AR), and some of its alleles are assumed to negatively affect male fertility. Our aim was (1) to study the variability of the length of CAG repeats of the AR gene and possible associations in the AR CAG genetic variants with semen quality and reproductive hormone levels in a population-based cohort of men and (2) to estimate distributions of AR CAG repeat alleles and associations with semen parameters in different ethnic subgroups. The cohort of 1324 young male volunteers of different ethnicities (median age 23.0 years) was recruited from the general population of five cities of the Russian Federation, regardless of their fertility status. Semen quality , reproductive hormone levels  and trinucleotide (CAG) n repeat polymorphism in exon 1 of the AR gene were evaluated. The semen samples were analyzed according to the WHO laboratory manual , serum hormones were measured by enzyme immunoassay, and the AR CAG repeat length was analyzed by direct sequencing of leukocyte DNA. The median AR CAG repeat length in men of our multi-ethnic population was 23 (range 6–39). In the entire study population, a significant difference (p ≤ 0.05) was found in the frequency distribution and the mean values for the CAG repeat length between the groups with normal (23.2 ± 3.3) and impaired semen quality (23.9 ± 3.2). Additionally, we demonstrated that the total sperm count, sperm concentration, progressive motility and normal morphology were lower in the category of long CAG repeats (CAG ≥ 25) compared with those in the category of short CAG repeats (CAG ≤ 19); however, hormonal parameters did not differ between the long and short CAG categories, with the exception of estradiol. Significant differences were observed in the AR CAG repeat length between the most common ethnic cohorts of Slavs (Caucasians), Buryats (Asians), and Yakuts (Asians). The Buryats and Yakuts had a higher number of CAG repeats than the Slavs . The range of alleles differed among ethnicities, with the Slavs having the largest range , the Yakuts having the smallest range  and the Buryats having the middle range . The longer CAG repeats were associated with an impaired semen quality within the Slavic (CAG ≥ 25) and Buryat (CAG ≥ 28) groups, but this effect was not found in Yakuts. Hormonal parameters did not differ between the three CAG repeat categories in men of all ethnic groups. This is the largest Russian study of the distribution of AR CAG repeats and the search for association between length of AR CAG repeat tract and impaired spermatogenesis in men from the general population. Our results confirmed the association of longer CAG repeats with a risk of impaired semen quality, but this association can be modified by ethnic origin. Identification of the number of AR CAG repeats can be an effective tool to assess the risk of male subfertility and the control of androgen hormone therapy of reproductive diseases.Male infertility is a multi-factorial and multi-genetic disorder, and the prevalence of male infertility in the world is estimated at 5–35%. The search for the causes of male infertility allowed for identifying a number of genetic factors including a single X-linked gene of the androgen receptor ( AR gene contains eight exons and seven introns, located on chromosome Xq11-12 [AR gene, code for polyglutamine and polyglycine stretches, respectively. It was found that longer CAG repeats are associated with a decrease in AR transactivation activity, and clinical studies have shown that longer polyglutamine tracts are a factor affecting spermatogenesis and male fertility [Male infertility is a multi-factorial and multi-genetic disorder, and the incidence of male infertility in the world is estimated at 5–35%, which may reflect real differences between populations . Current Xq11-12 . The AR,ertility ,7.AR gene CAG repeats expands from 6 to 40 trinucleotides; however, ethnic differences in the length of CAG repeats were revealed [AR CAG alleles suggest potential ethnic differences in the transcriptional activity of the androgen receptor and, consequently, in the sensitivity of target tissues, including reproductive ones, to androgens.The maximum range of the length of the revealed ,11,12,13revealed . SignifiAR CAG repeats had a different effect on male fertility depending on the ethnic composition of the study population: long CAG alleles were associated with the risk of male infertility mainly in Caucasian populations, but such relationships were occasionally observed in some Asian and African populations [Quantitative impairment of spermatogenesis accounts for approximately 75% of cases with male factor infertility . The assulations . Thus, tulations .AR CAG repeat polymorphism with impaired spermatogenesis is not clear and is often contradictory in different populations, it is assumed that knowledge of the population variability of AR CAG length may be extremely informative as a basis for understanding ethnic differences in semen quality associated with the androgen action.For the past decades, several population-based studies addressed the issue of geographic differences in semen quality. A significant amount of data available came from European and Asian