Background and purpose: With the emergence of linear accelerators in radiotherapy, it becomes necessary to accurately select new dosing regimens. The purpose of this study was to assess the morphological changes of spermatogenesis after radiation exposure. Materials and methods: Male Wistar rats (n = 40) were subjected to targeted ionizing radiation on a pulsed electron accelerator “NOVAC-11” with doses of 2, 8 and 12 Gy. Spermatogenesis was assessed a week later using light microscopy and immunohistochemical method . Results: A decrease in the number of normal germ cells was seen in all experimental groups, while radioresistant Sertoli and Leydig cells were barely affected. The most serious damage to the tubules and germ cells was observed in 8 and 12 Gy irradiation groups. IHC analysis of testes after irradiation showed a shift in the proliferative-apoptotic balance toward apoptosis of germ cells: a decrease in the expression levels of Ki-67 and Bcl-2, an increase in p53-positive and caspase 3-positive cells by the end of the experiment. Conclusion: Dose-dependent progressive pathomorphological changes in histoarchitectonics of the testes are traced, and a decrease in the number of germ cells is seen on the seventh day after irradiation with a pulsed electron accelerator “NOVAC-11”. Infertility is a major health problem worldwide and is estimated to affect 8–12% of couples in the reproductive age group. A Global Burden of Disease survey reported that between 1990 and 2017, the age-standardized prevalence of infertility increased annually by 0.370% in women and by 0.291% in men. The cause of infertility lies solely with the man in 20–30% of cases, and a male cause is contributory in a further 20% [Radiation exposure plays a crucial role in spermatogenesis disorders, along with such risk factors as bad habits, the influence of heat, chemicals, anatomical features, and hormonal misbalance. People who live in zones of radionuclide pollution and near nuclear facilities may endure the consequences of past man-made disasters .Radiation therapy is successfully used in medical practice for malignancies treatment. In particular, reproductive system malignancies are the leading cause in the structure of oncological mortality .For general or targeted effects in external beam radiotherapy, there can be used cobalt machines that emit high activity γ-rays, linear electron accelerators, which produce both high-energy and direct electron beam (β-irradiation), and other machines with ionic, proton and neutron types of irradiation. The advantages of targeted radiotherapy are the minimal damage risk to neighboring organs under the maximum possible radiation dose, high rates of effectiveness and favorable prognosis for patients.Nevertheless, with the emergence of new diagnostic and therapeutic methods of radiology, we face the problem of their safety. The selection of optimal radiation doses, their proper adjustment and leveling of their toxicity are the key points that have become an increasingly important task of modern oncology.The effect of radiotherapy on the human reproductive system is a controversial issue. It is known that radiotherapy is characterized by a gonadotoxic impact on the male organism and significantly reduces the number of dividing spermatogonia and spermatocytes and can lead to azoospermia . DisordeRecent research results report the more aggressive impact of targeted γ-ray irradiation compared to the general one; however, it is important to consider, for example, electron beam therapy, which is widely used nowadays because of its high success rate in the treatment of, for example, gynecological malignancies . At the γ-Rays are used in most of the existing experimental models of male infertility. Because of the appearance of the linear electron accelerators, there is a need for a precise selection of new dosing regimens and detailed studying of this perspective method. Hence, in our study, for the first time, we highlight the use of local electron irradiation on testes and estimate its effects on rats’ spermatogenesis.The objective of the study: The assessment of proliferation and apoptosis in testes of rats after local electron irradiation under different dosing regimens .The experimental study was carried out on the basis of the Medical Radiological Scientific Center, named after A.F. Tsyb.Male Wistar rats  were kept in a vivarium, two animals in each plastic cage under a controlled temperature (22 °C), with a 12 h light day, air conditioning at a temperature of 23 °C and humidity 40–60%, on a standard diet with water ad libitum. Rats were randomly divided into control  and experimental  groups depending on the dose of single targeted electron irradiation they were exposed to: II–2 Gy, III–8 Gy, IV–12 Gy. The rats were exposed to local abdominopelvic irradiation in the testes projection.Irradiation procedures were carried out in the Department of Radiation Biophysics of A. Tsyb Medical Radiological Research Center on the pulsed electron accelerator “NOVAC-11” —the first IOeRT mobile electron linear accelerator designed worldwide in 1997. It is the first example of a radiotherapy device that is flexible and easy to use, powerful and safe. It is able to perform the IOeRT treatment in only 100 s. NOVAC 11 ensures proper coverage of any anatomic region where the IOeRT treatment needs to be delivered. By selecting the correct applicator/energy combination, it is possible to treat any neoplastic disease effectively and safely. NOVAC 11 maximum energy is 10 MeV which allows treating targets with a thickness up to 2.6 cm inside the 90% isodose (3.0 cm inside the 80% isodose). According to the performed dosimetric studies, an electron beam with 10 MeV energy potential guarantees irradiation of the zone of interest with the required dose. Before irradiation, rats of experimental groups were treated with a single administration of ketamine  and xylazine . Anesthetized rats were placed on the object table one by one, in the supine position, with splayed legs, so that the radiation beam focused on the testes while the lungs and heart remained in the area of radiation shadow. The tube was applied to the irradiated region so that its end was not more than two millimeters above the skin in the perpendicular position to it.Animals of all groups (I–IV) were removed from the experiment by administration of high doses of anesthetic. After routine euthanasia, the testes were removed from the rats according to the design of the experiment. At the same time, the appearance was evaluated: weight , size and condition of the parenchyma on the section. Time of death: for all groups, after seven days.The study was performed in accordance with the “International Recommendations for Biomedical Research Using Animals” , the “European Convention for the Protection of Vertebrate Animals used for Experiments or Other Scientific Purposes”  and Guidelines for the conduct of biomedical research on the care and use of laboratory animals  and the Rules of laboratory practice and the order of the Ministry of Health of the Russian Federation No. 199n dated 01.04.2016 “On the approval of the rules of laboratory practice”, as well as approved by the Local Ethics Committee of Sechenov University .After extraction, the appearance of the testes and the state of the parenchyma on the cut were assessed and weighed (in grams). Then they were cut parallel to the sagittal plane every 2 mm, fixed in Bouin’s solution; after insertion , they were embedded in paraffin blocks, from which serial sections (2 µm thick) were prepared, dewaxed, dehydrated and stained with hematoxylin and eosin (H&E) for histological evaluation.Morphological and morphometric analysis was carried out in 10 randomly selected fields of view of the microscope at ×100 and ×200 magnification in 4 random sections from each sample, moving the slides at equal intervals along the X and Y axes, using a semi-automatic image analyzer. Light microscopy was performed using a video microscopy system , and morphometric data were obtained using software for image processing and analysis (Leica Application Suite (LAS) Version 4.9.0) . The spep < 0.01. In the absence of normal distribution of data, nonparametric F. Wilcoxon  criterion was used with significance level of p < 0.01.The data were statistically processed using SPSS Statistics version 10.0 for Windows . The arithmetic mean and the root-mean-square deviation were then calculated. The fact that the results corresponded to the normal distribution was proved using the Kolmogorov–Smirnov criterion. To compare the two samples, t-criterion was used with significance level of Immunohistochemical study was performed on paraffin sections according to a standard protocol in an automatic mode in a Ventana immunohistostiner . We used primary antibodies to Ki-67 , Bcl-2 , p53  and Caspase 3 . For each marker, control studies were performed in order to exclude pseudo-positive and pseudo-negative