With the introduction of the Diagnostic and Statistical Manual of Mental Disorders-5th ed. (DSM-5) autism spectrum disorders (ASD) fall into the category of neurodevelopmental disorders. ASD is characterized by the inhibitory mechanisms responsible for social adaptation and emotional expression being underdeveloped, causing a child’s recognition and understanding of emotions to be impaired. Our study hypothesizes that early intervention using behavioral interventions such as Applied Behavior Analysis (ABA) and reflexive functions (RF) training on parents can improve the development of joint attention (JA), a cognitive precursor to the theory of mind (ToM) and mentalization processes. We considered a sample of 84 children aged between 20 and 30 months who had received a diagnosis of risk of autism spectrum disorder (level 1). The sample was divided into two groups of 42 subjects, in the first group we carried out a weekly behavioral parent training (PT) based only on ABA principles, while in the second group we carried out a weekly PT aimed at improving reflective functions and parental awareness according to a model inspired by the model based on emotional mirroring and mentalization of Fonagy. Our study shows that parents who are able to make sense of both their own mental state and that of their child can serve as a protective factor for the child’s development even in atypical developmental situations such as in ASD. Autism spectrum disorders (ASD) fall into the category of neurodevelopmental disorders with the introduction of the Diagnostic and Statistical Manual of Mental Disorders-5th ed. (DSM-5) . Over thDespite the clinical heterogeneity, the core deficit of ASD is represented by a deficit in Social Reference and Mentalization. From the age of 8 months, typically developing children move from a self-referential learning situation to a form of socially mediated learning. Experiments such as Tronick’s Still Face ,5 show tThe tendency for mothers to attribute mental states to their children is related to the development of ToM skills through social processes such as the types of language used and the implementation of pretend play ,10. MeinUnderstanding and recognizing one’s own and others’ emotions, desires and mental states helps us to empathize with others. It promotes social exchange and fluid communication, and these abilities are compromised in people with ASD at multiple levels. JA’s capabilities are conceptualized into two types: initiation of joint attention (IJA) and response to join attention (RJA) ,20,21,22The Parental Reflective Function (PRF) refers to the caregiver’s capacity to reflect upon his/her own internal mental experiences as well as those of the child ,30. PRF In the present study, we investigate how important it is to not only work on skills and individual behaviors subject to imperative JA through ABA interventions, but also on reflective skills and awareness of the parents . From a neo-behavioral perspective, parental reflexive skills become a fundamental mark of the stimulation of some complex abilities. They require the integration of multiple simple behaviors and the development of complex cognitive functions. Moreover, working on reflective functions and emotional mirroring with the activation of a medium-intensity ABA intervention can improve the overall effectiveness of the treatment. This favors a faster emergence of some skills, including JA, which will then favor the development of other social skills. The hypothesis tested in our study is that if we intervene early using behavioral interventions such as Applied Behavioral Analysis (ABA) combined with parental training focused on Parental Reflexive Functions (RF), we can improve the development of joint attention (JA), cognitive precursor of the theory of mind (ToM) and mentalization processes.In this study, we included a sample of 84 children, aged between 20 and 30 months, who had been diagnosed with Mild Autism Spectrum Disorder (ASD-grade 1) risk (DSM-5) [All children underwent 15-hour weekly ABA behavioral treatment, split between home and school, and were supervised monthly by a Behavior Analyst Board Certified (BCBA). The sample was sufficiently homogeneous for age, diagnosis, severity of diagnosis and the absence of other related neurologists. Another element of homogeneity of the sample was the absence of psychopathological correlates in the parents ), the middle–high socio-cultural class of the families (presence of degrees in both parents with stable employment of at least the fathers) and the parental age. In particular, the mean maternal age was 30 years (SD = 0.40), and the mean paternal age was 32.3 years (SD = 0.65). The sample was divided into two experimental groups of 42 subjects. Since the subdivision was homogeneous for all the characteristics described and evaluated as inclusion criteria, the process of dividing the group can be considered random. Group 1 consisted of 10 Female and 32 Male, and had a mean age of 23 months (SD = 1.41), while group 2 consisted of 10 F and 32 M, and had a mean age of 24 months (SD = 1.25). The data was collected at the Italian Foundation for Neuroscience and Neurodevelopmental Disorders (FINDS) Child Neuropsychiatry Outpatient Clinic by qualified psychologists.The protocol used consists of the following tests: ADOS 2—Toddler Module (Autism Diagnostic Observation Schedule) , ADI-R  anomalies in mutual social interaction, (ii) qualitative anomalies in communication, (iii) patterns of repetitive behavior and (iv) stereotyped restricted behavior. It focuses on the systematic and standardized observation of behaviors that are rarely found in non-clinical subjects, and mainly on the three areas of functioning: language and communication, mutual social interaction and stereotyped behavior and restricted interests. ADI-R follows the structure of an interview protocol and consist of five algorithms, usable at various ages for diagnosis or intervention. If the purpose of the evaluation is to formulate a formal diagnosis, one of the two diagnostic algorithms  is adopted. If, on the other hand, the objective is the planning of therapy or an educational project, one of the three algorithms of current behavior is adopted .PRFQ: The PRFQ is a structured self-report questionnaire for parents of children between 0 and 5 years of age. It is developed to provide a short multidimensional evaluation of parental reflexive functions. The evaluation consists of 18 items and is split into three subscales: PM , CM  and IC . These sub-scales evaluate parental curiosity related to the child’s mental states, parental efforts to understand mental states and how they relate to children’s behaviors.ESCS-L: An in vivo observation tool that provides a time-efficient quantitative assessment of non-verbal social communication skills. ESCS measures the initiation of joint attention (IJA) and the response to joint attention (RJA), which are critical precursors to the development of social skills and ToM. It also measures the initiation and response to social interaction and demand behavior, part of the overall score of the scale to be considered for this study.VABS II: A semi-structured interview evaluating adaptive behavior (AB). It includes the activities that the individual usually carries out to meet the expectations of personal autonomy and social responsibility, such as activities typically demonstrated by people of the same age and cultural context. Specifically, semi-structured interviews measure AB with the subscales of communication, personal autonomies, socialization and motor skills. In line with the diagnostic criteria of DSM-5, they allow us to establish the severity level of the disorder.Subjects in both groups received a Mild Autism Spectrum Disorder diagnosis following a clinically confirmed neuropsychological evaluation with DSM-5  criteriaWe then divided the children into two experimental groups composed of 42 subjects in each. The two groups were exposed to different parent training (PT) and we analyzed the measured differences. In the first group (G1), we carried out a weekly behavioral PT based only on ABA principles. For the second group (G2), we carried out a weekly PT aimed at improving reflective functions and parental awareness, according to a model inspired by the model based on emotional mirroring and mentalization of Fonagy. The mothers of G1 followed a PT protocol for a total of 24 meetings in 6 months, with a frequency of 1 weekly meeting. Each meeting lasted 90 min, 15 of which were spent on decoding autistic symptoms, 30 on explaining the behavioral strategies to be used, 30 on explaining the objectives to work on and 15 on questions and doubts. The mothers of G2 also followed a PT protocol for a total of 24 meetings in six months, with a frequency of 1 weekly meeting. Each meeting had a duration of 90 minutes, of which 30 were for the enhancement of parental perception and the enrichment of parental reflexive function, 30 for the explanation of the behavioral strategies to be used, 25 for the explanation of the objectives to work on and 5 for questions and doubts.The treatment period between T0 (pre-test) and T1 (post-test) lasted 6 months. At T1, we re-administered the PRFQ questionnaire, the Vineland II and the ESCS to the mothers of the children in order to evaluate the quantitative and qualitative changes in test results.Data analysis was performed using SPSS 26.0  statistip = 0.706), the ESCS test (t (82) = −0.282, p = 0.779) and the VABS test (t (82) = −0.103, p = 0.918). These results indicate that the two groups at T0 (before PT) were homogeneous (The results of the T test showed a non-significance of the scores on the PRFQ test (t (82) = −0.379, ogeneous . –p < 0.05). This data indicates that there is a significant interaction between the time and the type of treatment. More specifically, both treatments have a positive effect on parental awareness, but this is even more true for PT based on reflexive functions (G2)  = 1637.699, ons (G2) .As regards the PRFQ test, the following results were highlighted:–p < 0.05). This data shows us that there is a significant interaction between the two subscales, those being the time and type of treatment. More specifically, both treatments show significant improvements in the two subscales of the ESCS test, but in G2, a more significant improvement in the IJA behaviors of children at T1 is noted  = 161.102, is noted .With regards to the ESCS test, the following results were highlighted:–p < 0.05). This data shows us that there is a significant interaction between the three subscales. Both treatments show significant improvements of the three subscales of the VABS test, but there is a more significant improvement in G2 on the communicative and social area of children at T1  = 424.871, en at T1 .With regards to the VABS test, the following results were highlighted:These results indicate that the PT intervention combined with ABA treatment and reflexive functions (G2) guarantees improvements both on the behavioral level of the child and on the reflexive and mentalizing abilities of the parents.In autism spectrum disorder, the development of ToM can be said to be markedly compromised. Baron-Cohen ,38 stateFrom our study, we note that early intervention with ABA behavioral interventions can positively impact the development of the cognitive precursors of ToM. This is evident from the significantly valid results that emerged from the re-administration of ESCS after PT treatment in both experimental groups. We note the increase in JA precursors are more significant if training on parental RF is added to behavioral training. In particular, we can say that through an enhancement of parental RF, those aspects that have been defined by Baron-Cohen ,39,40 asThese results allow us to say that the PT intervention focused on RF and mentalization skills allows us to observe an improvement in these skills, unlike the behavioral matrix PT, which showed less relevant changes between T0 and T1. This was also consistent and significant with the G2 children’s scores on the ESCS after the PT intervention, which were significantly better than G1. The presence of adequate parental care, congruent affective mirroring, the containment function and the establishment of a secure attachment become the sine qua non for the integration of body and mind, for the birth of the psychological self and the acquisition of the emotional self-regulation ,48. The Our study provided further evidence supporting the theoretical notion that parents’ mentalizing capacity (ToM) plays an important role in the ability to provide care and comfort to the child, in line with several previous studies. Findings indicated that RF is a central mechanism in the quality of the attachment relationship. Our study showed that parents who are able to make sense of both their own mental state and that of their child can serve as a protective factor for the child’s development, even in atypical developmental situations such as in ASD. Our study also highlighted that interventions focused on improving parental mentalization skills and on enhancing parental RF seem to increase the quality of mentalization, ensuring behavioral changes in children with ASD. However, the evidence of the effectiveness of the treatment is limited by the small sample size and the absence of a follow-up to verify the maintenance of skills.