countries, and United States of America showing large regional differences in sperm concentration, total count and motility ,17,18,19AR CAG repeats and the association between various AR CAG genetic variants with semen quality and reproductive hormone levels in a multi-ethnic male population from Russia. The distribution of AR CAG repeat alleles and the association of the AR (CAG) n polymorphism with semen quality or reproductive hormone levels were also evaluated in individual ethnic groups.The aim of this population-based study was to investigate the variability of the length of AR (CAG) n polymorphism in the entire study population revealed 29 alleles with the range of 6–39 repeats (p < 0.01) and was shifted to the right  among the subgroups with normal and impaired semen quality  in the participants with impaired compared to normal semen quality. The testosterone level did not differ between these subgroups.The search for associations between the CAG repeat polymorphism of the p < 0.05) in total sperm count, sperm concentration, progressive motility and normal morphology, and small but statistically significant differences (p < 0.05) in the estradiol level , Buryats (Asians), and Yakuts (Asians) represented significant ethnic cohorts; thus, we limited our comparative ethnic analysis to three ethnic subgroups, which accounted for 79.4% of our study population. The ethnic distribution was as follows: Slavs 52.5%, Buryats 16.1%, Yakuts 10.8%, other small ethnic groups and descendants from mixed marriages constituted 20.6%.AR (CAG) n, frequency distributions of the CAG repeat length and mean values for CAG repeat length for the Slavic, Buryat and Yakut ethnic subgroups are summarized in p < 0.001) and was the shortest in the Slavic subgroup and the longest in the Yakut subgroup, the Buryat subgroup ranked middle ; Buryats and Yakuts ; Yakuts and Slavs . The most common CAG alleles were 22 (16.3%), 22 (12.9%) and 25 (21.6%) triplets in the Slavic, Buryat and Yakut subgroups, respectively (The numbers of samples genotyped at the d middle . Accordid middle . In partectively . The ranp < 0.05) in the CAG repeat length between men with normal and impaired semen quality was found only in Slavs, remaining lower in the first group compared to the second. These results indicate a higher incidence of long CAG repeats in the Slavic subgroup with impaired semen quality. Additionally, higher LH and FSH and lower inhibin B level, and as expected, lower total sperm count, concentration, motility and normal morphology were demonstrated in men with impaired compared to normal semen quality in the Slavic ethnic group. In the Buryat and Yakut groups, the impaired semen quality was not accompanied by any changes in the CAG repeat length or hormonal levels, with the exception of the LH level in Yakuts. Testosterone and estradiol levels did not differ significantly between the subgroups with different semen quality in all ethnic groups.The CAG repeat length, semen and hormonal parameters in the Slavic, Buryat and Yakut subgroups with normal and impaired semen quality are presented in The next step in the search for possible associations between the AR CAG repeat polymorphism and reproductive parameters was the stratification of CAG repeat length into three categories: “short”, “medium” and “long” for each ethnic group. The lengths of short and long CAG repeats were calculated taking into account ethnic differences between the ranges and medians of the length of CAG repeats for Slavs, Buryats and Yakuts.p < 0.05) lower sperm progressive motility and normal morphology than the short CAG repeat category in Slavs, and the long CAG allele category had significantly (p < 0.05) lower sperm concentration and normal morphology than the short CAG repeat category in Buryats  n may have an increased risk of impaired spermatogenesis. More specifically, we revealed a significant difference in the frequency distribution and mean values for the CAG repeat length between subgroups with normal and impaired semen quality. Additionally, we found that all sperm indicators were lower in the category of long CAG repeats compared with those in the category of short CAG repeats.Our population-based cohort study represents the largest Russian study investigating the frequency distribution of CAG repeat length and the effect of the AR gene and semen parameters including sperm concentration, motility and morphology is the subject of intense discussion, since the available results are contradictory and often unreliable. In an earlier review, it was noted that longer AR CAG repeats were associated with infertility and impaired spermatogenesis of different severity [AR CAG polymorphism in male infertility was also clarified in a later meta-analysis [Currently, the supposed relationship between the CAG repeat length of the severity . The rolanalysis . The anaanalysis . More reanalysis . Later, analysis ,25,26.AR CAG repeats in infertile and fertile males are not always detected. The length of AR CAG repeats in the Israeli male population of mixed ethnicity (Jews and Arabs) did not differ between the group with idiopathic infertility and pathozoospermia and the