results. Cell nuclei were counterstained with Mayer’s hematoxylin; the sections were washed under running water, dehydrated and embedded in a balm.Evaluation of the results of immunohistochemical reactions was carried out according to the distribution of staining and using a 3-point system with calculation of the number of immunopositive cells in 10 randomly selected fields of view at magnification ×200.After electron irradiation, in all experimental groups (II–VII), a decrease in the area of the seminiferous tubules, their diameter, and the height of the spermatogenic epithelium compared to the samples of normal testes were found. Most of the seminiferous tubules corresponded mainly to types III, as well as types II and IV of the spermatogenic cycle.Light microscopy of sections of the testes of the control group showed normal histoarchitectonics with physiological spermatogenesis.In the samples of the experimental groups, a decrease in the number of germ cells, signs of degenerative changes and lysis of spermatids and spermatozoa, and a decrease in the number of spermatogonia, Sertoli and Leydig cells without changing their structure were revealed. The most serious damage to the tubules, up to aplasia of the spermatogenic epithelium and extensive vacuolization, was observed in the irradiation groups of 8–12 Gy. Destruction and wrinkling of the tubules, aplasia of the epithelium and its vacuolization progressed .2; diameter—189.083 μm; p < 0.01) with disorganization of the epithelium and loss of cell polarity accounted for up to 1/8 of the testis (1–2 transverse sections of tubules in sight).In the samples of the irradiation group with a dose of 2 Gy, the proportion of damaged seminiferous tubules . The area of single primary spermatocytes was 0.8 µm2 at p < 0.01, and the diameter was 0.9 µm at p < 0.01, which is 1.1 times more than normal.In the 8 Gy irradiation group, 1/3 of the seminiferous tubules showed the appearance of highly degenerated spermatids and spermatocytes combined into seminal balls  than in the previous groups, and therefore, an increase in the interstitial component was observed testes with edema. Damage to the seminiferous tubules was 3/4.The largest vacuoles were found in the seminiferous tubules of the irradiation group with a dose of 12 Gy; their area reached 3.71 µmIn all studied groups, positive IHC reactions to Ki-67, Bcl-2, p-53 and Caspase 3 were seen, but the degree of their severity correlated with the radiation dose, which indicates a disruption in the proliferative–apoptotic balance. For clarity, we present images of the seminiferous tubules’ sections of all irradiation groups .Ki-67-positive reaction in spermatogonia in the 2 Gy irradiation group was 1.2 times less than in the samples of the control group, and the degree of staining tended to decrease in a dose-dependent manner. Moderate marking of gametes was observed in the samples, especially in 8 Gy and 12 Gy irradiation groups, in which the intensity of staining decreased 1.7–2.7 times compared with the control one. Primary spermatocytes showed the maximum intensity of immunolabeling. There was an increase in the number of Ki-67-positive interstitial endocrinocytes in the 12 Gy irradiation group compared with the rest groups.The expression of apoptotic p53 in the spermatogenic epithelium of experimental groups (mainly spermatogonia and type I spermatocytes) tended to increase, though there could be seen an abrupt decline of its expression in group III. Such a decline was also noted in the expression of the Bcl-2 marker at high irradiation doses . However, a decrease in anti-apoptotic activity (Bcl-2) was still traced in experimental groups . A practically similar immunohistochemical pattern compared to the expression of the p53 protein was demonstrated by the expression of the anti-apoptotic protein Bcl-2: it was also noted that at high doses of irradiation, for the first time, an increase in staining of interstitial endocrinocytes was revealed compared to low doses.The number of Cas-3-positive germ cells a week after irradiation at a dose of 2 Gy was 2.46 times higher than in the control group. Then, a constant increase in the number of Cas-3-positive germ cells was observed depending on the dose increase: when irradiated at a dose of 8 Gy—5.46 times, when irradiated at a dose of 12 Gy—6.14 times compared with the control.In our experimental research, we, for the first time, studied the effects of targeted electron irradiation with energy 10 MeV in different dosing modes on the spermatogenesis of male rats.Our findings demonstrated a progressive decrease in all indicators and changes in the cells’ compound in experimental groups compared to the control one, especially while applying sublethal doses (8 and 12 Gy). According to the literature data, only premeiotic germ cells in mice’s testes, irradiated by targeted x-ray in moderate (less than 4 Gy) doses, go into apoptosis . HoweverHowever, in our research, using a pulse electron accelerator «NOVAC-11» did not cause such morphological changes of seminiferous tubules after targeted irradiation in a dose of 2 Gy. Pathological changes were limited by the disorganization of the epithelium, decreasing the number of spermatids and spermatocytes with the preservation of their normal cell structure. The observed effect can be explained by the softer effect of electrons on testis’ tissue and impulse impact, unlike γ-radiation. In comparison with existing experimental models, the observed pathomorphological changes at the dose of 2 Gy are less destructive than after γ-radiation exposure in other scientific works, and that is why our model could be considered an optimal one for spermatogenesis study.The rise in the dose to 8 Gy showed aggravation of aplasia. Since doses of 8 and 12 Gy showed extremely negative effects on spermatogenesis, the dose of 2 Gy could be considered borderline.We observed the emergence of degenerated spermatocytes and spermatids, which was described by some of the authors after general γ-irradiation in doses of 8 and 10 Gy . In our Some researchers’ data show the absence of germ cells in most seminiferous tubules or the detection of only undifferentiated spermatogonia in the basal compartment after cobalt γ-irradiation in a dose of 14 Gy (Shohada-E-Tajrish machine) . PracticThe course of physiological spermatogenesis is possible owing to the proliferation-apoptotic gametes’ balance. In our study, the IHC reactions confirmed that the level of proapoptotic p53 marker increases, while the level of the proliferation Ki-67 and antia-poptotic Bcl-2 markers decreases after germ cells’ damage, and these results agree with previous studies that show similar results unless the cells are radioresistant ,13,14. AMoreover, in our previous study, the results of the TUNEL reaction and the spectrum of caspase  indicate the internal  pathway of apoptosis of the germ cells of irradiated testes and a more pronounced increase in the expression of caspase-9 in the group with the highest radiation exposure (8 Gy)—on a possible correlation between the absorbed dose of ionizing radiation and the degree of damage to mitochondria .Thus, we can assume that the effects of irradiation and the damage level of spermatogenic epithelium depend on doses. Probably, a threshold dose is much higher for electron irradiation on the accelerator “NOVAC-11” due to a lighter dosing mode. Another important factor of irradiation efficiency is its type (β-irradiation (electron), γ-rays, x-rays etc.), its focus , activity and power of the machine and spermatogenesis stage. This assumption was confirmed by the results of the morphometrical analysis: decreasing of testes mass, seminiferous tubules diameter and height of the germinal epithelium due to a reduction in the number of differentiated germ cells, which depends on the type of radiational exposure at different dosing regimens ,18.The detected progressive pathomorphological changes of histoarchitectonics in testes are dose-dependent. These alterations manifest in a decrease in the number of spermatogonia and other germ cells and are already seen on the seventh day after irradiation. The basis of the revealed hyperplasia of Leydig cells as a reaction to irradiation is a sharp imbalance of pro- and anti-apoptotic factors toward dominance in most cells of the expression of the anti-apoptotic factor Bcl-2 (with less significant activation of Ki-67 expression) and extremely low expression of p53. With an increase in the radiation dose, the number of apoptotic bodies also increases. In all experimental groups, a dose-dependent decrease in Ki-67 positive and Bcl-2 positive germ cells was noted a week after irradiation and, on the contrary, an increase in p53-positive and Caspase-3 positive germ cells. The recorded morphological changes of spermatogenesis when exposed to the pulsed electron irradiation on the accelerator “NOVAC-11” are more gentle, in contrast to other applied machines.