Recycling scrap tyres as alternative aggregates of concrete is an innovative option. To clarify the dynamic properties of the pretreated rubberized concrete with some cumulative damage, the natural frequency, flexural dynamic stiffness, and damping ratio of the specimens under incremental stress level were investigated in this paper. The results indicated that the pretreatment of rubber particles improved the strength, ductility, and crack resistance of the rubberized concrete. The reduction of the flexural dynamic stiffness was clarified with the increase of concrete stress level. The addition of the pretreated rubber particles enhanced the concrete energy dissipation capacity during the destruction, and the specimen dissipated more energy with the increase of rubber content before its failure. In recent years, the continuous development of new raw materials has enriched the materials in the field of construction ,2,3,4, aThe rubberized concrete (RUC) with the appropriate amount and size of rubber particles featured better durability , thermalIn recent years, researchers have paid more and more attention to concrete’s dynamic properties ,33. The According to the above studies, rubber particles replacing coarse aggregate increased the concrete damping capacity more notably, but the strength reduced significantly. The concrete with fine aggregate replaced with rubber particles was relatively better. Muñoz-Sánchez et al.  reportedNumerous mentioned papers have drawn important conclusions about the damping capacity of RUC through different methods, and promote the development of research on the dynamic capacity of concrete ,42,43. AThe reported publications mainly studied the properties of original rubberized concrete with undamaged specimens. The dynamic properties of concrete with some damage directly affect the anti-vibration capacity of the structures. It is necessary to study the dynamic properties of PRC during its destruction. In this test, specimens of uniform size were used, and the dynamic properties of the PRC, including natural frequency, flexural dynamic stiffness and damping ratio under incremental loading, were investigated using free vibration tests on them. The raw materials and the test methods were stated first, and then a brief research on the concrete slump and mechanical properties was made. Finally, the concrete dynamic properties were investigated.The cement used in this test was the 42.5 Ordinary Portland Cement produced by Tianrui Group Cement Co., Ltd., Zhengzhou, China. The chemical compositions and properties of the cement are listed in 3, and the ash content, fiber content and moisture content are 2.7%, 0.5%, and 0.6%, respectively. The distributions of the rubber and aggregate particle size are shown in The coarse and fine aggregates were natural crushed stone and river sand. Their properties are listed in The pretreated solution of rubber particles was sodium hydroxide solution with a mass concentration of 5%. The rubber particles were washed with water three times and dried, then soaked in the solution for 30 min. The mass ratio of rubber particles and the solution was 1/2, and each batch of the solution was used only once on the rubber particles. Finally, they were washed with water again, and dried.The concrete mixtures were based on China Standards JGJ 55- contents of the rubber particles were 0%, 5%, 10%, 15%, and 20%. The dynamic 2011 . The volA total of nine groups of specimens, including four groups of RUC, four groups of PRC, and a group of NC as reference specimens were prepared. Each group contained 17 concrete specimens, three for the test of dynamic elastic modulus and flexural strength, two for the test of dynamic properties, and three for each other test. The six specimens for the test of cube compressive strength and splitting tensile strength were of the size of 100 × 100 × 100 mm. Another six specimens for the test of axial compressive strength and static elastic modulus were of the size of 150 × 150 × 300 mm. Three specimens for the test of dynamic elastic modulus and flexural strength were in the size of 100 × 100 × 400 mm. The last two specimens for the test of dynamic properties were in the size of 100 × 100 × 1000 mm.The structural form of the cantilever beam is simple, and the pre-loading damage is easy to operate ,50. The rM, 0.6uM and 0.8uM) with 0–0.8 of the concrete stress level –(4), the stress level  Natural frequencyξ, E, I, and f are the damping ratio, dynamic elastic modulus, the moment of inertial, and natural frequency, L are the mass per unit length and calculated length of cantilever beams. Based on Equation (6), the theoretical natural frequency of the specimens was determined byThe acceleration-versus-time data were obtained through the free vibration test of a cantilever beam under incremental loading. The test natural frequency of the specimens was determined with the attenuated curves of acceleration. For the concrete specimens without damage, their natural frequency can be calculated by(2) Damping ratioξ bya1 and a21 were the initial peak and the 21st peak in the given time history.The acceleration amplitude from the first 20 oscillation cycles was used to calculate (3) Flexural dynamic stiffnessEI is no longer an invariant constant. Based on Equation (6), once the natural frequency was obtained, Equation (9) could be used to approximately predict the flexural dynamic stiffness under various conditions, and the dimensionless flexural dynamic stiffness was calculated by Equation (10).EI)0 and f0 were the flexural dynamic stiffness and natural frequency about cantilever beams of reference, and (EI)i and if were the values of cantilever beams under different conditions. For each test of natural frequency, damping ratio and flexural dynamic stiffness, the average value of the two specimens was reported as the test value.The properties of concrete change with the increase of rubber content and cumulative damage, and The concrete workability was measured by the slumps obtained by the standard test. The slumps of RUC and PRC with the same rubber content were similar, as listed in In this test, the concrete slumps first decreased and then increased slightly with the increase of rubber content. This is understandable since both the amount of sand and water are the important factors of fresh concrete slumps. When some sand was replaced by rubber particles, the flowability of concrete decreased as the result of the reduction of sand and coarse aggregate ratio. Note that when the sand was replaced with enough rubber particles, the actual moisture content of RUC was higher due to the lower water absorption of rubber particles (1–2 mm) than that of sand, which may have increased the slump of concrete slightly. Also, the gas carried by rubber particles during the concrete mixing process can also improve the workability of the concrete.The test results for the concrete strength and static elastic modulus are listed in As most papers reported, the addition of rubber particles weakened the concrete strength significantly ,56,57,58ff, fcp and fts represent the dimensionless values of flexural strength, axial compressive strength, and splitting tensile strength. As shown in ff/fcp is larger than fts/fcp, and both the ff/fcp and fts/fcp increase first and then decrease with the increase of rubber content. To ff/fcp and fts/fcp of PRC, the values are larger than that of RUC. This indicated that the pretreatment of rubber particles with sodium hydroxide further enhanced the ductility of the rubberized concrete, and the recommended content of rubber was about 15%.The effects of rubber content and pretreatment of rubber particles on the ratio of splitting tensile and axial compressive strength, and the ratio of flexural and axial compressive strength, are shown in The surface of the routine and pretreated rubber particles, as well as their bonding interface with cement matrix, were obtained by scanning electron microscopy. As shown in ff/fcp and fts/fcp decrease when the content of rubber particles exceeds 15%.Gao et al.  and BompcM/uM) of the cantilever beams reflects the ductility of the specimens. When cM/uM is too high, the material lacks ductility and there will be a sudden brittle failure of the specimen without obvious warning signs. The test results of cM and uM are listed in The ratio of cracking and ultimate moment  about all the mixtures are listed in The dynamic elastic modulus  was 26.684 Hz, while the value reduced 2.8% and 2.5% for the specimens produced with RUC (RUCB) and PRC (PRCB), respectively, with 20% rubber content. To the simply supported beams in the similar test condition reported by Zheng et al. , the redThe natural frequency is a major dynamic parameter of concrete, which is mainly affected by dynamic elastic modulus and unit weight of the specimen. With the increase of rubber content, the natural frequency of concrete specimens decreases slightly since the reduction of dynamic elastic modulus is smaller than that of unit weight. The reduction of dynamic elastic modulus is the representation of development in concrete damage ,59. The d2E) can also be determined through the free vibration test of cantilever beams, as listed in d1E and d2E, but all of the differences are less than 11.2%, and less than 10.0% reported by Zheng et al. [d1E and d2E of PRCB with 20% rubber content reduced by less than 15.0% and 11.6%. The flexural dynamic stiffness calculated with d2E reduced by 41.8% for NCB with the stress level of 0.8 and about 43.1–47.2% for RUCB (PRCB).The concrete dynamic elastic modulus  of the specimen decreases with the increase of rubber content, and the concrete failure period is prolonged. Therefore, the specimen will dissipate more energy with the increase of rubber content before its failure.The increase of damping ratio is slight in the elastic and elastic–plastic stage before the specimen cracking  and rubber content, respectively. PRC is a good choice where the requirement of strength is not too high, and the deformation as well as dynamic properties are relatively strict.The research of dynamic properties presented in this paper is of great significant for reference to evaluate the dynamic properties of rubberized concrete with some damage. Moreover, considering the limitation of the test data, improvement of the test setup and specimens, to verify further applicability and validity of the method to measure the concrete dynamic properties, the extended numerical tests are the focus of our next work.


