time points of the experimental day , one of the groups of rats was randomly selected to be sacrificed, and the spinal cords were removed. The gamma-aminobutyric acid (GABA), glutamate (GLU), dopamine, serotonin, epinephrine (E), and norepinephrine (NE) levels in each extracted spinal cord were measured with high-pressure liquid chromatography (HPLC)-EQ and HPLC-fluorescence systems. Our results indicate that the spinal concentrations of GABA and GLU showed sinusoidal variation in a 24 h cycle, with the highest peak in the dark period (~20 h). Dopamine and serotonin also fluctuated in concentration but peaked in the light period (between 8 and 12 h), while E and NE concentrations showed no significant fluctuations. The possible relationship between neurotransmitter spinal concentration and sensitivity to pain and locomotor activity is discussed. It was concluded that most of the neurotransmitter levels in the lumbar spinal cord showed circadian fluctuations coupled to a light-dark cycle. Circadian rhythmicity is inherent to all organisms and is one of the main characteristics of biological systems. It is present in most biochemical and molecular processes, from the transcription of genes in cellular nuclei to the control of the cell cycle , and it Although the SCN is crucial for the generation of biological rhythms in mammals, the expression and regulation of clock genes are not unique to that nucleus. The rhythmic expression of the same clock genes that regulate the oscillator in the SCN is widely distributed among other areas of the brain and the spinal cord  and many5It is well established that communication between neuronal circuits in the central nervous system is based on the interaction of diverse inhibitory, excitatory and modulatory synaptic influences; these influences are mediated by a series of neurotransmitters and neuromodulators. In many neuronal nuclei and centers in the brain, it has been demonstrated that several neurotransmitters show rhythmic variations in concentration , but the10Male Wistar rats, provided by our institutional animal house, were accommodated in individual acrylic cages with ad libitum access to food and water and were maintained under a 12:12 h light-dark cycle (lights on at 6:00 AM) and controlled temperature (22 ± 1°C). At the age of 70 days, rats were sorted into six groups  and each group was randomly selected for sacrifice by cervical dislocation at 08:00, 12:00, 16:00, 20:00, 24:00 and 04:00 hours, day time, and subsequently decapitated. Then, their lumbar spinal cord was extracted by negative pressure injection of saline applied with a 20 ml-syringe through the vertebral central canal at the spinal sacral level (S2-S3). Experiments were conducted following the guidelines of the Mexican Official Norm (NOM-062-ZOO-1999) and National Institutes of Health Guide NIH Publication No. 8023 (revised in 1996) for the Care and Use of Laboratory Animals and approved by the Institutional Bioethical Committee for Care and Handling of Laboratory Animals .Samples of each spinal cord extract (10 mg) were subjected to sonication for 30 seconds, with an amplification of 30%, homogenized with 200 µL of 30% methanol and centrifuged at 3,500 rpm for 5 min. The pellets were washed and resuspended in 1 N NaOH, and the amount of total protein in each sample was determined by the Bradford method.The supernatant was analyzed with a high-pressure liquid chromatography (HPLC) system with electrochemical detection . The separation of GABA (inhibitory neurotransmitter) and GLU (excitatory neurotransmitter) was achieved with a 2.1 × 50 mm dC18 column . Then, each neurotransmitter was measured by precolumn derivation and ECD. Briefly, a derivation was achieved by mixing 12 µL of working derivation reagent  with 30 µL of filtered supernatant (nylon membrane/0.45 µm pore size). The derivation was detected with a glassy carbon electrode (VT-03 Antec Leyden) set at ± 550 mV.The spinal cord tissue was homogenized in 200 µL of perchloric acid per sample and centrifuged at 3,500 rpm for 5 min. The pellets were washed and resuspended in 1 N NaOH, and the amount of total protein in each sample was determined by the Bradford method. The catecholamine concentration in the sample supernatant was analyzed by HPLC and fluorescence detection . The samples were passed through a C18 column . The mobile phase was 9.48 mg/L monochloroacetic acid, 189 mg/L EDTA, 166 mg/L 1-octane sulfonic acid, and 4.5% acetonitrile, pH 3.2, adjusted with NaOH. The chromatograms were processed by using Empower Software, USA.Data are expressed as the amount of neurotransmitter (ng) per milligram of protein present in tissue sample extracts. The concentration of each neurotransmitter per hour was averaged . Rhythmic oscillation parameters  were determined with the cosinor test by using R software, v.3.6, which established the best adjustment of data to a sinusoidal waveform. Because the cosinor test adjusted the experimental data to a sinusoidal waveform, we decided to use the F test and Pearson’s correlation coefficient (r) to assess the fitness of the sinusoidal curve with the experimentally obtained concentration values of each neurotransmitter. In addition, to establish a possible synchronization between the day-night variations in the concentration of the different neurotransmitters analyzed, Pearson’s correlation coefficient was