The Scandcleft intercentre study evaluates the outcomes of four surgical protocols  for treatment of children with unilateral cleft lip and palate (UCLP) in a set of three randomized trials of primary surgery .To evaluate and compare dental arch relationships of 5-, 8-, and 10-year-old children with UCLP after four different protocols of primary surgery and to compare three dental indices. The results are secondary outcomes of the overall trial.n = 418), 8 (n = 411), and 10 years (n = 410) were analysed by a blinded panel of orthodontists using the Eurocran index, the 5-year-olds’ (5YO) index, and the GOSLON Yardstick. Student’s t-test, Pearson’s correlation, chi-square test, and kappa statistics were used in statistical analyses.Study models taken at the ages of 5  and in Trial 1 Arm B (at 5 and 10 years of age) than in Trial 3 Arm D. The mean Eurocran dental index scores of the total material at 5, 8, and 10 years of age were 2.50, 2.60, and 2.26, and those of the 5YO index and GOSLON Yardstick were 2.77, 2.90, and 2.54, respectively. At age 10 years, 75.8% of the patients had had orthodontic treatment.The results of these three trials do not provide evidence that one surgical method is superior to the others. The reliabilities of the dental indices were acceptable, and significant correlations existed between the indices at all ages. The reliability of the Eurocran palatal index was questionable.ISRCTN29932826. Controversy about primary cleft surgery has led to several variations in type, technique, and sequencing of lip and palate surgery in unilateral cleft lip and palate (UCLP). The aim of the Scandcleft intercentre study is to test the outcomes of four surgical protocols for treatment of children with complete UCLP. The project consists of three concurrent randomized trials of primary palatal surgery for infants born with UCLP. The study was developed and executed by ten North European cleft teams: Aarhus/Copenhagen (Denmark), Helsinki (Finland), Bergen/Oslo (Norway), Gothenburg/Linköping/Stockholm (Sweden), Manchester/Belfast (UK). Belfast withdrew at the beginning of the study.The principal outcome measures of the Scandcleft study are speech and dentofacial development. The hypothesis of the study is that variations in surgical methods, timing, and staging are not associated with difference in outcome in UCLP. The design, background, surgical methods, and comparisons of dental arch relationships with 5-year-olds’ (5YO) index, GOSLON Yardstick and Modified Huddart Bodenham (MHB) index at 5 and 8 years of age have been published earlier . This paOne of the challenges in orthodontic treatment and research of children with UCLP, in early adulthood, is the time lapse between primary surgery and dentofacial outcome. Regardless of the surgical method for primary palatal closure, facial growth in UCLP is characterized by a progressive retrusion of the profile relative to the cranial base involving the nasal bone, the mandible, and especially the maxilla , 8. AnteSeveral dental indices have been developed for comparison of surgical methods, auditing the results of primary cleft surgery, and prediction of outcome. The most commonly used index for evaluation of occlusal outcome and severity of crossbite in children with UCLP is the GOSLON Yardstick . Other cThe Eurocran index is a modification of the GOSLON Yardstick and 5YO index . It asseWhereas the 5YO, Eurocran indices and the GOSLON Yardstick were developed for categorizing the degree of malocclusion, the HB and MHB indices use linear scales to measure maxillary arch constriction. The MHB index scores each maxillary tooth and its opposing tooth based on presence and degree of crossbite. These scores are summed to produce the overall score. The more negative the score, the more severe the crossbite.The 5YO, Eurocran indices, and the GOSLON Yardstick have proven to be reliable and capable of discriminating the quality of dental arch relationships in children with ULCP within and between centres and treatment protocols , 17–20. The purpose of this Scandcleft paper was to evaluate the occlusal outcome of UCLP after four different protocols of primary surgery at 5, 8, and 10 years of age. The additional aim of the research was to compare the Eurocran index with the 5YO index and the GOSLON Yardstick. The hypotheses were that variations in surgical method, timing, and staging are not associated with different outcomes in UCLP, and the results do not differ between the different indices.Nine cleft centres in Denmark, Finland, Norway, Sweden, and the UK participated in a set of three randomized trials of primary surgery. Three groups of centres  tested surgical methods  against a common method (Arm A). The background of the study, material  had undergone orthodontic treatment . No presurgical orthopaedics was used. In Trial 1, seven of the children had used nasal elevators and eleven children used nasal plugs in early childhood.The goals of the bone grafting of the alveolar cleft in the mixed dentition are to separate the oral and nasal cavities, to stabilize and consolidate the maxilla with a bony union, to create adequate bone for region of the upper incisors and canines, and to support the alar base and nose. The orthodontic protocols of the different cleft centres after 8 years of age prior to alveolar bone grafting operation were rather similar. After multidisciplinary team assessment with the surgeons, and agreement to perform bone grafting, presurgical orthodontics was started in the upper arch with a removable appliance or quad helix and / or labial fixed appliances. The orthodontic treatment was planned individually. The goals of the orthodontic treatment included correction of crossbites and transverse and sagittal relationships; alignment, rotation and proclination of upper incisors to establish maxillary arch form and to create room for alveolar bone graft placement. In addition, the lesser dentoalveolar segment was usually rotated laterally as in UCLP the lesser segment is often mesially and distally displaced.In very poor cases with bilateral crossbite and with reverse overjet with proclined incisors, the crossbite was not corrected at this age as the later orthognathic surgical treatment was planned. Only one small centre in Trial 1 used Delaire face masks prior bone graft for protraction, if needed. Most of the orthodontic treatment was performed in the cleft centres. In one large centre in Trial 2, most of the orthodontic treatment was performed locally according to the instructions of the cleft centre.Ratings of duplicate dental models using the 5YO and Eurocran indices and the GOSLON Yardstick were done by cleft orthodontists of the participating cleft centres. The ratings were performed in three meetings during years 2012 (rating of the 5-year models), 2015 (rating of the 8-year models), and 2018 (rating of the 10-year models). The ratings were performed during several days by blinded panels of orthodontists who scored independently all models. Customized scoring sheets were used. During the ratings reference models of both palatal morphology  and dentThe research protocol was approved by all centres and local ethical approvals were obtained: Denmark (Aarhus and Copenhagen), Regional Ethics Committee 1997/4121, Finland (Helsinki) 4/9/97, Norway (Bergen and Oslo) S-97152, Sweden (Gothenburg) R257-97, (Stockholm and Linköping) 97-372, UK (Manchester) 99/197 (CM/96/197). Principles outlined in the Declaration of Helsinki were followed.T-tests were used in comparisons of the trials. Pearson’s correlation was used to compare the indices with each other. Chi-square statistics was calculated to compare orthodontic treatment between trials. Test statistics with P-values equal to or less than 0.05 were considered significant. Kappa values above 0.81 were regarded as very good, values between 0.61 and 0.80 as good, values between 0.41 and 0.60 as moderate, and values below 0.40 as poor , and the Eurocran and the GOSLON Yardstick scores at 8 and 10 years of age .Significant correlations existed between the Eurocran dental index scores and the 5YO scores at 5 years of age . The palatal scores varied from 1.62 (Arm D) to 1.73 (Arm A). As the reliability of the palatal index was questionable, no further statistical comparisons were performed.P < 0.001), with those in Trial 1 having most orthodontic treatment.Comparisons within each trial showed no significant differences in the mean 5-, 8-, and 10-year index scores or their distributions between the common method A and methods B, C, and D. A trend of slightly more unfavourable scores with increasing age from 5 to 8 years was noted with all indices . At the Comparisons between trials detected significantly better mean index scores in Trial 2 Arm C  and in Trial 1 Arm B (at 5 and 10 years of age) than in Trial 3 Arm D . In addiMost of the models of the total sample (31.7–48%) were rated to category 2 with all indices at all ages, except for the Eurocran index at 8 years of age . At thatA trend of a slightly more unfavourable distribution of index scores with increasing age from 5 to 8 years was noted. At the age of 10 years, the proportion of index scores 1 and 2 increased with both the Eurocran index and GOSLON Yardstick. The proportions of all patients at all ages in Eurocran and 5YO indices as well as in GOSLON Yardstick categories excellent and good  between Most of the palatal scores (56.9%) were moderate (grade 2), 36.8% were good (grade 1), and 6.3% were poor (grade 3).These are secondary reports of the dental arch relationships of the Scandcleft randomized trials in which three groups of centres tested four surgical protocols. This is the first follow-up study from 5 to 10 years of age and the first comparison of the dental indices used in the rating of the study models. The follow-up will continue until early adulthood.The results at 10 years of age agree with earlier findings of the Scandcleft study on dental arch relationships at the age of 5 and 8 years; none of the three trials revealed any significant differences within the trials between the common method and the alternative protocol regarding the dental arch relationship . HoweverWhen comparing the results of primary surgery on later dentofacial development, technique, staging, and sequencing of the palatal surgery are important. Other key variables that may affect surgical iatrogenesis are the skill and experience of the operating surgeon. A limitation of the Scandcleft study is the number and varying experience of the surgeons and the predominance of common method Arm A . There wBesides surgical iatrogenesis, genetics, congenital dysmorphology of the midface, other variations intrinsically associated with the cleft and functional adaptations may interfere with the normal growth pattern in individuals with clefts . An inteOrthodontic treatment and correction of a crossbite may influence the indices positively. The greatest influence on the GOSLON score is from the anteroposterior assessment or overjet. The second and third determinants are vertical and transversal assessment, respectively. If there are dental compensations present such as proclination / retroclination of maxillary incisors or mandibular incisors, the score may shift to the next higher or lower score, depending on the magnitude of the compensation . In addiAccording to the World Health Organization, the ideal index should meet all of the following criteria: reliability, validity, and acceptance by the profession; lend itself to statistical analysis; and be administratively simple . In larget al. , whereas the prediction of cases in group 3 was very poor at all ages. Of the 5-year-olds allocated to group 3, 4, or 5, 60% had a good or fair dental arch relationship at 19 years. The use of the 5YO index before orthodontic treatment may help to predict outcome and clinical need for orthognathic surgery, especially in patients with the lowest and highest index scores (The predictive validity of the dental indices has been questioned. A systematic review of the predictive validity of the GOSLON Yardstick in UCLP  highlighx scores .The strength of this randomized study is the large patient material of more than 400 Caucasian children with UCLP. The selection and allocation bias are reduced because of the randomization. Study limitations include the high number of operating surgeons, individual surgical learning curves and skill, lack of evaluation of the initial extent of cleft, agenesis of teeth and the amount of secondary surgery, and the type, length, and effect of orthodontic treatment. In addition, the age range of the patients at the time of the models, and the reliabilities of the individual ratings of the models varied. The final outcomes of dentofacial relations of the Scandcleft randomized trials can be evaluated after the growth is complete. Appraisal of other parameters, such as speech, facial appearance, patient satisfaction, and burden of care, should also then be included.The results of the three trials do not provide evidence that one surgical method is superior to the others when dental arch relationships are evaluated. The dental indices in this study could be used in evaluating, categorizing, and comparing the results of the surgical methods in children with UCLP. The results did not differ between the indices. The reliabilities of the dental indices were acceptable, and significant correlations existed between the dental indices at all ages. The reliability of the Eurocran palatal index was questionable.This study was supported by the Finnish Dental Society.