Wistar rats were exposed to laser-driven UPEB irradiation, after which different immunological parameters were studied on the 1st, 3rd, 7th, 14th, and 28th day after irradiation. According to the results, this type of irradiation induces alterations in the rat immune system, particularly by increasing the production of pro- and anti-inflammatory cytokines and elevating the DNA damage rate. Moreover, such an immune response reaches its maximal levels on the third day after laser-driven UPEB whole-body irradiation, showing partial recovery on subsequent days with a total recovery on the 28th day. The results of this study provide valuable insight into the effect of laser-driven UPEB whole-body irradiation on the immune system of the animals and support further animal experiments on the role of this novel type of irradiation.The development of new laser-driven electron linear accelerators, providing unique ultrashort pulsed electron beams (UPEBs) with low repetition rates, opens new opportunities for radiotherapy and new fronts for radiobiological research in general. Considering the growing interest in the application of UPEBs in radiation biology and medicine, the aim of this study was to reveal the changes in immune system in response to low-energy laser-driven UPEB whole-body irradiation in rodents. Forty male albino  The application of electron beams in radiobiomedicine and the commercial development of medical electron linear accelerators goes back to the early 1950s, when a huge amount of research in the field of radiation therapy conducted by different leading centers provided reliable evidence of the benefits and advantages of this kind of irradiation for the treatment of certain types of cancer , includi11 Gy/min) than those provided by conventional beams [However, new laser-driven electron linear accelerators are now available, providing unique ultrashort pulsed electron beams (UPEBs) with ultrashort picosecond pulses of low repetition rates of just several Hertz, but with doses several orders of magnitude higher , while no difference was observed in the percentage content of the cells .The irradiation of animals with the laser-driven UPEB resulted in dramatic changes in the population content of blood after 24 h of irradiation. Thus, compared to both control groups , a statistically significant decrease was observed in the amount of white blood cells (17 × 103 cells/mL). Moreover, there was a 10–13 times statistically significant decrease in the percentage content of the lymphoid cells. Meanwhile, no changes were detected in the absolute number of neutrophils, while their relative abundance increased due to the loss of lymphoid cells . It should be mentioned that this type of irradiation also resulted in the appearance of a large population of pathological myeloid cells. Although there was a slight decrease in the absolute values of cell content in the population of monocytes, their relative abundance was increased 2 times (compared to control groups) due to the loss of the main amount of lymphocytes.On the third day after irradiation, a termination in the progress of lymphopenia was observed, and the levels of lymphocytes did not differ from those observed on the first day after irradiation. Nevertheless, there was still a progress of leukopenia, which in this case was caused first of all by the loss of myeloid population resulting in the increase of the content of destroyed cells.From the seventh day of irradiation, recovery processes of the cell populations were observed, including both absolute (the absolute content of blood cells was increased) and relative (the lymphoid population started to recover) recovery. This process continued up to the 14th and 28th day after irradiation, when almost all the indicators of white blood cells reached control levels. It should be noted that this distinctly expressed lymphopenia was not accompanied by the shift to the left, since the amounts of lymphoblasts in all the irradiation groups did not exceed those in the control group, i.e., no expressed regeneration processes took place.The morphological changes of peripheral blood cells are presented in The total quantities of cells per unit of area are presented in The immune cells were represented by lymphoid (30–35%), myeloid (32–35%), and monocytic (5%) populations. All other cells overall constituted less than 1%. The analysis of the myelogram in both control groups of rats  revealed that in these groups the content of red blood elements was about 25–30% of all the parenchymal cells .In the bone marrow cellular populations, a significant decrease in the content of mature lymphocytes was observed on the first day after irradiation, with a simultaneous increase in the content of pathological lymphocytes and the appearance of a large number of destroyed cells. Meanwhile, there was a statistically significant increase in the content of late myeloid cells, from metamyelocytes to segmented neutrophils b. Then, As in the bone marrow, a statistically significant decrease in the content of mature lymphocytes was observed in the lymph nodes  on the fThe concentrations of interleukin-1β (IL-1β) and interleukin-10 (IL-10) in the plasma samples of the control rats and those exposed to the laser-driven UPEB irradiation  were alsThe results of laser-driven UPEB irradiation-induced DNA damage in the cells obtained from spleen, thymus, and bone marrow are presented in The viability of cells, assessed in parallel to each experimental point, was not significantly changed compared to that of the corresponding controls, being 75.8 ± 3.21% in the spleen, 89.2 ± 1.56% in the thymus, and 86.3 ± 2.78% in the bone marrow cell populations. A visible but not statistically significant decrease in the weight of the thymus and spleen was observed. Particularly, the mean absolute weight decrease of approximately 16% in the thymus was detected on the 3rd day (435 ± 40.8 mg) and by 9.9% on the 28th day (472 ± 24.53 mg) of the study, compared to that in the control group (524 ± 68.12 mg). The mean absolute weight of the spleen was slightly decreased (5.9%) on the seventh day (785 ± 56.5 mg) of the study, as compared with the control value (834 ± 93.4 mg). The DNA damage recovery was most effective in the thymus, then in the bone marrow, and to the least extent in the spleen cells. However, on the 28th day of the study, the DNA damage levels reached the control (spleen) or even lower (thymus and bone marrow) values.Wistar rats. We tested the effect of laser-driven UPEB whole-body irradiation on the cells of peripheral blood, bone marrow, and lymph nodes, as well as cytokine levels in peripheral blood plasma and DNA damage and repair in the spleen, thymus, and bone marrow of rats. The number of animals involved in each of the seven experimental groups of this study allowed us to obtain statistically reliable and significant data on the dynamics of the alterations of the studied parameters of the immune system during 28 days after radiation exposure, to assess the radiosensitivity of different organs and blood and bone marrow cells to the laser-driven UPEB whole-body irradiation. The data obtained describes for the first time the adaptive reactions of the immune system of rats in response to the irradiation with laser-driven UPEBs. This study could be used as a basis for future, more detailed investigations of the molecular and cellular mechanisms of these reactions, as well as for the assessment of the effect/efficiency of different radioprotective agents on the immune system of the animals.In this manuscript, we studied the laser-driven UPEB whole-body irradiation-induced radiation adaptive response in the immune system of In general, whole-body irradiation studies are important for understanding the extent to which the radiation causes different effects in the organism, whether it is whole-body irradiation-induced injury from a radiation accident or a radiological terrorism event (including atomic bombings or serious nuclear power plant accidents), or if it is a conditioning therapy before hematopoietic stem cell or bone marrow transplantation for the treatment of several types of cancer, including leukemia, lymphoma, and multiple myeloma ,27. ThusWe revealed statistically significant immune cell depletion and recovery consequent to total body irradiation by laser-driven UPEB. First of all, we studied the changes in the main indicators of the immune system after the laser-driven UPEB irradiation. Such studies are limited technically due to the importance of laser-driven UPEBs produced by linear accelerators. In a linear accelerator, electrons are accelerated to a high energy and can be used in some medical areas. There are several properties which make the laser-driven UPEBs promising for future therapeutic approaches. First of all, this new kind of electron beam irradiation is very promising in different fields of clinical oncology, not only because of the smaller penetration rate of electron beams into deeper situated tissues, but also because laser-driven electron linear accelerators provide an opportunity to regulate the penetration rate by modifying the energy of the incident electrons .The studies of the immune cell pathology via investigation of blood cell populations revealed a statistically significant pancytopenia in peripheral blood with relative neutrophilic leukocytosis with a