calculated between pairs of neurotransmitters. For graphic construction, we used the data processed with Chronos Fit software . Differe(GABA) = 0.87 and r(GLU) = 0.88, respectively; Figure 1A). The level of GABA began to increase at the middle part of the light phase and reached its maximal value in the dark phase at ~20:00 h, almost one hour after the lights went off, while the lowest level of GABA was determined at the 08:00 h time point . The mesor and zero-amplitude values were 33.68 and 26.02, respectively.GABA and glutamate in the lumbar spinal cord showed fluctuations in their levels that followed a sinusoidal curve during a light-dark cycle , variations in the detected level adjusted well to a 24 h sinusoidal curve , which was similar to that of GABA . The highest value was obtained at 20:00 h, and the lowest level was obtained at 08:00 h. GLU had an acrophase value of 19:31 h and mesor and zero-amplitude values of 214.4 and 160.6, respectively. Significant differences in GLU levels were determined between 08:00 h and 16:00 h and between 08:00 h and 20:00 h . These results indicate that the concentration of GABA and GLU in the spinal cord varied during the light-dark cycle.When comparing the GABA levels between time points, a significant difference between the values obtained at 08:00 and 20:00 h was found . In the case of GLU . The DA concentration reached its highest level during the light phase at 12:00 h, while the lowest level was observed at 20:00 h . The rhythmic parameter acrophase reached a value of 2:44, and a mesor value of 2.67 and zero-amplitude of 2.04 was determined. Significant differences in DA levels were found between time points 12:00 h and 20:00 h .DA and SER followed a similar sinusoidal function (r(SER) = 0.91; Figure 1D). With the acrophase at 10:18 h, almost four hours before the lights turned on, a mesor value of 55.06 and zero-amplitude of 23.72 were observed. These data indicated that the levels of DA and SER in the spine also showed a circadian variation of almost 24 h, but they appear to differ with respect to GABA and GLU sinusoidal curves by approximately 12 h . In contrast, the levels of E and NE in the spine showed no significant variations during the light-dark cycle , and as a consequence, they did not follow any sinusoidal variation.In the case of SER, the variation in its level in the spinal cord during a 12:12 light-dark cycle also followed a sinusoidal function , while the rest of the neurotransmitter pairs had relatively smaller r values (r < 0.6). These results could suggest that the levels of GABA-GLU and DA-SER neurotransmitter pairs fluctuate in a synchronized manner in the spine during a day-night period.Due to the time course similarities shown by the changes in the levels of GABA and GLU, DA and SER, but not E and NE, it is assumed that these neurotransmitters are correlated and highly synchronized. To explore the latter concept in this study, we determined the Pearson’s coefficient of correlation (r) of the averaged light-night variations in the levels of the aforementioned pairs of neurotransmitters in the spine in this study. In this study, we found that the level of GABA, GLU, DA, and SER in the lumbar spinal cord appears to fluctuate in a sinusoidal oscillation during a 12:12 light-dark cycle, while the levels of E and NE do not seem to fluctuate in a circadian day-night cycle. GLU and GABA are two of the most abundant neurotransmitters in the central nervous system . SeveralIn the case of GABA, it has been reported that the number of GABA-A receptors in the cerebral cortex fluctuates rhythmically, with their maximum value occurring at night . Most im16Brain monoamines, such as DA and SER, are involved in the regulation of several essential physiological functions, including the circadian rhythm. It has been shown that DA receptors exert direct or indirect influences on circadian clock genes and proteins in several brain areas . Schade Serotonergic descending axons originate from several nuclei in the brainstem, particularly from the ventrolateral periaqueductal gray nucleus and the nucleus raphe magnus, whose activation induces an antinociceptive effect in the spinal cord .It is well known that neuronal ensembles in the lumbar spinal cord participate in an ample repertoire of sensory and motor processes, such as sensorial integration, pain, voluntary and nonvoluntary movements, posture and control of locomotion ; in the In rats, sensitivity to pain is modulated by neuronal and neurotransmitter ensembles in the spinal dorsal horn . The expSince our results were obtained by using the entire lumbar spinal cord, which prevented us from discerning the occurrence of regional variations in the levels of neurotransmitters in the spine, we assume that the levels of neurotransmitters in the spine determined at each time point along a light-dark period was due to the sum of several sensory and motor neuronal processes that occur in the spinal cord at each time point. Therefore, we consider that it is not feasible to establish a close relationship between light-dark variations in the level of neurotransmitters of the entire spinal cord and variations in individual physiological processes or behaviors. To establish this relationship, it would be necessary to use an experimental paradigm different from the one used in the present study.Regarding E and NE, to the