control group with a normal semen analysis [AR CAG repeats and the risk of male infertility or defective steroidogenesis was found in the studies of Egyptian [Statistically significant differences in the length of analysis . No signEgyptian ; IranianEgyptian  and BaltEgyptian . An assoEgyptian , China [Egyptian , Turkey Egyptian , and JorEgyptian .AR CAG number, but results were controversial. A positive correlation between the number of CAG repeats and serum testosterone levels or, in some cases, estradiol levels was shown in large cohorts of healthy European men, which suggested that the AR CAG repeat polymorphism explains 6.0–8.5% of interindividual testosterone variations [In our entire study, there were no hormonal changes between the stratified subgroups with different CAG repeat length, except estradiol. To date, several studies have examined the possible relation of reproductive hormone levels with riations ,35,36,37riations ,38. A lariations ,40,41. Triations ,43.AR gene becomes important for understanding the variability of alleles of this gene and differences in sensitivity to androgens. In connection with the above, the second aim of our study was to investigate the distribution of AR CAG repeats and possible associations of the length of AR CAG repeat tract with spermatogenesis in men from different ethnic groups of Russia.Currently, genetic testing of the population is becoming relevant, providing information about the prevalence and influence of certain genetic factors on male fertility and allowing predicting and planning preventive, diagnostic and clinical work on the male reproductive health. The assessment of ethnic differences in the length of CAG repeats of the AR CAG repeats with the risk of impaired semen quality can be modified by ethnicity. More specifically, the ethnic differences were revealed in the allele distribution of the AR (CAG) n polymorphism and the medians of the GAG repeat length between the groups of Slavs (median 23), Buryats (median 24) and Yakuts (median 25). Longer CAG repeats were associated with an impaired semen quality in the Slavic (CAG ≥ 25) and Buryat (CAG ≥ 28) groups, but this effect was not found in Yakuts. In a previous study, we found that Slavic men had a higher semen quality compared to Buryats and Yakuts, apparently due to the higher testicular function in the Caucasian compared to Asian race [We have found significant ethnic differences in the frequency distribution of CAG repeat length and confirmed that the association of longer ian race . A greatAR (CAG) n polymorphism have been revealed between Caucasian, Thai, Afro-Caribbean, Hispanic populations [AR CAG alleles is remarkably different between South Europeans, North Africans and Sub-Saharan Africans [AR gene between races or ethnicities, but also associated reproductive differences, including male infertility, impaired spermatogenesis and diseases of the reproductive system.Our ethnic findings confirmed the previously obtained facts about ethnic differences in the (CAG) n repeat polymorphism. It is well documented that the mean CAG tract length in men of African descent possess significantly shorter alleles than in men of non-African populations . Signifiulations . In the Africans . PopulatAR CAG alleles with impaired semen quality found only in Slavs and Buryats, while this effect was not detected in Yakuts. Consequently, the number of CAG repeats can have different effects on the male reproductive function depending on ethnic background. It was noted that the association of long AR CAG alleles with male infertility or poor semen quality was confidently established for populations of the Caucasian race, but such associations were rare in Asian and African populations, which was confirmed by the conclusions of several meta-analyses [The most interesting result of the current study was the association of longer analyses ,15.AR CAG alleles affect spermatogenesis are insufficiently studied. As is well known, testosterone plays a key role in spermatogenesis, but its functions are mediated by the AR. The effect of testosterone on spermatogenesis is carried out mainly through Sertoli cells, which express the androgen receptor and provide output of mature spermatozoa, and through the androgen-dependent and androgen receptor-expressing epididymis, where spermatozoa acquire motility and higher concentration [AR CAG repeat length, reducing the sensitivity of Sertoli cells and epididymis to androgens, can attenuate the physiological effects of androgens on the sperm output. From the information presented above, we could suggest that the receptor’s sensitivity to testosterone is involved in the impairment of semen quality in the Slavic or Buryat carriers of longer AR CAG repeats. Additionally, it should keep in mind that mainly intratesticular testosterone plays a major role in spermatogenesis, and the testicular testosterone content is 25–125 times higher than the blood serum [The mechanisms demonstrating how long or short ntration ,45,46. Lod serum . The serAR gene, the compromised transcriptional activity of AR was not connected with signals of impaired spermatogenesis or with hormonal levels. Although the