slight shift of the leukocyte formula to the left, indicating absolute and relative lymphopenia. The reason for the progress of leukopenia, caused by the loss of myeloid population and resulting in the increase of the content of destroyed cells, may be apoptosis. These results are in compliance with the first stage of acute radiation syndrome (sickness) to the primary acute reaction on the acute radiation injury. In general, there are four phases of acute radiation injury  based on which the course of the syndrome varies from several hours to several weeks depending on the irradiation type , the dose, the part of the body exposed to irradiation , and different individual characteristics  .Young and rapidly growing tissues are the most radiosensitive ones to radiation exposure . RepreseThe lymph node is a dynamic organ, composed of transient lymphocytes, a small number of monocytes and/or macrophages, several lymphoblasts, and very rarely erythroblasts. After electron beam exposure, a severe lymphopenia and a significant number of destroyed and pathological cells occurred. Partial recovery occurred on the 7th day after electron beam exposure, which continued up to the 14th and the 28th day.According to the obtained results, it can be concluded that after the whole-body irradiation by the laser-driven UPEBs, a partial recovery of the immune system took place in a shorter time, unlike with X-ray or gamma ray radiation ,36,37,38The formation of bilobed lymphocytes after electron beam exposure is an interesting phenomenon, which was first described by cyclotron personnel at the University of Rochester in 1948–1951 , and theAs described previously , the forThe delayed immune reconstitution is known as a major cause of morbidity. It has been shown to be associated with myelosuppression, which is one of the detrimental effects of cytotoxic radiation therapy. Therefore, the reconstitution of all immune cells using hematopoietic stem cells, as well as T-cell recovery via the effective thymopoiesis, could be an efficient therapeutic strategy for the restoration of the functional immune cell repertoire .It is well-known that ionizing radiation leads to the alterations in the expression of proinflammatory and anti-inflammatory cytokines, which is highly time-dependent and occurs as a response of the organism to the radiation in the form of cytokine storming with a subsequent total or partial recovery to baseline levels ,51, depeDecreased amounts of IL-10 from the seventh day after laser-driven UPEB exposure revealed in this study can explain the phenomenon of the removal of mature neutrophils (bands and segments) from the bone marrow since the seventh day, as well as the absence of the rise of these cells in the blood on the same day. It is known that anti-inflammatory interleukins, such as IL-10, inhibit the expression of endothelial adhesion molecules and prevent the leukocyte extravasation, thereby preventing leukopenia .Wistar rats induced by whole-body laser-driven UPEB irradiation. The hematopoietic system, including the bone marrow, spleen, thymus, and lymph nodes, is highly sensitive to ionizing radiation. Since DNA is the primary target of ionizing radiation, we performed comet assay on the cells obtained from the spleen, thymus, and bone marrow, to compare the tissue sensitivity and possible adverse effect of low-energy UPEB irradiation. While the spleen was the most sensitive to low-energy UPEB radiation, demonstrating a higher level of DNA damages and slower recovery kinetics, a drastic increase in DNA damages in the thymus and bone marrow cells was also observed. It is well known that hematopoietic organs are mutually related, which is expressed by the consequent cell migration from the bone marrow to the thymus and then to the spleen and lymph nodes. Thus, the slight decrease in the mean absolute weight of the spleen with the persistent high level of DNA damages, detected on the 7th day, can be explained by the death of highly damaged cells, whereas the delayed recovery of the spleen, observed on the 14th day of the study, can be associated with the recovery kinetics of bone marrow and thymus (day 7–14), the time period until irradiated bone marrow can seed the spleen with the sufficient number of non-damaged cells. The biphasic pattern of thymus weight change (on the 3rd and 28th day) can be explained by the death of highly damaged cells during the first three days after irradiation, with the consequent proliferation of surviving cells in the thymus. However, the second slight decrease of spleen weight, while the cells’ DNA was shown to be undamaged, can be associated with the insufficient supply of cells from the bone marrow, which itself demonstrated a significant decrease of DNA damages in cells during day 14–28 of the study. The non-detected changes in cell viability in the parallel experiments could be due to the loss of dead cells during cell isolation procedures  or apoptosis. Overall, DNA damages, observed in the bone marrow, thymus, and spleen, achieved the maximal level on the 3rd day of the study, with further recovery on the 7th day (in the case of the bone marrow and thymus) and on the 14th day (in the case of the spleen) after irradiation. The lower levels of DNA damages compared to those of the corresponding controls, observed in the spleen and bone marrow cells, on the 28th day after irradiation, can be explained by the full repopulation of those organs with healthy cells.The comet assay applied in this study is widely used in different biomedical fields, including radiation biology, toxicology, and oncology. It is a single-cell gel electrophoresis tool for the determination of DNA damage and repair kinetics ,57. X-raThe interplay between ionizing radiation-induced DNA damages and immune system alterations has been shown earlier, which was explained by the activation of DNA damage response machinery leading to the immunogenicity of the damaged cells. The reciprocal/reverse situation was also observed. For instance, the prolonged expression of cytokines, such as interferon β (IFNβ) or interferon γ (IFNγ), led to the accumulation of DNA double-strand breaks . The rolWith regard to the development perspective, experiments have to be done to understand the effect of this novel type of irradiation on the biological level, pathological and molecular alterations, and different systems and organs of the animal, including the oxidant/antioxidant system, lipid metabolism, cell death mechanisms, DNA alterations, and so on.Wistar rats weighing approximately 180–200 g were involved in this study. The rats were housed and cared for under controlled colony conditions providing 12-h reverse light–dark cycles in a temperature-regulated animal facility (22 °C). Rats had access to food and water ad libitum. All the rats were fed a standard laboratory chow diet . The rats were randomly divided into seven groups: a control group of six non-irradiated animals , a sham group of six non-irradiated anesthetized animals (necessary for the assessment of the effect of the anesthesia only on the studied parameters in the non-irradiated rats), and five irradiation groups each consisting of six animals . The number of animals was chosen and designed as n = 6 to correspond to the minimum amount of the animals necessary to conduct an adequate and well-controlled study to perform the accurate and reliable statistical analysis of the data obtained.Forty-two male albino The animal protocol was approved by the Animal Ethical Committee of the Institute of Molecular Biology NAS RA and the experiments were carried out in accordance with its relevant guidelines and regulations. All the rats except those in the control group were anesthetized with an intramuscular administration of 20 mg/kg aminazine . The adequate depth of the anesthesia was ensured by testing the pedal withdrawal and palpebral reflexes. All the rats were euthanized by carbon dioxide overdose followed by blood collection from the carotid artery and subsequent decapitation in the morning of the 1st, 3rd, 7th, 14th, and 28th day after irradiation and on the 1st day after anesthesia in the sham group.As an irradiation source, the laser-driven UPEB from AREAL (Advanced Research Electron Accelerator Laboratory) facility was used developed at CANDLE  Synchrotron Research Institute, Yerevan, Armenia. Radiation treatment was carried out using an electron beam generated by a laser-driven radiofrequency gun-based linear AREAL accelerator. To extract the electron beam from the accelerator, a 20 μm thick titanium window was mounted at the end of the experimental beamline. The characteristics of the AREAL accelerator, as well as the description of the dose calibration measurements and uniformity quality assurance, have previously been described ,24,47,63The one-time irradiation was delivered at 10:00–11:00 AM with a dose of 2 Gy and a repetition rate of 2 Hz corresponding to the lethal dose 50 (LD50) for this type of irradiation and animal, determined previously . The expThe rats were divided into seven groups with six rats in each group. There were five irradiated groups exposed to laser-driven UPEB irradiation and euthanized on the 1st, 3rd, 7th, 14th, and 28th day after irradiation, as well as the control non-irradiated group. Peripheral blood, bone marrow, and lymph node profiling was conducted. The levels of proinflammatory