best of our knowledge, there are practically no experimental reports in the literature about the circadian rhythmicity of E and NE in the spinal cord of mammals. Our results reveal that the spinal levels of these monoamine neurotransmitters do not have significant differences between time points during a light-dark cycle, which is indicative of the possible absence of circadian rhythmicity in the level of E and NE in the spine. However, as mentioned above, our results do not allow us to exclude the possibility of the activation of two different sensory and/or motor processes that use NE and E as neurotransmitters in such way that their algebraic sum may alter or cancel the light-dark variation in NE and E levels, as observed in this study. These processes would be located in the dorsal and ventral regions of the spinal cord, and occur in parallel but would be completely desynchronized . This possibility needs to be evaluated with experimental approaches other than those used in this study.As pointed out in the Results section, the time course similarities and high correlation values for the changes in spinal concentrations of GABA and GLU and of DA and SER are notable. Due to the latter, temporal synchronization between the circadian clocks of both pairs of neurotransmitters could be proposed. It is possible that such temporal synchronization of neurotransmitter pairs could be related to the dynamic balance of excitation and inhibition that occurs in neuronal circuits, which has been considered essential for the proper functioning of the brain and spinal cord 30. HowevFinally, it is important to highlight the need to evaluate the day-night variation in the expression of clock genes, neurotransmitter receptors, and transporters at the lumbar spinal cord level to characterize the possible role of genetic and synaptic mechanisms in the physiological function of each of the neurotransmitters analyzed; moreover, such an evaluation could also reveal the possible routes of interaction between different circadian rhythms, such as those between the nucleus accumbens and the spinal cord.The results obtained in this study demonstrate that the spinal concentration of GLU, GABA, DA, and SER vary during the 24 h day-night period, indicating that they are subjected to synchronized circadian rhythms coupled to a light-dark cycle. In contrast, E and NE levels do not seem to follow a circadian oscillation. Further studies are needed to evaluate the functional role of these light-dark fluctuations in the level of neurotransmitters in the spine that may influence the essential activity of spinal neuronal ensembles and the expression of sensory and motor-related behaviors during a light-dark cycle.The additional file for this article can be found as follows:10.5334/jcr.215.s1Supplemental Data.Data corresponding to the article: Day-night variations of neurotransmitters content in the rat lumbar spinal cord.


This article was republished on April 27 2021, to correct numerous errors in the content and placement of references and text. The following errors were addressed: values in the Table 1 References column, the number and content of two references (now [34] and [35]) in the Statistical analysis section, Fig 1 caption appearing incorrectly in the main text of the Statistical analysis section, an incorrect number and content of a reference (now [36]) in the Computations using GRS and wGRS values subsection of the Materials and methods sections, and lastly, the first and last pages missing in S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


To report the one and two year outcome of cycles of three, monthly anti-VEGF injections given upon reactivation of the disease in eyes with neovascular age-related macular degeneration (nAMD).Retrospective study of naïve nAMD cases with more than one year of follow-up, treated with a protocol of cycles of three monthly injections of anti-VEGF drugs upon reactivation. Visual acuity (VA) and central macular thickness (CMT) are the main outcome measures.p = 0.013, 95% CI= 0.60 to 12.65) and 0.77p = 0.4, 95% CI: –5.65 to 7.2) letters at 12 and 24 months. CMT at baseline, 12, and 24 months was 403.55 Twenty-six patients with a mean age of 78.15  This protocol can stabilize or improve vision in 87.5% of nAMD patients and can reduce the number of visits. Less than monthly protocols have been examined; quarterly injections of ranibizumab did not meet the non-inferiority criteria compared to the monthly injections in PIER and EXCITE studies.,5 Pro-re-nata (PRN) dosing based on pre-specified criteria of activity showed more promising results.,7 Treat and extend (T&E) was another protocol first suggested by Spaide to lessen the burden of injections and visits. PRN and T&E are among the most popular ones.Intravitreal injection of anti-VEGF drugs is currently the standard treatment of the neovascular form of age-related macular degeneration (nAMD). The randomized clinical trials that proved efficacy of different anti-VEGF drugs administered monthly or bimonthly intravitreal injections of these medications.,2,3 As a loading dose seems to be an accepted logic for the initial suppression of choroidal neovascular membrane (CNV) activity, it may also be more effective than just one injection in reactivations of CNV. Based on this logic, instead of giving just one injection, we gave a cycle of three successive