size of this group was not big enough for the final interpretation of the results, the negative results obtained can confirm that long (CAG) n repeats do not always impair the semen quality. In addition, in other studies, no association between the expansion of AR CAG repeats and impaired spermatogenesis was found, including Iranian, Swedish, German, Dutch, Belgian, Danish, and Japanese populations [In the Yakut group with longer (CAG) n repeats in the ulations ,14,15,48ulations . There iulations . The assAR CAG repeat polymorphism currently has low diagnostic value due to the lack of clearly defined thresholds assessing the risk of male infertility. A question of choosing the threshold repeat length for diagnostic purposes is of fundamental importance. In most cases, the division into “short”, “medium” and “long” CAG repeat length categories is carried out empirically or based on the results of previously published studies. For example, the threshold value of the length of AR CAG repeats as 21 triplets is given in the meta-analysis [AR CAG repeats into three categories, taking the number of CAG repeats equal to 22–23 triplets as a reference limit of the norm as the highest AR activity. The repeat lengths less than 22 or more than 23 led to an increased risk of infertility [AR CAG repeats for Russian males. In our study population, the (CAG) n length ranges between 6 and 39 repeats, with a median value that varies according to the ethnicity and longer CAG repeats increased the risk for an impaired semen quality only in men of two ethnicities from three. Based on these data, it is impossible to find a threshold value of CAG repeats, above which the risk of poor spermatogenesis increases. Thus, there are disagreements in determining the threshold values of AR (CAG) n length, which, apparently, need to be eliminated, at least for large ethnic groups, before introducing this polymorphism into the diagnostic protocol.It is believed that genetic testing subfertile or infertile males for the (CAG) n repeat polymorphism has diagnostic and prognostic value, since it allows for assessing the risk of androgen-dependent disorders, an effectivity of testosterone therapies and the likelihood of the development of reproductive pathology in offspring. Some authors acknowledge that genotyping of the analysis , based oanalysis , it was ertility . A numbeertility . In anotertility , the intAR CAG repeat alleles on semen parameters. Firstly, more research is needed to evaluate the effects of CAG repeats in the Yakut ethnic group to confirm or reject our pilot observations. Our multi-ethnic research may also serve as a starting point for further studies, taken into account a wide diversity of nationalities in Russia. Secondly, some studies suggest that GGN repeats in the AR gene or specific combinations of CAG and GGN triplets can modulate the AR function [In our study, there are few limitations associated with effects of function ,39,40. AFor the present study, 1324 males were recruited from five Russian cities: Archangelsk, Novosibirsk, Kemerovo, Ulan-Ude, Yakutsk, and all samples were collected during a 5-year period (2009–2014). The city of Archangelsk is located in European North of Russia within the circumpolar area, the cities of Novosibirsk and Kemerovo in Western Siberia; all three cities have a predominantly Slavic population (approximately 90–95%). The cities of Ulan-Ude and Yakutsk are located in Eastern Siberia, and Yakutsk is located near the Arctic Circle. Buryats make up 32% of the total population of Ulan-Ude, and Yakuts make up 43% of the total population of Yakutsk.In all cities, the study design and standardized recruitment protocol were the same, which were described earlier in more detail ,21. Brie2) was calculated. Testicular volume (mL) was estimated by a Prader orchidometer and was presented as bitesticular volume (paired testicular volume). Age was calculated as the difference between year of attendance in study and self-reported year of birth.All the participants were examined by the same experienced andrologist and the results of examination were recorded. During the examination, some current urogenital disorders were diagnosed. Body weight (kg) and height were measured in all participants. Body mass index , Buryats (n = 208) and Yakuts (n = 134). Participants were stratified into the ethnic groups according to information obtained from the self-reporting questionnaires, taking into account self-identified ethnicity and ethnicities of their mothers, fathers and grandparents. The participant of the Slavic, Buryat or Yakut ethnicity was eligible if the ethnicity of himself, his mother, father and both grandparents was the same. Our research cohort consisted of Slavs living in all five cities, and Buryats and Yakuts living compactly in the cities of Ulan-Ude and Yakutsk, respectively.Our study population was multi-ethnic and consisted of men of 20 ethnicities and descendants from ethnic mixed marriages living in five Russian cities. To investigate ethnic differences in the p = 0.69).Differences in semen parameters between these ethnic groups were presented elsewhere with higher semen quality among Slavs, average