Pandanus amaryllifolius Roxb.). This research incorporates ultrasonic extraction to extract the aromatic compound in pandan leaves. The parameters varied in this study are the extraction time, sonicator amplitude, concentration of solvent and the mass of pandan leaves. The experiment was conducted using a central composite design (CCD) model generated by the response surface methodology (RSM). From the extraction process, it can be deduced that the effect of leaves’ mass is comparably higher than other parameters, while sonicator amplitude gives the most negligible impact on the process. The obtained p-value was 0.0014, which was less than 0.05. The high R-squared 0.9603 and adjusted R-squared 0.8809 indicate the model is well agreed with the actual data. The optimal control variables of ultrasonic extraction of 2AP are at an extraction time of 20 min, 60% of solvent concentration, amplitude of 25% and 12.5 g of pandan leaves, which produced 60.51% of yield of the extract and 1.43 ppm of 2AP. It is found that the mass of pandan leaves and the concentration of solvent have a significant impact on the extraction process of 2AP.2-acetyl-1-pyrroline (2AP) is the compound that gives out the typical aroma and flavour of pandan leaves ( Pandanus (Pandanaceae family), palm-like evergreen trees or shrubs. They are widely distributed from Africa to the Pacific lands in the moist tropics. There are over 600–700 species, and the sizes vary from less than 1 m tall to 20 m tall. The leaves are strap-shaped , varying from 30 cm long to 2 m or longer and 1.5 cm up to 10 cm broad, depending on the species [Pandanus amaryllifolius Roxb. It holds commercial interest in the flavour industry. This species is abundant in Southeast Asia, such as Malaysia, Thailand, Indonesia, and India [Pandan leaves belong to the Pandan leaves are of a few volatile compounds in a group of alcohols, aromatics, carboxylic acids, ketones, aldehydes, esters, hydrocarbons, furans, furanone and terpenoids. Other volatile compounds in pandan extracts include ethyl formate, 3-hexanol, 4-methylpentanol, 3-hexanone, 2-hexanone, trans-2-heptenal, β-damascenone, 4-ethylguaiacol and 3-methyl-2-(5H)-furanone .In this study, despite many conventional extraction techniques available, a greener technique, ultrasonic-assisted extraction (UAE), is favoured to extract 2AP from pandan leaves. The benefit of the UAE is mainly due to a reduction in extraction time and solvent consumption compared to conventional methods. The UAE working principle depends on acoustic cavitation that happens when significant negative pressure is applied, forming microbubbles in liquid. The bubbles will grow to various sizes, collapse violently, and release intense local energy with significant chemical and mechanical effects. Temperature and pressure changes from these implosions cause shear disruption, thinning of cell membranes, and cell disruption, resulting in enhanced solvent penetration into cells and amplifying the mass transfer of target compounds into the solvent. The direct outcome of these processes is that the mass transfer from plant material to bulk solvent is significantly improved, and the extraction yield is raised ,6,7,8.The solvent to be used in this process is a mixture of ethanol and water. Water is known to be abundant and its properties are non-toxic and polar and will not bring harm to the environment. The compound to be extracted in this study is 2AP, which is a polar compound; therefore, 2AP should be able to dissolve in water. Ethanol is also a polar solvent; therefore, it can dissolve 2AP. The polarity of water and ethanol are higher compared to n-hexane which makes it a better solvent to extract 2AP .Despite the advantages of the process, there has been no available literature on the use of UAE to extract 2AP from pandan leaves. Literature on the extraction of 2AP from pandan leaves has been focused on supercritical carbon dioxide and solvent extraction methods. Ultrasonic extraction has never been reported as one of the methods to extract 2AP. Hence, optimization of the extraction parameters needs to be carried out to obtain the maximum yield of 2AP from pandan extract. The parameters involved in this study include sonication amplitude, extraction time, solid to solvent ratio and concentration of solvent. Therefore, this study aimed to find the optimum conditions of ultrasonic extraction to obtain a maximum concentration of 2AP from pandan extract. This method may allow for the substitution of natural pandan flavours for artificial pandan flavours, which may serve to raise the value of various low-quality food products, such as old and non-aromatic rice, as well as contribute value to the plant as the source of the pandan flavour.Pandanus amaryllifolius Roxb.) were collected randomly from their trees grown in Perak, Malaysia on 4 October 2020. The fresh green leaves were then weighed using an electronic balance. The leaves were put on a tray, and the tray was put into an oven at a temperature of 105 °C for 24 h. After 24 h, the tray containing the leaves was taken out of the oven, and the leaves’ weight was measured using an electronic balance [2 [Pandan leaves  were used. Both reagents were purchased from Sigma Aldrich. The purity was 99%. All the solvents and chemicals used in this study were analytical grade.In this study, four parameters were manipulated to observe their effect on yield extract and the concentration of 2AP obtained from the extract. The parameters involved were extraction time, the concentration of solvent, sonication amplitude and mass of leaves. This study used an ultrasonic probe because it allowed direct contact with the sample and allowed it to develop a power up to 100 times more than that provided in the ultrasonic bath. After the extraction process was done, the solution was filtered using vacuum pump and the solvent was removed using a rotary evaporator. Then, the yield of the pandan extract was then calculated before the extract was analysed using GC-MS and GC-FID.v/v)%. 200 mL of solvent was poured into the vessel. The vessel was then connected to a refrigerated water bath at a temperature of 35 °C to help to maintain the process temperature. The setup for the ultrasonic system is shown in Dried pandan powder was weighed according to the desired mass using an electronic balance and stored in a 500 mL ultrasonic vessel. The solvent used in this process was ethanol and distilled water  and M2 is the mass of the pandan leaves.Whereby MGas chromatography–mass spectrometry (GC-MS) was used to analyse the compounds present in the extract from pandan leaves. The operating parameters involved in doing GS-MS analysis were as follows: injector temperature is 250 °C, carrier gas used was helium at a flowrate of 1.0 mL/min operated in the split mode 1:5, the mass spectrometry parameters were electron impact of 70 eV, ion source temperature of 200 °C, line of transference is 250 °C, electron multiplier voltage of 2500 V, and the scan rate of 5 scans/s and mass interval of 35–500 m/s . After tA method used to minimize the errors when an accurate weight or volume of samples is required to determine the amount of 2AP present in the extract ,12,13, wUltrasonic extraction of 2AP from pandan leaves was carried out using RSM coupled with CCD, which is a particular set of mathematical and statistical methods for designing experiments, building models, evaluating the effects of variables, and searching for optimum results conditions of variables to predict targeted response. A CCD with four factors was performed to investigate and confirm the process parameters affecting the extraction of pandan extract. Four independent variables: extraction time, the concentration of solvent, mass of pandan leaves, and sonication amplitude were studied with the yield of pandan extract as the response. Optimum conditions for extracting pandan extract were then determined. Design-Expert 12.0, a statistical software, was used for regression analysis and graphical analysis of the data obtained during the ultrasonic extraction experiment. The statistical significance of the full quadratic model predicated was evaluated by the analysis of variance (ANOVA) and least-squares techniques. The ANOVA lies in determining which factors significantly affect the response variables in the study.The moisture content of the pandan leaves was carried out to determine the effect of the drying pre-treatment on the yield and concentration of 2AP in pandan leaves extract. The moisture content of fresh pandan leaves was calculated to be 81.4% after the procedure of oven drying at 105 °C for 24 h was done. The result was typical for plant materials where about 80% of water is removed. The previous study included the moisture content of pandan leaves at 86.5% [In this study, four factors were used to evaluate the effect of process variables on the response, namely the yield of pandan extract. A total of 26 batch experiments were carried out, as shown in p-value < 0.05 for models to be significant and p-value > 0.05 for insignificant lack of value. The regression analysis of the responses was carried out using a second-order polynomial equation for the evaluation of the best fit model: A second-order polynomial equation (modified) was developed to understand the interactive correlation between the yield of pandan extract and the process variables. The final model obtained in terms of coded factors is given below,A second-order polynomial equation (quadratic) was developed to understand the interactive correlation between