monthly injections of anti-VEGF drugs with every reactivation of the disease (PRN protocol). This report is the visual and anatomic results at 12 and 24 months of using this protocol in the treatment of naïve nAMD eyes.In major randomized clinical trials of anti-VEGF agents in nAMD, a loading dose of three monthly injections has usually been applied followed by fixed interval dosing protocol of injections.This is a retrospective study of naïve eyes with nAMD treated with injection of anti-VEGF drugs in cycles of three injections upon reactivation of the disease. Data of patients who presented to one of the three retina clinics of University of Texas Medical Branch at Galveston (UTMB) from October 2014 to December 2016 and treated by a single surgeon (JE) were retrieved from the EMR of the UTMB. The study was approved by the IRB and followed the tenets of the Declaration of Helsinki. Patients who were followed for more than one year were included in this study. One eye per patient was included and in case of bilateral AMD the first involved eye was studied. There was no threshold for the level of vision for inclusion in the study.Patients had complete ophthalmic examination including determination of best-corrected visual acuity (BCVA) with the Snellen chart, tonometry, slit lamp examination, and dilated fundoscopy at presentation and at each follow-up visit. Fluorescein angiography (FA)  was performed at presentation for all patients. Repeat FA was at the discretion of the treating physician. Optical coherence tomography (OCT)  was performed at the initial and each follow-up visit. with the formula: approximate ETDRS letters = 85 + 50X log (Snellen fraction). LogMAR VA was calculated and used for comparison of the baseline and 12 and 24 months data. VA of counting fingers was considered equal to 1/200 or three letters of ETDRS chart.Visual acuity (VA) was measured with Snellen chart, and converted to ETDRS letters based on the protocol previously describedAnti-VEGF drugs used were aflibercept , bevacizumab , and ranibizumab . All patients were started with bevacizumab and in case of inadequate response were switched to one of the other two medications. The criteria for switching were: At the commencement of treatment and upon discovery of disease activity at any visit, patients were treated with three monthly doses of anti-VEGF drugs. Follow-up examinations after each series of injections were scheduled monthly for the first two months; extended to bimonthly and after two bimonthly visits to every three months and continued as such if there was no activity of the disease. Patients were also instructed to check their central vision at home and return promptly in case of any new metamorphopsia, scotoma, or change in vision. Disease activity was judged based on the presence or absence of intraretinal or subretinal fluid in OCT scans, or the presence of intraretinal or subretinal blood on fundoscopy.P-value Descriptive statistics are provided as mean Twenty six patients with a mean age of 78.15 VA at baseline was from 3 to 80 ETDRS letters with a mean of 53.87 p = 0.013). Twelve eyes (46.15%) had a vision of The range of VA at 12 months was 3 to 85 with a mean of 60.54 p = 0.4). Eleven (45.83%) patients had a vision of The range of VA at 24 months was 0 to 85 with a mean of 53.68 Patients gained a mean of 6.67Changes in logMAR VA and CMT are presented in Figure 1.Of the six patients with baseline VA of 20/40 or more (70 ETDRS letters), all had a vision of 20/40 or more at 12 months. Only one eye had a vision of Of the eight patients with baseline VA of 20/200 or less (35 ETDRS letters), four in the first year and five in the second retained the vision of p = 0.013, 95% CI = –164.81 to –14.77) and –103.16 pThe mean CMT at baseline, 12, and 24 months was 403.55 Seven patients (27%) achieved long-term periods of inactivity of All patients were first treated with bevacizumab and six of them were switched to aflibercept after a mean of nine injections; one was further switched to ranibizumab, so we can consider them to be practically treated with bevacizumab during the first year. Of the total of seven switching episodes in six eyes, vision improved in three eyes .,20,21 Contribution of all the aforementioned factors results in real-world treatment to be mostly preventive of further visual deterioration instead of increasing vision as expected by results of clinical trials.,13,14 Physicians have devised newer strategies to improve the results in routine clinical practice, such as the “observe and plan” protocol.,23 Reports of observe and plan protocol seem very promising, however, results are still not available in a real-world setting, besides the protocol seems too complicated to be followed thoroughly in a routine clinical practice.Although clinical trials that proved efficacy of intravitreal injection of anti-VEGF drugs reported vision gains of 6.5–11.3 letters in the first and second years,,6,24 which has been shown to result in better visual outcomes compared to PRN dosing from the outset. We speculate that for an active CNV, there is a need for continuous suppression of VEGF for at least three months until the CNV becomes quiescent and we generalized this concept to every reactivation, so we treated every reactivation of the disease with another three monthly series of injections with the hope that adoption