and anti-inflammatory cytokines were determined in peripheral blood plasma samples, and DNA damage and repair was assessed in the spleen, thymus, and bone marrow of the rats.g for 15 min at 4 °C; they were then put in storage at −30 °C until further use. Immediately prior to use, the plasma samples were thawed and centrifuged at 10,000 rpm for 5 min at 4 °C to remove any precipitate. For complete blood cell count analysis, conventional blood smears were made by spreading a blood drop on a clean slide with another one with subsequent air drying and Giemsa staining. The films were viewed under the light microscope using the 100× objective for counting.Blood samples (4 mL) were collected from the tail vein of the rate into ethylenediaminetetraacetic acid-containing (EDTA-containing) tubes (to prevent coagulation) for the isolation of plasma samples and a complete blood picture and blood film morphology. For the isolation of plasma samples, the blood samples were kept on ice for 60 min with the following centrifugation at 3000× 2) using a light microscope at 1250× magnification. In total, at least 3000 cells were analyzed and classified at a given day before and after radiation.Bone marrow was obtained from the humerus, as described previously , and theg for 10 min, aspirated, and washed with bone marrow medium. The cell count and viability were determined by Trypan blue exclusion tests [Bone marrow cells were isolated according to the previously described procedure . Brieflyon tests .2) using a light microscope at 1250× magnification. In total, at least 3000 cells were analyzed and classified at a given day before and after radiation.Fresh lymph nodes  were cut with a sharp blade through the hilum, and tissue imprints were made by gently touching the freshly cut surface of the tissue with a clean glass microscope slide. For cell analysis, slides were fixed in pure methanol and stained with modified Giemsa solution  according to the manufacturer’s protocol . Cells were analyzed and counted in 100 randomly selected fields  kits  according to the manufacturers’ instructions. The concentrations of IL-1β and IL-10 were expressed in pg per mL of plasma (pg/mL), and the concentrations of thrombopoietin were expressed in ng per mL of plasma (ng/mL).6 cells/mL were mixed with low-melting-point agarose (0.5%) (20:180 μL) at 37 °C, placed on slides pre-coated with a layer of normal melting agarose (0.5%), and allowed to polymerize at 4 °C for 5 min. After removing the cover slips, the slides were placed in Coplin jars with 50 mL of lysis solution  at 4 °C. After 1 h of incubation in lysis solution, the slides were placed in a horizontal gel electrophoresis tank side by side, leaving no spaces. The slides were incubated in a fresh alkaline (pH 13) electrophoresis buffer (300 mmol/L of NaOH and 1 mmol/L of EDTA-Na2) for 20 min to allow for the unwinding of DNA, and then electrophoresis was run at 25 V and 300 mA for 20 min at 4 °C. Slides were neutralized three times for 5 min in 0.4 mol/L of Tris, pH 7.5, and DNA was stained with 20 μL of 20 μg/mL ethidium bromide. Cells were analyzed using a Nikon Labophot 2 fluorescence microscope  at 200 × magnification and computer-aided image analysis. Images of at least 100 cells (50 cells from each of the two slides) were evaluated by the use of the software program Komet 5 . The percentage of DNA in the tail region of each cell was determined .The alkaline comet assay was performed according to the previously described method . Bone map < 0.05 was considered as the statistically significant value.Statistical analysis was performed using GraphPad Prism 5.01 . Depending on parametric or non-parametric data, one-way ANOVA or the Kruskal–Wallis tests were used, and the differences between groups were determined with Bonferroni or Dunn’s post-hoc tests, respectively. Values are expressed as mean ± standard error of the mean (SEM), and In conclusion, the studies of the biological effect of laser-driven UPEB whole-body irradiation showed that this type of irradiation induces alterations in the rat immune system, particularly by increasing the production of pro- and anti-inflammatory cytokines and elevating the DNA damage rate. Moreover, such an immune response reaches its maximal levels on the third day after laser-driven UPEB whole-body irradiation, showing partial recovery on subsequent days with the total recovery on the 28th day. The adaptive reactions of the immune system of rats are described for the first time in response to the irradiation with laser-driven UPEBs. This study could be used as a basis for future, more detailed investigations of the molecular and cellular mechanisms of these reactions, as well as for the assessment of the effect/efficiency of different radioprotective agents on the immune systems of animals. The results of this study provide valuable insight into the effect of laser-driven UPEB whole-body irradiation on the immune systems of animals and supports further animal experiments on the role of this novel type of irradiation. This understanding will serve as a background and will be necessary for future studies of possible applications of laser-driven UPEB irradiation in cancer treatment strategies as a promising new radiotherapeutic approach, mitigating the risk of the harmful side effects of radiation therapy and providing early recovery of the organism afterwards, as well as for ensuring that the adoption of this promising technology is appropriate and evidence based.


A three-dimensional (3D) finite element (FE) model of the mandibular bone was created from 3D X-ray CT scan images of a live human subject. Simulating the clinical situation of implant therapy at the mandibular first molar, virtual extraction of the tooth was performed at the 3D FE mandibular model, and 12 different implant diameters and lengths were virtually inserted in order to carry out a mechanical analysis. (1) High stress concentration was found at the surfaces of the buccal and lingual peri-implant bone adjacent to the sides of the neck in all the implants. (2) The greatest stress value was approximately 6.0 MPa with implant diameter of 3.8 mm, approx. 4.5 MPa with implant diameter of 4.3 mm, and approx. 3.2 MPa with implant diameter of 6.0 mm. (3) The stress on the peri-implant bone was found to decrease with increasing length and mainly in diameter of the implant. Dental implants are prosthetic devices which restore masticatory function or improve aesthetic appearance. A metal post is surgically implanted in the bone at an area of a missing tooth or external trauma, and this acts as the support for a superstructure. In many cases of tooth deficiency with minimal intervention, implants have been selected due to favorable and reliable results, with a 10-year survival rate of over 95% ,3,4.For long term function of implants, there should be no bone resorption in response to the repeated force exerted through mastication, and for the patient to maintain physiologically sound periodontal tissues. Furthermore, mechanical problems may occur at the bone–implant interface in implants that are subjected to occlusal force , and theMoreover, with a wide range of implants currently in clinical use, it is difficult to select the type of implant best suited to the individual needs of the patient. At present, in the absence of any clear selection criteria for implants, selection is almost always made on the basis of the clinician’s experience with mandibular bone size and previous clinical outcomes. For this reason, three-dimensional visualization techniques using dental X-ray computed tomography (CT) scanning devices have become effective methods of examination for making accurate diagnoses ,9,10,11.Here, a 3D FE model that reconstructs the bone morphology and material properties of the mandible was created from X-ray CT scans of a live subject. The model was used for mechanical simulations to evaluate the effects of thickness and length of implants on the stress exerted on the peri-implant bone, in order to investigate the usefulness of virtual implant diagnoses in clinical settings.The mandible selected for scanning was that of a healthy 20-year-old male with no caries, who gave his informed consent. The study protocol was approved by the Ethics committee of Nippon Dental University.A CT scanner  was used to scan the data needed for the study, and the 3D X-ray CT scanning conditions are shown in Integrated digital image processing and finite element analysis software  were used to reconstruct the 3D FE model on a computer from the data converted to DICOM files. The procedure for image processing and creation of the analysis model is shown in First, the mandibular bone and the teeth were classified separately in order to create a 3D FE model capable of virtual implant therapy simulation. Virtual extraction of the first molar was performed on the constructed mandibular 3D FE model, and Standard Triangulation Language (STL) data were created for the extraction site by a 3D CAD program . Three-dimensional FE models with virtual placement of implants of different shapes were then created using the STL data. A total of 12 one-piece implants  were used, combining three diameters  and four lengths . The shape data of implants with each of the 12 shapes were converted to 3D models on VX and then imported to MF, and 3D FE models of the implants were