concentration of 2AP and the process variables. The final model obtained in terms of coded factors is given below,1 is the yield of pandan extract, Y2 is the concentration of 2AP, A is extraction time, B is the solvent concentration, C is sonication amplitude, and D is the mass of pandan leaves. YAmong all the variables studied for ultrasonic extraction, it can be affirmed that the mass of dried pandan leaves has the most significant impact on the extraction process. The solvent concentration plays an essential role since higher concentration favoured better extraction, as there is higher availability of such compounds with the higher loading of the solute. Another control variable that influences the measured responses is the extraction time. However, operation time is critical during the process as it would significantly reduce electricity consumption due to reduced operation time .The optimization procedure was performed and the optimum conditions of the ultrasonic extraction process of pandan extract yield from pandan leaves were found to be as mentioned in The optimum conditions were repeated three times and the average value of pandan extract yield was found to be 60.51%, as mentioned in From this process, it can be concluded that the experimental results were in good agreement with the predicted value. The optimum extraction time was 20 min. A study of the extraction of carotenoids  mentioneThe optimum solvent concentration is in agreement with previous research conducted. According to a study in ultrasonic extraction of polyphenols from apple pomace , the higNext, the optimization procedure was performed and the optimum conditions of the ultrasonic extraction process of 2-acetyl-1-pyrroline (2AP) from pandan leaves were found to be as mentioned in The optimum conditions were repeated three times, and the average value of the concentration of 2AP was found to be 1.43 ppm, as mentioned in From these experimental results, it is found that the optimum conditions of ultrasonic extraction produced the average concentration of 2AP (1.43 ppm) which is higher compared to the previous study using supercritical carbon dioxide extraction (0.16 to 0.19 ppm) . MeanwhiAnother research has been conducted to extract 2AP from pandan leaves using a solvent extraction process. The solvents used were ethanol, methanol and propanol [This is understandable given that the presence peak in the ethanol chromatogram was higher than in the methanol chromatogram. Ethanol is an extremely polar molecule. This is due to ethanol’s hydroxyl (OH) group. Because of the high electronegativity of oxygen in this alcohol, hydrogen bonding with other molecules is possible. As a result, ethanol attracts polar and ionic molecules, making ethanol have the ability to dissolve both polar and non-polar substances. Furthermore, ethanol has been identified as one of the most important solvents during solvent extraction. Ethanol solvent is also more environmentally friendly than pure alcohol . p-value (p < 0.05) demonstrated that the developed mathematical model was highly significant. The model’s goodness of fit was evaluated by the determination coefficient (R2), adj-R2 and Pre-R2. The high R2 values revealed that only slight variations are not explained by the developed mathematical model and demonstrate a significant and intense correlation between the observed and predicted values [The statistical significance of the developed model equations was evaluated using Pareto analysis of variance (ANOVA). The higher model F value and lower d values .Meanwhile, the yield of pandan extract shows a declining pattern as the mass of pandan leaves increases. The yield of pandan extract declines from 60% to 40% as the mass of pandan leaves increases from 7.5 g to 12.5 g. The amount of sample will determine the number of compounds that can be released into the solvent. In the case of extracting too much of the sample, the solvent can be readily saturated, avoiding the complete compound extraction. In general, a higher volume of solvent can effectively dissolve more target components and leads to higher extraction efficiency. A high solid to solvent ratio could promote an increasing concentration gradient, which causes an increase in diffusion rate and ultimately increases extraction efficiency. The chance of bioactive compounds coming in contact with extracting solvent is higher when the amount of solvent is increased and causes a higher rate of leaching out. However, it should be noted that once equilibrium is reached, the yield of active compounds will no longer increase.p value is higher than 0.05. This can be supported by a study to extract total polyphenols and antioxidant activity from mango residues [p value greater than 0.05. As frequency increases, the cavitation yield drops because the cavitation bubbles become smaller and less energetic thus lowering the yield [As for the amplitude, the yield of pandan extract decreases from 64.8% to 58% as the amplitude increases. According to residues . Accordihe yield .Next, the effect of interaction between the mass of leaves and extraction time is illustrated in On the other hand, the 3D surface of the yield of pandan extract against amplitude and concentration of solvent is displayed in Finally, The coefficient of variance (CV), the ratio of the standard error of estimate to the mean value of the observed response, was a measure of the reproducibility of the model. CV was reproducible when it is not greater than 10%. The value of CV for the response of concentration of 2AP is greater which suggests that it is a reproducible error of 17.64%.p value of 0.0054, according to p value of 0.008 along with the mass of pandan leaves. Hence, the interaction between the concentration of solvent and extraction time becomes significant as the p value is 0.0002. From the figure, the increment of concentration of 2AP can be seen as the concentration of solvent used and extraction time applied during the extraction process increases. A similar result was reported in a study to extract antioxidant compounds from Moringa oleifera leaf [3D surface plot of concentration of 2AP against the effect of concentration of solvent and extraction time is illustrated in era leaf . The reaera leaf . A similera leaf .Next, the effect of interaction between the mass of leaves and amplitude towards the concentration of 2AP is displayed in Other the other hand, the 3D surface plot of concentration of 2AP as a function of amplitude and concentration of solvent is shown in Finally, the effect of interaction between the concentration of solvent and mass of leaves towards the concentration of 2AP is illustrated in Sonication enhances the mass movement of bioactive chemicals through the combined influence of three processes, namely cavitation, mechanical agitation, and thermology . UltrasoThe solvent concentration is also an essential factor that affects the extraction process. A higher concentration of solvent will result in higher viscosity. The initiation of cavitation in a liquid requires that the negative pressure during the rarefaction cycle overcome the cohesive forces between molecules composing the liquid. A rise in viscosity or surface tension increases these molecular interactions, raising the cavitation threshold. The formation of a cavitation becomes more difficult when the solvent viscosity and surface tension are higher. Ultrasonic energy produced to aid the cavitation process will also increase . In thisThe solute-to-solvent ratio is one of the most critical factors during the mass transfer because a larger solvent volume helps accelerate the diffusion process . An incrThis study applies a CCD-based response surface methodology to obtain valuable insights and optimize the process parameters. While RSM may not achieve substantial gains in process parametric values, it is a very useful technique for the chosen system because the methodology implies evaluating the linearity and non-linearity of specific response variables in terms of process variables, which cannot be used deduced using trial and error approaches. As a result, parametric interactions and the identification of specific parameters and their interactions on the selected response variables can be understood. It is worth noting that the CCD-based RSM design required fewer experimental runs to accomplish an optimal set of process parameters.This study proved that UAE of pandan leaves facilitated excellent extraction profiles of 2AP due to sonication. Extraction time and solvent consumption in the conventional methods can be reduced by applying UAE method. It also proved that the inexpensive and non-toxic ethanol could be used as an alternative solvent for the optimal extraction of 2AP from pandan leaves. The optimal control variables of ultrasonic extraction of 2AP are at a 20 min extraction time, 60% of solvent concentration, amplitude at 25% and 12.5 g of pandan leaves, which produced 60.51% of the extract yield and 1.43 ppm of 2AP. RSM-based design and analysis provided essential insights as non-linear and quadratic profiles existed for several measured responses with variations in control variable values. As a result, RSM enabled a greater comprehension of the combined effect of the various control variables on the response variables. In conclusion, the study is highly promising in furthering the use of ultrasound to extract pandan leaves to produce natural functional foods. The finding of this study can be used to enhance the rice flavour as well as to be used as one of the flavour enhancers for cooking or baking.