of this strategy within an as-needed protocol may improve the results by replacing some of the non-injection visits with injection visits. This is similar to the protocol used in the IVAN study.,21 The difference between our and IVAN's protocols is in the follow-up visits, which were kept monthly in the IVAN study but were extended upon inactivity in two successive visits in the current study, that is, incorporation of a T&E philosophy within the PRN protocol. Another major difference of the current study with IVAN is that IVAN was a prospective randomized controlled clinical trial, whereas this study is a retrospective real-world description of the results of the protocol. In spite of being a real-world study, the results of this study compare to those of IVAN [Table 2].In nearly all major RCTs of anti-VEGF agents and also in clinical practice, treatment is started with a loading dose of three monthly injections,,7PRN treatment of AMD with anti-VEGF drugs was first examined in the PrONTO study, which was not a controlled study, and reported visual results comparable to the results of MARINA and ANCHOR trials. In this prospective study, patients with nAMD were treated based on visual, angiographic, and OCT objective criteria of reactivation. The study followed a strict monthly follow-up of patients and treatment was started with three monthly injections followed by an as-needed treatment. There was a +9.3 (11 median) letter improvement in the first year and 11.1 P = 0.046).,26 IVAN study similarly showed that the difference in visual gain at 12 and 24 months was neither non-inferior nor inferior in PRN treatment when compared to monthly treatment.,21PRN treatment was compared with monthly regimens in some prominent RCTs. For example, in CATT study, monthly and PRN treatment of ranibizumab had a comparable visual results at 12 months. Analysis was inconclusive for bevacizumab monthly versus as needed in that trial. Worse visual results were reported at 24 months for eyes receiving PRN treatment of either ranibizumab or bevacizumab  registry, compared the one-year outcomes of the injections of bevacizumab, ranibizumab, and aflibercept. Patients gained –0.04 to –0.053 logMAR in the first 12 months with a mean of 5.9–6.4 injections of bevacizumab, ranibizumab, or aflibercept. Similarly, Ciulla et al reporting results from Vestrum Health Retina Database from the US stated that there was no change of vision  at 12 months with 7.3 injections of the three drugs. Lotery et al found a change of In the routine clinical practice, physicians mostly use the PRN or T&E protocols for the treatment of nAMD, The number of injections in the first and second years in the current study was 7.65 Although different studies cannot be directly compared with each other, with a mean gain of 6.67 The number of non-injection visits in the first and second years was 4.92 ,21 In CATT study, 19 and 13.9% of eyes in the PRN bevacizumab group and 23.9 and 22.3% of eyes in the PRN ranibizumab group were without any fluid in OCT at 12 and 24 months, respectively.,26 The monthly treatment groups of both IVAN and CATT had higher rates of absence of fluid in OCT at 12 and 24 months: 44 and 54% for the continuous treatment group – IVAN study; 26 and 30.2% in the bevacizumab monthly – CATT study; and 43.7 and 45.5% for ranibizumab monthly – CATT study. In a study by Hatz et al, which was a prospective study comparing the T&E and PRN protocols, the rate of absence of any fluid in OCT at 12 months was 67% in the T&E and 47% in the PRN group. The results of our study seem to be comparable to the results of this study, sitting somewhere between the PRN and T&E groups.In the current study, OCT was fluid-free in 52% and 72.7% at 12 and 24 months, respectively. The corresponding rates in the IVAN study were 31 and 37% in the discontinuous treatment group.There is a worse visual outcome in the second year in spite of less activity of the disease in OCT [Figure 1]. As we did not restrict inclusion of patients based on VA levels, lesion size, or components, it is possible that the development of atrophy or scar in eyes with more advanced lesions during the second year of the study have skewed the results toward lower vision range.,17,34,35 Thus, by not including patients lost to follow-up before one year, we may have introduced a selection bias toward patients with better visual results. The small number of patients is another limitation of the current study. As previously stated, because of the unavailability of same-day injections, the number of non-injection visits may have been overestimated.This study is a retrospective study and limitations of retrospective studies apply to it. One of the major limitations is that VA is not measured by the ETDRS chart and is reported as approximate ETDRS letters which is a calculated number. Some real-world studies have provided that patients who are lost to follow-up have worse vision at their last visit compared to those who are followed-up.In conclusion, the visual results of the current study in the first year are very promising. The protocol is easy to implement and the compliance rate is very high. In the second year of the study, there is a decrease in the gained letters of VA in spite of a higher rate of inactivity of the lesions in OCT. Continuing monthly follow-ups may reduce the vision loss in the second year.