created by element breakdown. The 3D FE models of the implants are shown in The space in the mandibular ridge following virtual extraction was set to be automatically replaced by bone, so there was no bone in the space when the implant was placed.The material properties of the bone, teeth, and implants are shown in The analysis was linear static analysis, the material properties of the bone were set as non-linear material, and the results were evaluated as equivalent stress. The models each had a total of 54,728 nodes and 302, 335 4-node tetrahedral elements. The models were created, and FE analysis was performed on a PC Workstation .The buccolingual cross-sectional view showed equivalent stress in the peri-implant bone surface on the buccal side. The equivalent stress was approximately 3.0–6.0 MPa with implant diameter of 3.8 mm, 3.0–4.5 MPa with implant diameter of 4.3 mm, and 1.0–3.0 MPa with implant diameter of 6.0 mm. The occlusal view of the implant and the horizontal cross-section at the cervical region show concentration of the stress in the peri-implant bone, and this is particularly high on the buccal side. With the buccolingual cross-section, equivalent stress values tend to decrease in the same way with increasing diameter and length of the implant. In the implant, equivalent stress was found from near the load-bearing site toward the base of the implant, and the equivalent stress decreased with increasing length and diameter of the implant. The stress distribution was such that stress appeared in peri-implant bone but tended to decrease in the central and lower regions.A 3D FE model is created of the mandible, comprising cortical and trabecular bone from data obtained non-invasively from CT scan images, and the implant, which was designed by CAD. Using this model, virtual implants of different diameter and length are placed in a tooth extraction socket and examined using the finite element method. Mechanical simulations using CT scans, CAD, and FEM in this way were shown to be useful.The finite element method is a technique for stress analysis that was developed as computers grew more sophisticated. This technique separates solid bodies, which are continuous, into countable elements. By calculating the mechanical balance of each element, the displacement or stress of the body as a whole can be determined. Although creating the model entails considerable difficulty, the method permits accurate measurement of internal stress and offers considerable versatility in the methods for evaluating the results. In the present study, a mechanical evaluation was performed by stress analysis using the finite element method.Principal stress and Von Mises stress  are often used as methods for evaluation of stress in finite element analyses of bodies with complex shapes such as teeth or mandible ,22. PrinA 3D FE model is needed on the computer when a finite element analysis is performed, and this sort of analysis is problematic in terms of efficiency when using conventional methods because of the time and the labor involved in creating a 3D FE model. Furthermore, it is extremely difficult to create 3D FE models of live subjects, and previously it was common to use 3D FE models created from mean anatomical values or dry skulls. However, there is a need in clinical implant practice for the development of diagnostic systems that incorporate mechanical analysis in order to ensure therapy with a high degree of predictability. For this reason, it is essential to be able to create 3D FE models within a short space of time that accurately reflect the living body. X-ray CT devices are currently used for a wide variety of cases, and have become indispensable scanning devices in the field of implant therapy.Here, we created a 3D FE model directly from 3D X-ray CT images in a short space of time that accurately reflected the inherent bone shape and bone quality of the living body, and we performed virtual implant placement on a computer using 3D FE models of implants of various different shapes created by CAD. We showed that if it were possible in clinical practice to draw up a mechanically optimal therapeutic plan for living individuals in this way, objective predictions of the prognosis can be made from a mechanical perspective, which is not possible with the conventional method of judging bone morphology only from data obtained by visual examination of scans. This could become a revolutionary diagnostic system in the field of dentistry in the future.MF is a finite element analysis program capable of creating a 3D FE model from 3D X-ray CT scan data. A major advantage of this program is that the 3D FE model can be moved and parts can be added or deleted at will comparatively easily. This functionality means that MF has wide applicability, not just in the field of research but also in clinical practice ,24. FurtAnalysis was performed with implants of different combinations of diameter and length. There have been reports that slender implants have weak mechanical strength and short implants give little support to the teeth ,27,28,29Comparing equivalent stress, the stress was concentrated in the neck of the implant and the surface of the peri-implant bone. Nearer to the base of the implant, the stress diminished. Comparing stress values, longer, thicker implants tended to have less stress. However, looking at the stress distribution shown in The results of the present study were roughly the same as when maximum principal stress was evaluated, and implant selection criteria suited to the shape and the quality of the bone were shown when virtual implant therapy was carried out on the subject who had been scanned for the study.A 3D FE model of a mandibular bone was created from 3D X-ray CT scanned images taken of a live subject. Simulating the clinical situation of implant therapy at the mandibular first molar, virtual extraction of the tooth was performed on the 3D FE mandibular model and 12 different implants diameters and lengths were virtually inserted in order to carry out a mechanical investigation. High stress concentration was found on the surfaces of the buccal and lingual peri-implant bone adjacent to the sides of the neck of all implants. The greatest stress value was approx. 6.0 MPa with implant diameter of 3.8 mm, approx. 4.5 MPa with implant diameter of 4.3 mm, and approx. 3.2 MPa with implant diameter of 6.0 mm. The stress on the peri-implant bone was found to decrease with increasing length and mainly in diameter of the implant.


Pancreatic lipase is critical for the digestion and absorption of dietary fats. The most abundant lipolytic enzymes secreted by the pancreas are pancreatic triglyceride lipase (PTL or PNLIP) and its family members, pancreatic lipase-related protein 1 (PNLIPRP1or PLRP1) and pancreatic lipase-related protein 2 (PNLIPRP2 or PLRP2). Unlike the family’s other members, PNLIPRP2 plays an elemental role in lipid digestion, especially for newborns. Therefore, if genetic factors cause gene mutation, or other factors lead to non-expression, it may have an effect on fat digestion and absorption, on the susceptibility to pancreas and intestinal pathogens. In this review, we will summarize what is known about the structure and function of PNLIPRP2 and the levels of PNLIPRP2 and associated various pathological states. Effective digestion and absorption of dietary fats is important, which begins in the stomach by preduodenal lipase with a small amount of dietary triglyceride . Then, tHSA149D17) is a member of the classic triglyceride lipase family. PNLIPRP1 and PNLIPRP2, respectively, have 68 and 65% homologous amino acid sequence with PNLIP  is about seven-fold greater than HPNLIPRP2 when against long-chain triglycerides and 10-fold higher than HPNLIPRP2 when against short- and medium-chain triglycerides. RPNLIPRP2 and MPNLIPRP2 have activity in the presence of bile salt micelles, and their activity can be increased by colipase . It was significantly lower in patients with chronic calcifying pancreatitis (CCP) than in the control group, and the ratio of HPNLIPRP2 to HPNLIP was 23.96% (W/W) and 28.3% (W/W) in CCP patients and controls, respectively . On the PNLIPRP2 was secreted not only from the pancreas but also from various tissues and cell types under certain conditions, such as cytotoxic T lymphocytes (CTL). It may play an auxiliary role in some types of cytotoxic T-cell-mediated lysis . Rabbit The structural parameters are responsible for the substrate specificity among these structural enzymes, and the degree of activity depends on the substrate and PNLIPRP2 species. These points remain, however, speculations and will deserve further structural studies to determine the conformational state of the PNLIPRP2 lid more precisely and also further investigations to elucidate the molecular mechanisms of PNLIPRP2 processing along with detailed analysis of the digestion products. It might pave the way for exploiting the different expressions and functions of PNLIPRP2 among species, different mutations of PNLIPRP2 among various races, and the relationship between PNLIPRP2 levels and various pathological states and for providing a new drug target to modulate lipase activity.DH and CY conceptualized and supervised the manuscript. GZ, QF, and FZ contributed to the drafting and editing of the manuscript. All 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.