The interaction of host molecules with water molecules is of primary importance in astrophysical and atmospheric studies. Water-binding interactions continue to attract a broad interest in various fields, especially those related to the formation of assembly structures. Using the physical vapor deposition (PVD) method and a two-beam interferometer with a wavelength of 406 nm, the refractive indices of thin films of a water and nitrogen (argon) mixture were calculated in the range from 15 to 35 K. The results of temperature transformations of the obtained films from a two-beam interferometer, and thermal desorption characteristics from the temperature of condensation to the temperature of evaporation of water (15–180 K), are presented. The relationship between the signal of the interferometer, the refractive index, and the film thickness during glass transition is demonstrated. The increased interest in molecules that are cold enough to form ice has led to a wealth of useful information on various properties. The review of articles helps to understand the significance of the studied parameters  of amorphous structures at low temperatures obtained in the experiment. At present, various methods have been described for studying the refractive index for a wide range of substances , at low The gradual and reversible transition in amorphous structures  from a solid and relatively brittle state to a viscous or elastic state with increasing temperature is called the glass transition process. In nature, clathrates and other amorphous structures such as glass are often present in sediments containing both natural gas and water. Clathrates are widely distributed in permafrost regions and in thermodynamically stable regions of the ocean, as well as in interstellar space, and therefore are important gas reserves. The appearance of clathrates in liquid inclusions was first described by Roedder in 1963 [Unlike , we usedAt present, interest in the properties of mixtures at low temperatures with substances such as water ,10,11,12To study the optical properties of thin films of cryocondensed mixtures, the following problems were formulated:Firstly, what structures are formed during the glass transition of mixtures of water and various gases .Secondly, what types of hydrogen bond vibrations occur during the interaction of a guest molecule and a host molecule of an amorphous structure.Thirdly, how does the concentration of water in a mixture of water and nitrogen/argon affect the optical properties of the IR spectrum.The aim of this work is to study the effect of gaseous impurities on the dependence of the refractive indices and on structural-phase transitions inside the sample during heating. We carried out several cycles of experiments with mixtures of water, methanol, and ethanol with nitrogen and argon. In this work, we begin with a detailed description of the study of thin films of solid water with nitrogen and argon obtained in the temperature range from 15 to 35 K.2, N2, CO, Ar, and CH4 that have been adsorbed in amorphous or crystalline ice are often used as an IR-mark to study the properties of ice and glass. When water molecules dissolve in matrices of simple molecules at low temperatures, very clear discrete bands of monomeric, dimeric, and polymeric water appear. The infrared spectrum of water, in various solid matrices of simple molecules, has been the subject of several studies [Simple molecules such as H studies ,21,22,23The components of ice, which are mixtures of different gases, have different sublimation temperatures, which, when the temperature rises, can lead to drastic changes in their structure. For example, for a two-component sample, when the temperature reaches above the sublimation temperature of one of the components of the mixture, it will evaporate, resulting in recondensation of the second component on the substrate. A schematic diagram of the recondensation process is shown in Our studies have shown that the properties of recondensates, including optical ones, differ significantly from the properties of single-component thin films. Thus, the recondensation process will be radically different from classical physical vapor deposition (PVD), because in the recondensate, the structure will be affected not by individual gas molecules, but by its polyaggregates, which were previously formed in the film during the condensation of the initial two-component sample . This isIn this work, we study thin films of mixtures of water with argon and nitrogen and their properties during structural-phase transformations. What follows is a description of the experimental methodology and setup, followed by a description of the results that were obtained during the study.Three-times-distilled water, argon (highest purity grade (99.993%) argon gas with maximum oxygen fraction not exceeding 0.0007%, water vapor not exceeding 0.0007%, and containing no more than 0.0005% of nitrogen  GOST 10157-79), and nitrogen (first grade nitrogen (99.999% purity) with maximum oxygen fraction not exceeding 0.0005% and water vapor not exceeding 0.0007%  GOST 9293-74) were used as the test substances. The purity of chemicals was evaluated by measuring their saturated vapor pressure at a temperature of 293.15 K and comparison with literature values. The mixtures of the systems under study were freshly prepared by mixing carefully selected volumes of pure liquids and gases at a temperature of ~293 K in the inlet system volume. The mixtures were mixed uniformly. Extreme care was taken to minimize preferential evaporation during the filling process by creating the mixture at pressures below saturated vapor pressures. During gas puffing into the chamber and condensation, the mass spectrum of the mixture was measured using an Extorr XT Series RGA Model XT100 . The main unit of the experimental setup see  is a higInside the vacuum chamber, the substrate is in thermal contact with a closed-cycle helium Gifford–McMahon refrigerator, due to which the temperature can be varied in the range of 12–200 K. This copper substrate, 60 mm in diameter (6), is covered with a flat silver film. Temperature control is carried out by a silicon diode TS 670–1.4 connected to an M335/20 thermostat, which maintained the temperature constant with an accuracy of 0.5 K. A two-stage cooling system cooled the substrate to 12 K. A resistor heater was connected to the end of the second stage.The Extorr XT100, mounted in a vacuum chamber, is a quadrupole residual gas analyzer that also includes an advanced Pirani probe as well as a hot cathode Bayard/Alpert (B/A) ion probe. The Pirani pressure gauge measures vacuum based on the thermal conductivity of the gaseous medium. The B/A ion sensor uses electron impact ionization of residual gases to measure pressure-related ion current. The quadrupole gas analyzer uses precision mechanics and electronics to measure the ion currents due to the partial pressures of the residual gases in the vacuum chamber. Pirani starts at atmospheric pressure, B/A ionometer at 10 mTorr nominal pressure, and quadrupole starts at 0.1 mTorr nominal pressure. The XT100 operates from 1 to 100 amu.A special protective screen see  ensures This insulation is leaky, but at operating pressures of 1–10 µTorr it is quite effective because the mean free path of molecules at these pressures is several orders of magnitude greater than the distance between the protective screen and the substrate. Therefore, during our experiments, all the gas is deposited on the substrate when the pumping line is closed. The mixture of water and nitrogen (argon) is pre-uniformly mixed in the mixture production system (11). Then the homogeneous mixture is fed into the cryovacuum chamber for condensation.In Condensation of the water-nitrogen and water-argon mixtures took place at a temperature of 16 K, and the thickness of the cryofilms was the same and amounted to 3.9 μm.The refractive index is calculated by the following Equation (1):t1 and t2—interference periods of laser 1 and laser 2;α1 and α2—angles of incidence to the normal of the substrate of laser 1 and laser 2: α1 = 1°; α2 = 45°.The refractive index was measured using a two-beam laser interferometer. The laser had a wavelength of (406 ± 0.5) nm. The total error in measuring the refractive index was estimated to be no more than 1.5%.It should be noted that each refractive index value in the table below corresponds to a separate experiment carried out at a given dew point. After completion of each experiment, the experimental setup was repeatedly prepared for a new cycle of experiments.n was 1.293 for water-nitrogen and 1.305 for water-argon. Experiments on the condensation of thin films of mixtures of water-nitrogen and water-argon were carried out at a pressure of 5 μTorr. The mixture was deposited in a cryo-vacuum chamber at a temperature of 16 K, at which a thin film 3.9 µm thick was formed . The filThe concentration of argon in the mixture has a greater effect on the change in the refractive index than the deposition temperature. After the mixture is deposited onto the substrate, the IR spectrum of the film is measured every 2 K during heating . The figTo compare the IR spectra and determine the effect of concentration on different modes of the spectrum, the amount of water after the sublimation of nitrogen and argon should be the same for different concentrations of the mixture. For this purpose, a series of experiments were carried out with different concentrations of water content in the mixture  in comparison with a pure water sample.−1; −1), which disappear after heating to 170 K. In At a water concentration of 25%, peaks appear (−1), which begins to disappear when the cryofilm is heated from 42 K. At temperatures above 50 K, the peak is completely smoothed out and disappears, but as mentioned earlier, the nitrogen content of a small concentration is retained up to a temperature of 171.6 K.In Full 3D IR spectra for a mixture of water and nitrogen, obtained every 2 K during the heating process, are presented in 2 with water. There is also a peak at 3650 cm−1, together with the modes of CO2 vibrations at 3600 and 3700. However, the peak at 3650 cm−1 persists to temperatures above 110 K and disappears only when the guest molecule leaves the film, which does not happen for argon and nitrogen.We have carried out similar experiments for CO−1 . Peaks at 1284, 2149, 2228, and 2340 cm−1 a,b are aum fixes b the preThe thermogram of a water and argon mixture in the temperature range from 20 to 180 K  shows chThe dependence of the IR spectrum on temperature from 20 to 180 K for a mixture of water and argon is presented in As mentioned earlier, a series of experiments were conducted to determine the influence of mixture concentrations on the escape of nitrogen from the film. During the defrosting process at a temperature of 172 K, the remaining nitrogen in the film completely leaves the film . In this−1 is associated with the presence of water polyaggregates of different compositions formed in the nitrogen matrix during condensation. This process could occur either in the adsorbed layer immediately before condensation or in the nitrogen matrix during the diffusion of water molecules.Firstly, the peak that was obtained at a frequency of 3685 cm2-water (Ar-water) interactions. Of particular interest is the change in O-H vibrational modes during N2 or Ar binding. An optical study showed that the destruction of the cryofilm structure affects the same vibration frequencies for a water-nitrogen mixture and a water-argon mixture. When water molecules in clathrates form an internal network of hydrogen bonds, there are several free O-H groups on the surface that do not participate in any hydrogen bonds. Obviously, the key parameters describing these systems are the interactions of internal hydrogen bonds and N−1). A strong influence during the destruction of the sample structure is present at the frequencies 1284 cm−1 (bending vibration), 2228 and 2340 cm−1 . Amorphous ice turns into crystalline ice as the temperature rises, while crystalline ice remains stable as the temperature falls. This can be achieved by considering both the profile of the feature of dangling O-H bonds present in porous amorphous ice and on the surface of large accumulations of crystalline ice, and the profile of the vibrational band of adsorbed particles of nitrogen and argon on the frequency 3685 cm−1.Secondly, a change in the temperature of the sample has a weak effect on the frequency of the combination (bending + libration) type of vibrations (2149 cmThirdly, the admixture of nitrogen increases the RI and adds porosity to the ice structure, which also leads to an increase in the RI. The concentration of argon in the mixture has a greater effect on the change in the refractive index than the deposition temperature.−1 are a source of information about the structural properties of amorphous water ice and especially icy mixtures of water and other frozen gases. A similar spectral effect was found by our authors in a series of other experiments conducted at the Laboratory of Cryophysics and Cryotechnology at Al-Farabi Kazakh National University for various concentrations of mixtures of water with nitrogen, methane, ethanol, CO2, or CCl4 [Dangling bonds in IR spectra near 3700 cm or CCl4 ,27,28,29