among Buryats and lowest in Yakuts . There w6/mL), sperm motility and normal morphology (percentage) according to the WHO laboratory manual for the examination and processing of human semen [The semen samples were analyzed for semen volume (ml), sperm concentration , follicle-stimulating hormone (FSH), luteinizing hormone (LH), and inhibin B (InhB) were 0.2–50 nmol/L, 0.1–20 nmol/L, 2.0–100 mME/mL, 20–90 mME/mL, and 12–105 pg/mL, respectively. The sensitivities for T, E2, FSH, LH, InhB were 0.2 mmol/L, 0.025 nmol/L, 0.25 mME/mL, 0.25 mME/mL, 2.6 pg/mL, respectively. The intra- and interassay coefficients of variation were as follows: for T < 8.0%; E2 < 8.0%; FSH < 8.0%; LH < 8.0%, InhB ≤ 6.8%.Serum hormone concentrations were determined by enzyme immunoassay using commercially available kits “Steroid IFA-Testosterone-01”, “Gonadotropin IFA-LH”, “Gonadotropin IFA-FSH” , “Estradiol-IFA”  and “Inhibin B Gen II ELISA” . The ranges of evaluated concentrations for total testosterone (T), estradiol . We obtained AR (CAG) n genotypes for 1313 of 1324 men. The method allows for determining the relative length of the product in relation to the length standard and is based on the separation of DNA into fractions by molecular weight. Among the azoospermic participants (n = 31), samples with Y-chromosome deletions as known genetic causes of infertility have been excluded. The evaluation principle of the Y-chromosome deletions is available in a For genotyping of the ® Size Standard (dilution 1:100). The sizes of fragments were determined by using a “Nanophor-05” sequencer and Fragment software . The number of CAG repeats was calculated using an allelic ladder of marker fragments, which consisted of eight fragments of different lengths and were used as an internal standard for calculations , which were confirmed by Sanger sequencing.Ten nanograms of DNA were amplified in 15 µL of reaction mixture containing 2.5 pmol each of fluorescently labeled forward, non-labeled reverse primer and RT-PCT master mix . The primer sequences were as follows: forward, 5′-(FAM)-TCCAGAATCTGTTCCAGAGCGTGC-3′ and reverse, 5′-GCTGTGAAGGTTGCTGTTCCTCAT-3′. Each cycle included a denaturation step at 95 °C for 15 s, a primer-annealing step at 63 °C for 15 s and a primer extension step at 72 °C for 15 s with a subsequent of initial denaturation step at 95 °C for 2 min and a final extension step at 72 °C for 7 min. The amplified samples of 0.7 µL each were mixed with 10 µL of formamide and the GeneScan™ 500 LIZ2 test was used to compare frequency distributions of the CAG repeat length among different semen quality or ethnic groups. A p value < 0.05 was regarded as statistically significant.Statistical analysis was performed using the statistical package “STATISTICA” (version 8.0). The results are presented as mean (SD) as well as median with 5th and 95th percentiles. The Kolmogorov–Smirnov test for normality estimated that the values of the studied anthropometrical, semen and hormonal parameters, as well as bitesticular volume were not normally distributed. The Kruskal–Wallis ANOVA test for comparing multiple independent groups was carried out to find the differences in the CAG repeat length, semen and hormonal parameters among different semen quality or ethnic groups. χThe first way to identify associations of the CAG repeat length with semen and hormonal parameters was as follows: the participants of the entire study population or each ethnic group were divided into two semen quality subgroups using the WHO reference limits for sperm concentration, motility and morphology . Men werThe second way to identify associations of the CAG repeat length with semen and hormonal parameters was as follows: the participants of the entire study population or each ethnic group were stratified into three CAG categories based on the CAG range restriction with a frequency below 5% for short or long CAG repeat length. The rest of the CAG repeat range represented the medium CAG repeat category. The categorization of the CAG repeat length for the entire study population is presented in AR gene on spermatogenesis and its role in the regulation of fertility in young men from Russia. Our results support the opinion that men with longer (CAG) n may have an increased risk of impaired spermatogenesis. In particular, the CAG repeat length in the AR gene can affect semen quality in an ethnic-specific fashion. However, more studies are required to understand a role of the CAG repeat polymorphism in different ethnicities, specifically a range of allele variants in clinic aspects should considered. Additional epidemiological studies are also needed to validate these findings on the AR function and its correlation with spermatogenesis and male fertility for other ethnic groups.This study highlights the influence of the CAG repeat polymorphism in the