The research covered the process of nanofiltration of low molecular weight organic compounds in aqueous solution. The article presents the results of experiments on membrane filtration of compounds containing amino groups in the aromatic ring and comparing them with the results for compounds without amino groups. The research was carried out for several commercial polymer membranes: HL, TS40, TS80, DL from various manufacturers. It has been shown that the presence of the amino group and its position in relation to the carboxyl group in the molecule affects the retention in the nanofiltration process. The research also included the oxidation products of selected pharmaceuticals. It has been shown that 4-Amino-3,5-dichlorophenol—a oxidation product of diclofenac and 4-ethylbenzaldehyde—a oxidation product of IBU, show poor separation efficiency on the selected commercial membranes, regardless of the pH value and the presence of natural organic matter (NOM). It has been shown that pre-ozonation of natural river water can improve the retention of pollutants removed. The recent developments in more advanced analytical methods have revealed new threats associated with the presence of various micropollutants in drinking water sources. One of such micro-pollutants are N-nitrosamines (NA). In many industrialized countries, they attract attention because of their high carcinogenicity and frequent occurrence in disinfected drinking water, in which chemical compounds initiating their formation are used . Bei et As recently assessed by Platzek , exposurFiltration processes are widely used in many areas of life, in the food and pharmaceutical industries, as well as in environmental protection ,16,17,18Surface waters, which are the main resources of drinking water in many parts of the world, contain a number of pollutants, including natural organic matter (NOM). NOM is a complex matrix of organic matter derived from the decomposition of living terrestrial and aquatic organisms and consists mainly of humic and fulvic and non-humic fractions, which are a mixture of carbohydrates, amino acids, and proteins ,20. The The removal effectiveness of the compounds containing an amino group in their structure during nanofiltration process depends on the additional functional groups in the molecule. Amino acids that have, in addition to the amino group, also a carboxyl group may have a different charge depending on their pH ,24. ThisThe aim of the study was to investigate the effect of the presence of amino groups on the effectiveness of nanofiltration. The study also includes tests for selected oxidation products of diclofenac (DCF) and ibuprofen (IBU). Due to the serious risk associated with the emission of amines to the environment, such tests are justified. The article does not cover all the needs related to the lack of knowledge in the field of degradation of aromatic amines and some pharmaceutical oxidation products. However, it makes an important contribution to the field of nanofiltration and the chemistry of hazardous environmental pollutants.Twelve compounds were tested in the study: amines, amino acids and pharmaceutical oxidation products. −1 were used. The selected compounds were dissolved in pure deionized water or, if the experiment required it, in natural raw or ozonated (15 min) water from rivers.Ibuprofen and diclofenac oxidation products: 4-Amino-3,5-dichlorofenol , sodium salt 2-Aminophenylacetic acid (2-APA) and 1-(4-isobutyl-phenyl)ethanol (MPPE) were synthesized and provided by SIA Chemspace . Ibuprofen (IBU) sodium salt, salicylic acid (SA), acetylsalicylic acid (ASA), 4-ethylbenzaldehyde (4-EBA), p-aminobenzoic acid (PABA), phenyloacetic acid (PAA), 5-amino-o-kresol (KR) and anthranilic acid (AA) were purchased from Sigma Aldrich , and 1-hydroxyibuprofen (OH-IBU) was obtained from AKos GmbH . For the tests, solutions with a mass concentration of the active substance of 5 mg·L−1 at 30 °C. The membrane filtration unit was built as model prototype by Danish Technological Institute . The flow rate was regulated by rotameter (6). The flat sheet membranes were posted in a tight cylindrical pressure chamber (4). The tangential inlet of the feed caused a spiral flow of liquid in the pressure chamber (4). The pressure in the chamber (4) was regulated by two valves (3). The tests were performed at a pressure of 1.0 MPa. The thermostated feed solution was pumped from thermostat (1) through the pressure chamber. The permeate was collected in a separate vessel (7), which caused a gradual concentration of the feed stream (retentate) (The nanofiltration process was carried out with the cross-flow method at a constant flow rate of liquid inside the bypass of 2 L·mintentate) . The con2 each were selected for the tests, the parameters of which are summarized in Four flat sheet membranes with an area of approximately 314 cmPC and RC are the concentrations of a specific compound in the permeate and retentate, respectively.The experimental results are expressed in terms of the retention by the membrane of the compounds used in the present work. Retention is determined by employing the substance concentration in the permeate with reference to a representative value of the substance concentration in the retentate solution, as follows:−1.All chemical compounds were monitored by determining their concentration using a Shimadzu Nexera-i LC-2040C 3D plus an apparatus equipped with a Kinetex C18 column (2.6 µm). The 0.1% formic acid–water solution (A) and the 0.1% formic acid–acetonitrile solution (B) were used as eluents. The columns were thermostated at 40 °C. The injection volume was 10 µL. Flow rates were from 0.4 to 0.5 mL·minA series of tests was performed in the natural water matrix from the Dobrzynka River (RW). The natural water samples were collected in September 2020 and stored at 6 °C. A series of tests were performed for various aromatic compounds with an amino group, including the oxidation products of the pharmaceuticals IBU and DCF. The pH values of the tested solutions ranged from three to six, depending on the type of the dissolved compound  due to their high polar nature .There was an increase in the retention efficiency of the 2-APA compound A relativThe test results were compared with the experiments carried out for natural river water and river water ozonized for 15 min. The results for the DCF and IBU products are shown, respectively, in 2/L) and a relatively high pH 8. Theoretically, these factors usually increased the retention of the tested compounds [The river water contained natural organic matter  and the pore size . Hence, different results were obtained for different membranes. In general, the lowest values of retention were obtained for PABA, which has an amino group in the para position to the carboxyl group. The higher values of retention were obtained with AA, which has an amino group in ortho position to the carboxyl group. It should be noted that, in the case of the TS80 membrane, no very marked differences were observed between the initial retention value and the in-process values, which was typical for an HL membrane. A clear decrease in retention during the filtration process usually indicates the dominant adsorption process in the separation mechanisms.Aromatic amines and their degradation products may contribute to the formation of n-nitrosamines, the carcinogenicity of which has been confirmed. Further research is needed on the effectiveness of the removal of compounds with amino groups in membrane filtration processes. The low efficiency of the separation of these compounds in the wastewater treatment process may pose a serious threat to the environment and human health.It was shown that the presence of the amino group and its position in relation to the carboxyl group in the aromatic ring affect the retention in the nanofiltration process. The experiments showed that the presence of an amino group in the molecule can significantly reduce the separation efficiency, even in the case of dense aromatic polymer membranes.The oxidation products of some pharmaceuticals, such as 4-Amino-3,5-dichlorophenol—the oxidation product of diclofenac or 4-ethylbenzaldehyde—the product of IBU oxidation showed poor separation efficiency in the selected commercial membranes, regardless of the pH value and the presence of natural organic matter (NOM).Further research is needed on the effectiveness of the removal of compounds with amino groups in membrane filtration processes. The low efficiency of the separation of these compounds in the wastewater treatment process may pose a serious threat to human health and the environment.It has been shown that the pre-ozonation of natural river water can improve the retention of pollutants removed therefrom. It is supposed that this is due to the formation of low-molecular-weight oxidation products, which constitute an additional spatial barrier inside the pores of the membrane.


The impact of the kinetic parameters (temperature and refining time) on the behaviour and the degree of removal of impurities was evaluated. Chemical and metallographic analysis of the obtained ingots is also discussed.The current study presents the electron beam melting (EBM) efficiency of copper technogenic material with high impurity content  by means of thermodynamic analysis and experimental tests. On the basis of the calculated values of Gibbs free energy and the physical state of the impurity (liquid and gaseous), a thermodynamic assessment of the possible chemical interactions occurring in the Cu-Cu Copper continues to be one of the most important metals that are at the basis of the economic development of society and the human efforts to achieve a higher standard of living . This isThe impurities content in secondary copper raw materials varies from 0.8% to 1.5% . They usAccording to , copper-In traditional metallurgy, the process of refining Cu scrap is carried out by either pyrometallurgical or hydrometallurgical method . In the The requirements for the quality of copper, its chemical composition and structure are constantly increasing with the development of new branches of energy and electronics . This neMethods such as laser, plasma and electron beam melting (EBM) are successfully applied following the development of modern metallurgy and effective methods for refining metals and alloys ,10,11. OThere are a number of publications in the literature related to the thermodynamics and kinetics of copper refining from impurities at atmospheric pressure ,17,18,19This work is a continuation of the research conducted so far by the team . In 20]20], the −3 Pa.The experiments for copper technogenic material melting were performed using EBM installation with power 60 kW (ELIT-60) at the Physical problems of the EB technologies laboratory of the Institute of electronics, Bulgarian Academy of Sciences. ELIT-60  is equipped with one electron gun (accelerating voltage of 24 kV), a feeding mechanism for horizontal input of the raw material, an extraction system (pulling mechanism), a water-cooled copper cylindrical crucible (a diameter of 50 mm) with moving bottom, where the molten metal solidifies and a circulation water cooling system–The copper content in the investigated technogenic material is 99.45% Cu (anode residues after an electrolysis process). The impurities with higher concentrations are: O (0.2251%), Ni (0.1%), As (0.07%), Se (0.0382%), Pb (0.021%), Bi (0.015%) and Ag (0.014%). With lower content are: S (0.0028%); Sb (0.0095%), Te (0.0068%), Sn (0.0034%) and Zn (0.0023%). The lowest is the content