The expression of B7-H4 in the placental chorionic villus was significantly higher in PA samples in relation to samples from healthy controls  but not in relation to RPT samples . This finding suggests that B7-H4 might play an important role in mechanisms restoring reproductive tract homeostasis. Further research is necessary in regard to the role of B7-H4 in PA.B7 homolog 4 protein (B7-H4), a member of the B7 family, is a immunomodulatory membrane protein. The aim of the study was to evaluate the expression of this protein in the decidua and placental tissues in case of placental abruption (PA) compared to cases of retained placental tissue (RPT) and controls. Tissue samples were obtained from 47 patients with PA, 60 patients with RPT, and 41 healthy controls. The samples were stained for B7-H4 expression, analyzed by an expert pathologist, and a semi-quantitative scale was applied. A statistical analysis revealed that the expression of B7-H4 was significantly higher in the decidua in PA samples compared to samples from patients with RPT ( The state of pregnancy is a unique balance between the activation and inhibition of the immune system in the female reproductive tract, which supports the existence and development of the fetus inside the mother’s body . RemarkaOne of the most important regulators of this balance is the decidua. The human decidua is a highly specialized tissue with many unique regulatory properties. The decidua provides complex immunological protection as well as wide nutritional support to the newly developing life . The decPlacental abruption (PA) is defined as a complete or partial separation of the placenta from the uterine wall during pregnancy, with the fetus still being present in the uterine cavity. It is a serious perinatal complication and one of the leading causes of second- and third-trimester bleeding ,18,19. PB7 homolog 4 protein–B7-H4 (AKA B7S1 or V-set domain containing T-cell activation inhibitor–VCTN1) is a costimulatory transmembrane molecule and a member of the regulatory membrane molecules B7 family ,51,52,53B7-H4 presents with two functional isoforms—the soluble form (sB7-H4) and the membrane-bound form. The source and functional mechanisms of the soluble form of B7-H4 remains obscure ,71,72,73Importantly, B7-H4 is involved in immunological changes associated with the spontaneous onset of labor as well as with several adverse perinatal outcomes. Its expression is higher during labor than during pregnancy, but it does not change during the course of labor . There iThe aim of this study was to explore B7-H4 expression in patients with PA and its significance to the process of placental detachment.The study was performed retrospectively at the II Department of Obstetrics and Gynecology, Centre of Postgraduate Medical Education—a tertiary perinatal care center based in Warsaw, Poland. The local Ethics Committee approved the study .We included cases from January 2017 to December 2019, for which the tissue material was available in the archives of the pathology department. We divided the identified cases into three groups: samples from patients diagnosed with PA, samples from patients diagnosed with retained placental tissue (RPT), and samples collected for other reasons . The necessary clinical characteristics of the patients were extracted from the available hospital database. Patients with incomplete medical history were excluded from the study.We included a total of 148 patients; 47 of them were patients with PA, 60 of them were patients with RPT, and 41 were healthy controls. PA diagnosis was based on the clinical symptoms including rapidly developing uterine tenderness, abdominal pain, severe vaginal bleeding/hemorrhage, and/or fetal distress. In all cases, the diagnosis of PA was first confirmed by the presence of a retroplacental clot and then was retrospectively confirmed in the tissue examined by a pathologist. All cases of diagnosed placental abruption were taken into consideration, despite the fact of the presence of obvious external bleeding , the perAll the participants were of Polish ethnicity. The characteristics of the studied groups are presented in The selected tissue samples were retrieved from the Department of Pathology, Bielański Hospital, Warsaw, Poland. The paraffin-embedded placental chorionic villous and decidual tissue samples were evaluated by an expert pathologist who subsequently selected material sufficient for further analysis. The chosen samples were cut using the microtome into 3 μm slices and sent for immunohistochemical processing.Immunohistochemical analysis was performed manually in the Department of Pathology, Bielański Hospital, with the Ultravision LPValue Detection System . The visualization of reaction products was performed with 3,3′-diaminobenzidine (DAB+) chromogen  used for 10 min at room temperature, receiving a gold–brown color of the final product. In the next step, the sections were counterstained with Meyer’s hematoxylin and mounted in glycergel. According to the recommendations of the producer, the specificity of the B7-H4 antibody was tested with a specimen of ductal breast cancer, constituting a positive control for B7-H4. The results obtained from the control were in accordance with the producer’s specification.2O2 in TBS for 15 min. Subsequently, the application of the secondary enzyme-conjugated antibody diluted in TBS with 1% BSA took place. The incubation lasted for 1 h at room temperature. The slides were developed with chromogen for 10 min at room temperature and then rinsed in running tap water and, subsequently, counterstained with hematoxylin.Afterwards, the slides were washed in tris-buffered saline (TBS) plus 0.025% Triton X-100 and blocked for 2 h at room temperature in 10% normal serum with 1% bovine serum albumin (BSA) in TBS. The slides were drained and incubated with the primary antibody, rabbit polyclonal B7-H4 , in 1:100 dilution in TBS with 1% BSA. The incubation with the primary monoclonal antibody took place in a humidified chamber overnight at 4° Celsius. Then, the slides were rinsed twice for 5 min with TBS plus 0.025% Triton and submitted to 0.3% HThe decidua is the endometrial tissue of the uterine cavity remodeled during the pregnancy under the influence of hormones. The tissue consists of monomorphic stromal cells closely adjacent to each other, expressing certain features of the epithelial tissue. All of the examined placental samples were collected from patients in the third trimester of pregnancy. The villi of the placenta in the last trimester of pregnancy are composed of connective stromal tissue surrounded by a layer of syncytiotrophoblast with thin-walled blood vessels located on the circumference of the villi under the syncytiotrophoblast. Sinus-type vessels are present in the stroma near the syncytiotrophoblast. All the obtained samples were evaluated by an expert pathologist and qualified as typical and appropriate for further analysis.A semi-quantitative scale based on the previously published studies ,82,96 wap < 0.05. The R version 4.1.0 software was used for statistical analysis.The normality of variable and group characteristics distributions were tested with the Shapiro–Wilk test. Regarding the group characteristics data, only the age was normally distributed. The ANOVA test and Kruskal–Wallis test were applied to analyze the differences between group characteristics. B7-H4 expression levels were found to be non-normally distributed. Statistical differences between groups were estimated using standard non-parametric tests (the Dunn’s test with Benjamini–Hochberg adjustment). Significance was accepted at A total of 148 women were enrolled in this study. The groups did not differ in terms of age, parity, and BMI, but inevitably differed in terms of gestational age and newborn weight and length. The control group had the highest rate of live births . The prep-value < 0.001) and in case of PA compared to healthy controls . The difference between the control group and RPT group was not significant . The expression of the B7-H4 molecule in the placental chorionic villus did not significantly differ in case of PA compared to RPT , but the difference remained significant in case of PA compared to healthy controls  and in case of RPT compared to the control group .The results are presented in According to available data, labor might be considered to be the result of the cessation of maternal immune tolerance toward the fetus. The augmented decidual cytotoxic activity is one of the most important components of the processing cascade leading to the expulsion of the fetus ,98. At the molecular level, the process of the development of PA takes place locally on the maternal–fetal interface and is not reflected in the peripheral blood ,103,104.2 domination  or TAn important limitation of the presented study is its retrospective character and the unavoidable fact that we could use only the available data and tissue material. Ideally, we would like to obtain samples from healthy controls without any comorbidities, nor pregnancy or labor complications, to avoid the bias of this issues. However, tissue samples are not routinely collected in such cases. It would also be beneficial to have a control group at a more precisely matching pregnancy age, as the difference may exert some influence on the immune response in the decidua. Nonetheless, according to many previous data, the changes of the immune response in the decidua seem mainly related to pregnancy complications or the state of labor. Thus, we assumed the level of bias acceptable ,108,110.The analyzed samples confirmed the involvement of the immunomodulatory molecule B7-H4 in the processes occurring on the maternal–fetal interface. B7-H4 seems to justify the prematurely increased decidual cytotoxic activity present in PA and is responsible for restoring reproductive tract balance. A focused look at the molecular basis of this clinically important pregnancy complication validates, with greater reliability, the significance of this investigated molecule in the placental detachment process. Considering the fact that PA results in perinatal deaths and numerous serious complications for both the mother and the child, it is critical to put effort to thoroughly understand the underlying mechanisms. Any progress that leads to the reduction of mortality and morbidity through the improvement of PA management is valuable.Our study revealed the abnormal expression of B7-H4 in cases of PA in women of Polish ethnicity. According to this finding and previous data, a significant role of this molecule might be indicated in maintaining maternal immune system balance, which is disrupted in case of PA. Our results also indirectly confirm increased cytotoxic activity on the maternal–fetal interface in case of PA, which starts at the early beginning, far before the actual detachment of the placenta. More clinical and molecular studies are still necessary in this matter. The molecular mechanisms underlying PA pathogenesis are an interesting target for further research to better understand and, thus, develop the appropriate management of this serious perinatal complication.


Tank level measurement is an important research area in radar technology. Level measurement using radar technology is a safe solution even under extreme process conditions, such as high pressure, temperature, and vapors. Special antennas are required to meet electromagnetic requirements such as high gain, low sidelobe level, and high bandwidth. Another requirement is a small size and good manufacturability of the antenna. One promising solution is the use of microstrip antennas for tank-level measurements. This paper presents a special microstrip antenna, that meets the main requirements in addition to a circular antenna layout, which fills optimally the tank hatch. To do this, we created a special antenna layout with a suitable feeding network and added an optimized ring around the antenna system, which significantly reduces the sidelobe level. The center frequency of the antenna is 25 GHz with a 1 GHz bandwidth. Constant product quality, operational safety, and economic efficiency can only be ensured by continuous measurements and intervention and control systems based on these measurements. Liquids, pastes, bulk solids, and liquefied gases are most often stored in tanks, silos, or mobile containers. These tanks are used in the chemical and petrochemical industries, the pharmaceutical and life sciences industries, the water industry, the chemical and petrochemical industries, the water and wastewater, and the food and energy industries.There are several classical and modern methods for measuring the product level in process and storage tanks. Applications are in the chemical, petrochemical, pharmaceutical, water, and food industries, mobile tanks on vehicles and ships, and natural reservoirs such as seas, dams, lakes, and oceans. Typical tank heights range from 0.5 m to 37 m. ,2continuous level measurement, i.e., level indication,level detection, i.e., detection of an alarm limit to prevent overfilling.Two main measurement tasks can be distinguished :continuoMany level devices are mounted on top of the tank and measure primarily the distance between their mounting position and the product’s surface  Figure .For level measurement, a significant number of different principles measurement techniques are available , and it We developed a sensor antenna for radar-level pulse  measuremA promising solution is the use of microstrip antennas ,7,8,9 foMany other antenna types are promising for tank-level radar measurements, such as conical horn antennas , parabolRecently, theoretical work is conducted applying metamaterial-based antennas  also forIn this paper, a special microstrip antenna was designed, that meets the main requirements, i.e., high gain, low sidelobe level, high bandwidth, small size, and good manufacturability of the antenna. The antenna was realized on a low-loss RT5880 substrate. As opposed to the traditional N × N microstrip antenna layout, we used a special 24-element layout. With this arrangement, the side beam suppression has been significantly reduced. The feeding network of the 24-element antenna system is made up of three main power-divider elements, with three different power-division ratios. CST Microwave Studio was used to simulate and fine-tune the antenna array. The measurements were performed in an anechoic chamber, where we measured input reflection and antenna radiation patterns in the 23–27 GHz frequency range.The special microstrip antenna satisfies two main requirements, namely a circular antenna layout and an optimized ring around the antenna system, which significantly reduces the sidelobe level. The center frequency of the antenna is 25 GHz with a bandwidth of 1 GHz.In this chapter, the design of the feeding network for a microstrip antenna system with a 24-element circular confinement geometry is presented in detail as the optimization of the sideband suppression ring. The electromagnetic design of the antenna was carried out using the Ansys Designer 2D planar  and CST To fulfill the requirements an antenna system will be introduced, which optimally fills the standard circular opening of the tanks. The sizes of the antenna designed were chosen to fit into and optimally fill the standard circular opening of the tanks .The feeding network is designed for the antenna system for optimal illumination. We introduce the detailed design process of the microstrip antenna element, placing, and feeding network. With the development of modern wireless communications and radar technology, there is an increasing demand for compact antennas. Microstrip Patch Antennas (MPA) are lightweight, low cost, and easy to integrate into microwave circuits, and are therefore widely used in mobile and wireless applications. The use of MPAs in mobile wireless communication systems allows the implementation of small, low antenna sizes ,19.The basic geometry of the antenna is a dielectric substrate located between the ground plane and the radiating patch. The size and shape of the MPA radiator, as well as the height and the type of substrate material, are the most important design parameters for evaluating the performance of an MPA. The substrate, in addition to providing mechanical strength to the entire antenna, also allows the propagation of surface waves. The dielectric substrate layer separates the radiator from the ground, its material and thickness are of primary importance for the antenna bandwidth. Since the antenna dimensions are finite, the fields along the antenna are finite, and the fields along the edges are frictional .Several authors have investigated the effect of substrate thickness and material on the bandwidth of MSA. We simulated a single MPA  to test The substrates investigated were Rogers RT5880, RO4003, and RO3010 with substrate heights of 5, 10, 20, 31, and 62 mils. The simulation results are summarized in The single MPA antenna, simulated on 31mils RT5880 substrate, input reflection, and radiation pattern are shown in The single radiator has 8 dB gain at 25 GHz and 80.7° and 72.7° beamwidth in E and H planes b.The next design step is the radiator array structure design.The excitation of each radiator, which can be of uniform or non-uniform amplitude, is adjusted by the feeding network. We first examined a 6 × 6 planar array of uniform rectangular shape and a special 24-element array. The directional characteristics of the two arrays were compared using the CST array simulator. The radiation pattern for the 6x6 uniform and 24 elements arrays can be seen in The main point of comparison was the Sidelobe Level.  The 24 eThe final arrangement of microstrip radiators in the Ansys Designer simulator can be seen in The antenna-connected transmission lines in The feeding network is made up of three main elements. We implemented three different power division ratios .The design of the power dividers was based on the simple T-junction.The power dividers are the following:Power divider for 1 to 4 power ratio ,3-way power divider from 1 to 4:2:4 power ratio ,4-way power divider from 1 to 2:10:10:2 power ratio .After calculating the elements of the power dividers, we used Ansys Designer/Nexxim  for the The purpose of this network is to feed four identical radiators with the same power and phase. The single microstrip radiator has a 120 ohms input impedance at a center frequency of 25 GHz.Finally, at the planar feeding network design, a transmission line step was introduced between the microstrip radiator and the T-matching element b.The resonance frequency of the power division network for the 1 to 4 power ratio was set to 25 GHz .The upper part of the network feeds 2 × 4 + 2 antennas with the same power. The 2 × 4 feeding part consists of two power division networks for a 1 to 4 power ratio.The design of the three ways power dividers was based on two equations, the impedance matching equation, and the power ratio equation.The equation for impedance match:The power sum equation:R:The power ratio Solving Equations (2) and (3) givesFor matching, we applied a 141.2 ohms quarter wavelength transformer between 100 ohms, 200 ohms, and 109.55 ohms quarter wavelengths transformer between 120 ohms and 100 ohms a.The circuit model of the power division network from 1 to 4:2:4 power ratio can be seen in The resonance frequency of the power division network for 1 to 4:2:4 power ratio was set to 25 GHz and the input reflection is shown in The The final part of the feeding network design was the 24 elements’ feeding structure. This power divider is a four ways circuit with a 1 to 2:10:10:2 power ratio.The design of the power divider was based on two equations, the impedance matching equation, and the power ratio equation.The equation for impedance matching:The power sum equation:The power ratio:Solving equations givesThe 600 ohms microstrip transmission line is difficult to implement because of its small width, therefore we implemented two-stage quarter wavelength transformers between 120 ohms and 600 ohms. Our technology makes it possible to realize less than 200 ohms of microstrip transmission lines. The two-quarter wavelength transformers’ characteristic impedances are 77 and 173 ohms .The whole system is symmetrical in the horizontal plane and is completed by an additional 2 × 2 elements left and right .The resonance frequency of the power division network for 1 to 2:10:10:2 power ratio was set to 25 GHz and the input reflection is shown in After the design and optimization of the feeding network, the CST Microwave Studio was used for the simulation and fine-tuning of the antenna array . Our mosTwo versions of antennas were investigated. After the first simulation was found, that the SLL is not acceptable in some directions, therefore an optimized ring with ground vias was implemented, to increase the antenna sidelobe level (SLL) b.Examination of the radiation patterns showed that the SLL could be reduced by up to 5 dB in some directions by using the ring. The antenna gain is not changed significantly. The SLL reduction was significant in the Phi = 0 plane in the elevation angle range of 50–180 degrees . Here thAfter simulation verification of the antenna’s functionality, the antenna was built .For the measurements, an SMA connector was connected to the antenna input with a semirigid coaxial transmission line, and a radome was applied .The measurements were performed in the antenna measurement room of the Department of Broadband Infocommunications and Electromagnetic Theory of the Budapest University of Technology.The antenna input reflection in the 23–27 GHz frequency band shows a good agreement between simulated and measured values .The antenna radiation pattern was measured in the two main planes and the results show SLL better than −17.3 dB .Finally, we compared and summarized the most important antenna radiation pattern characteristics in It can be concluded that the parameters of the antenna have been measured and verified to meet the specification. The parameters were achieved by using a special feeding network and a sidelobe suppression ring.We presented a systematic design of a microstrip antenna array and its feeding network. A new antenna layout was proposed for the tank level meter radar sensor. The microstrip antenna elements optimally fill the opening of the tank, therefore maximal gain can be reached. The optimized outer ring ensures the improved sidelobe level to reduce the side reflections from tank sidewalls. The correctness of the design was verified by measurements.We plan to investigate the antenna further, mainly to improve the feeding network with non-uniform illumination. Another possibility for further development is the use of artificial material as the sidelobe suppression ring.