of Co, Cd, Fe, Au, etc. (<20 ppm), therefore these impurities are not taken into consideration in the further analysis.The raw  material was melted under single processing at melting powers of 6 kW (T = 1460 K), 7 kW (T = 1500 K), 13 kW (T = 1700 K) and 19.5 kW (T = 1800 K). At T = 1500 K the lengths of melting time are 15 min and 35 min, while at T = 1700 K the retention time is 20 min and 45 min. The melting time for T = 1460 K is 20 min and for T = 1800 K is 25 min, respectively. The raw materials mass was about 500 g (each sample). The chemical composition of the copper samples before and after EBM is determined with ARL 4460 OES Thermo Scientific spectrometer . The spectrometer is equipped with a Paschen-Runge vacuum polychromator working in argon atmosphere. Oxygen analyses of the samples were performed with ELTRA OH-900 oxygen/hydrogen determinator .A 4% solution of nitric acid in ethyl alcohol was used to etch and reveal the microstructure of the obtained metal specimens. The etching time was 30 s.A light microscope Leica DM2500  with a digital camera Leica EC3  was used for the metallographic study of the macro or micro-structure of polished and etched surfaces of copper samples. The image processing was performed using the Leica LAS software .The thermodynamic analysis of the possible chemical interactions occurring during the refining of copper from impurities such as Se, Te, Bi, As, Sb, Pb, Sn, Ni, Zn, Ag under EBM conditions is performed on the basis of the Gibbs free energy (ΔF) and the physical state of the impurities. The analysis was carried out using the professional thermochemical calculation programme HSC Chemistry ver.7.1, module “Reaction Equation” , taking Since there is a constant pressure in the vacuum chamber during EBM, the main parameters that affect the refining process are the temperature of the metal and its physical state . Another2O, Pb and Bi2O3 slightly exceed the operating temperature of 1800 K, which allows us to assume that they will also be in gaseous state under vacuum conditions.It can be seen that under vacuum conditions and the studied temperature range, the metal impurities present in Cu such as Pb, Bi, Sn, Sb, Ag and their oxides will be in a liquid state. Impurities such as Zn, As and its oxide have a boiling point significantly lower than the melting point of copper and they will be in the gaseous state. 2Se and Cu2Te. These compounds will be present in liquid state as they have a low melting point.Phase diagrams Cu-Se and Cu-Te show that both impurities are present in copper in the form of intermetallic compounds: copper selenides and tellurides ,24. At lNi and Ag remain in liquid state. Nickel has complete mutual solubility in copper and at 1358 K (the melting temperature of copper) it completely passes into liquid phase ,26. The 2O, metal impurities and their oxides, and they are in liquid, solid or gaseous state depending on the thermodynamic conditions of refining and the type of impurity.Therefore, under EBM conditions, the liquid metal is a complex system of Cu, Cui): (pRiO) > (pRi) > (pCu2O) > (pCu).Under e-beam melting conditions, the refining processes take place mainly on the reaction surfaces of the liquid metal -Ri is made on the basis of the following equations:T,Cu/Cu2O, ΔFT,Ri/RiO and ΔFT are the Gibbs free energies of the respective processes. The indices (s), (l) and (g) mean that the substance is in a solid, liquid or gaseous state, respectively. The calculations were performed under melting conditions: at temperatures of 1460 K, 1500 K, 1600 K, 1700 K and 1800 K and operating pressure in the vacuum chamber of 10−3 Pa. These parameters correspond to the actual conditions of melting and refining of copper technogenic material in the EBM plant.Thermodynamic evaluation of the possible chemical interactions in the system Cu(l)-CuT,Cu/Cu2O) and metal-impurities . The analysis of the obtained dependences shows that the probability of oxidation of ZnO(g) to ZnO(s) is the highest and as the temperature increases, the affinity of Zn(g) to oxygen decreases significantly. In the whole temperature range, the values of ΔF of formation of SnO2(l), Sb2O3(l), As2O3(l) and PbO(l) are higher than that of the oxidation of Cu(l) to Cu2O(l). The thermodynamic probability of oxidation of bismuth to Bi2O3(l) is almost the same as that of copper to Cu2O, while the probability of oxidation of copper telluride to TeO2(l) and copper selenide to SeO2(l) is significantly lower than that of copper.2(g) and Pb(l) → PbO(g) increase significantly but they are significantly lower than those of oxidation to liquid oxides.It is observed that as the temperature increases, the values of the Gibbs energy of the oxidation of impurities: Sn(l) → SnO2O3(g), As → As2O3(g), Cu2Se → SeO2(g) and Cu2Te → TeO2(g) is almost independent of temperature.Energy of oxidation of Bi(l) → Bi2Se shows a higher thermodynamic probability of oxidizing to SeO2(g) rather than to SeO2(l). This trend is also observed in Cu2Te when increasing the temperature above 1500 K.Out of the impurities present in copper, only Cu2O and metal impurities is described by Equation (3). The calculated values of ΔFT are shown in 2O(s)) as the melting temperature of the copper oxide is higher (1508 K).The possibility of chemical interactions between Cu2O regardless of its phase state. The calculated values of ΔFT are high, which indicates a high thermodynamic probability of the process. The chemical interaction of copper oxide with gaseous zinc is most likely to occur in this case as well. Zinc is oxidized to a stable ZnO compound which floats to the reaction surface and Zn will be separated by degassing [It is observed that in the studied temperature range all impurities will interact with Cuegassing .T are positive within the investigated temperature range. According to the thermodynamic laws, the course of a given reaction is possible only when the calculated value of the free energy is negative (ΔFT < 0) [The oxidation reactions of Ni and Ag impurities are not shown in ΔFT < 0) .2(l), Sb2O3(l), PbO(l), Bi2O3(l).The analysis of the reactions with formation of gaseous phase shows that at temperatures above 1600 K, Se, Te and As will be removed mainly in gaseous state, while Sn, Sb, Pb, Bi will be oxidized mainly to oxides–SnOFollowing the performed thermodynamic analysis, it can be concluded that in the studied temperature range impurities such as Se, Te and As will be oxidized to gaseous oxides, while Sn, Sb, Pb, Bi will be oxidized mainly to liquid oxides.The influence of the temperature (beam power) and the duration of the retention time (τ), during which the melting metal is in liquid state, on the degree of removal of the impurities present in copper technogenic material is evaluated as well. Data about chemical analysis of the impurities concentration of the starting copper material (before EBMR) and of the specimens after e-beam refining of Cu, material losses  and structure of the melted samples are obtained and analyzed under each of the technological regimes studied.loss) which are mainly due to evaporation and also to splashes is presented in loss  is also presented in Ri and CRi are the initial and final impurity concentrations, respectively.The influence of the temperature on the degree of impurity removal (α) is presented in 2O. The lower removal values of As (84.9%) and Sb (84.2%) can be explained by the formation of complex compounds between these impurities and Ni  during EBMR of copper.It can be observed that increasing the temperature from 1460 K to 1600 K leads to the intensive removal of impurities such as Bi, Pb, Sn, Zn and As. The degree of removal of these impurities with the exception of As is more than 93%. In this case, the degree of oxygen removal is 99.3%. This means that during EBM the removal of impurities takes place mainly as a result of the chemical interaction with CuThe removal rates (degree of refining) of Se and Te at 1600 K are 62.6% and 82.4%, respectively which can be explained by the fact that these impurities form intermetallic compounds with copper, which are more difficult to oxidize. This fact is consistent with the significantly lower values of the Gibbs energy of the oxidation reactions of these impurities .The removal values of oxygen and sulphur at 1600 K are 99.33% and 96.43%, respectively and they increase to 99.56% and 99.64% at 1800 K. Silver and almost all of the nickel remain in copper. Nickel losses can be explained by the total copper losses.The influence of the refining time on the degree of impurity removal (α) is evaluated for 1500 K and 1700 K and the calculated values are presented in The highest removal efficiency of oxygen is 99.6%  and is obtained at T = 1700 K and T = 1800 K , Table 1At a temperature of 1500 K extending the refining time increases the rate of sulphur removal from 75% to 82.1%. At higher temperatures, the removal rate is 99.6% regardless of the retention time. The same trend is observed with oxygen.2O), E(Cu-Cu2S), the presence of loose eutectic melts of insoluble in copper impurities  and crystallization of intermetallic phases of Se and Te. The dark stripes in the micrograph of the transverse section of the sample (The microstructure observed on the upper surface of the sample a is dende sample b show thThe effects of the e-beam power and melting time on the microstructures of the copper ingots are presented in By increasing the beam power (temperature) for 20 min refining time a–d, form2O-Rx system in the studied temperature range 1460–1800 K and the conducted experimental studies, the influence of the kinetic parameters-temperature (beam power) and melting time on the degree of removal of non-metallic  and metallic  impurities, the refining efficiency and the structure of the resulting copper was evaluated. The conclusions can be summarized as follows:The results obtained show that the electron beam melting method can be successfully applied for refining copper technogenic material with a high content of impurities, which in the conditions of EBM are in a gaseous state  and reach nearly 100% removal degree and ~97% for Sb.Oxygen and sulphur also reach a high degree of removal (≥99%). Under the studied conditions, the maximum degree of refining of Se and Te is 73% and 85.3%, respectively, which is due to the fact that Se and Te form intermetallic compounds with copper, which are more difficult to oxidize. This corresponds to significantly lower values of the Gibbs energy of the oxidation reactions of these impurities.Silver and most of the nickel remain in copper. Under vacuum conditions and at the temperature range studied, silver does not oxidize or evaporate. The low degree of refining of nickel (34–42%) from copper can be explained by the good solubility of this impurity in copper. In addition, nickel is also not oxidized in the studied temperature range. It was found that raising the temperature above 1700 K, as well as extending the melting time over 20 min hardly change the purity and structure of the resulting refined metal.At temperatures in the range 1600–1800 K, the achieved refining efficiency is 78–85% and the purity of copper after EBM is 99.9%. The highest total refining efficiency of 84.6% is seen at a beam power of 19.5 kW for 20 min melting time and the best purification of copper technogenic material (99.92%) is achieved.The paper investigates the possibility of removing impurities from technogenic copper material (99.45%) using EBM. On the basis of the thermodynamic analysis of the possible chemical interactions occurring in the Cu-Cu