Transcranial electrical stimulation (tES) can modulate the neurovascular unit, including the perivascular space morphology, but the mechanisms are unclear. In this perspective article, we used an open-source “rsHRF toolbox” and an open-source functional magnetic resonance imaging (fMRI) transcranial direct current stimulation (tDCS) data set to show the effects of tDCS on the temporal profile of the haemodynamic response function (HRF). We investigated the effects of tDCS in the gray matter and at three regions of interest in the gray matter, namely, the anodal electrode (FC5), cathodal electrode (FP2), and an independent site remote from the electrodes (PZ). A “canonical HRF” with time and dispersion derivatives and a finite impulse response (FIR) model with three parameters captured the effects of anodal tDCS on the temporal profile of the HRF. The FIR model showed tDCS onset effects on the temporal profile of HRF for verum and sham tDCS conditions that were different from the no tDCS condition, which questions the validity of the sham tDCS (placebo). Here, we postulated that the effects of tDCS onset on the temporal profile of HRF are subserved by the effects on neurovascular coupling. We provide our perspective based on previous work on tES effects on the neurovascular unit, including mechanistic grey-box modeling of the effects of tES on the vasculature that can facilitate model predictive control (MPC). Future studies need to investigate grey-box modeling of online effects of tES on the neurovascular unit, including perivascular space, neurometabolic coupling, and neurovascular coupling, that can facilitate MPC of the tES dose-response to address the momentary (“state”) and phenotypic (“trait”) factors. The estimation of the HRF height parameter was not found to be different between the “canonical HRF” with time and dispersion derivatives and the “FIR HRF” model. However, when we investigated the HRF temporal profile, as shown in We analyzed an open-source fMRI-tDCS data set  that hadvia insulin independent mechanism  of the different neuronal subpopulations , i.e., or study . Therefoor study  containior study . Based oor study , neuronsulations  to changulations  and OGI ulations . Then, iwhere λ is arbitrary gain from the lead field matrix and τ is the time constant . The gre+) can act as a potent vasodilator signal  was perturbed by tDCS, IBK is current through big potassium (BK) channel, Ileakis leak current, ITRPVis electrical current through the TRPV channel, and IKΣ is the electrical current carried by the K+ influx at the perisynaptic process:Astrocytes are susceptible to small variations in their membrane potential, and their long processes are sensitive to polarization by tDCS . Therefotrocytes , 82. In A mitochondrial sub-compartment can be added from Jolivet et al.'s study  to accou+ concentration by the imbalance in their membrane polarity and can affect K+ spatial buffering, affecting tDCS modulation , into the perivascular space can be perturbed by tDCS. Astrocytic role in neurovascular coupling may be related to the strength of stimulation where high strength can lead to vasoconstriction . Vasoco+ concentration in the perivascular space. The K+ flow from the astrocyte and SMCs is JBK and JKIR, corresponding to BK and inward rectifying potassium (KIR), respectively. VRpa and VRps are the volume ratios of perivascular space to astrocyte and SMC, respectively. Rdecay is the rate at which perivascular K+ concentration decays to its baseline state.In this study, + channels are important in penetrating arterioles that control arterial diameter by exerting a major hyperpolarising influence  of SMCs and inwardly rectifying Knfluence . Therefonfluence  can pert+ that nested other tDCS perturbation Pathways 2–4, which results in vessel oscillations that critically depend on the parameters, including vessel stiffness, in the ordinary differential equations.In this study, the four tDCS perturbation pathways are nested , that isThe therapeutic effects of tES on brain metabolism are relevant in dementia  since thvia the perivascular pathway, where vasoconstriction can increase while vasodilation can decrease the volume of the perivascular space (refer to refer to . In the refer to , an immerefer to  due to trefer to , 54. Therefer to  can modurefer to . In our refer to , we founrefer to  for immerefer to . MPC userefer to  as well refer to . Acute mrefer to  that shorefer to , 50. Sinrefer to  when comrefer to , 46, porrefer to . Here, trefer to  in pathorefer to , 3, 100 refer to  due to eThe limitation of the current study includes the unavailability of the fMRI-EEG data to capture the long-term effects of tES on neurovascular coupling. Our fNIRS-EEG data has been shown to be feasible for computational modeling of neurovascular coupling , 41. AlsThe original contributions presented in the study are included in the article/YA: data curation, formal analysis, investigation, methodology, software, validation, visualization, and writing—original draft. AD: conceptualization, formal analysis, investigation, methodology, project administration, resources, supervision, validation, visualization, and writing—original draft and review and editing. Both authors contributed to the article and approved the submitted version.


Compounds 3f, 3d, and 3i exhibited promising nootropic activity by enhancing acetylecholine (ACh) release in A549 cell line. Moreover, the carbamate hybrid 3f was found to exhibit higher in vitro potency than donepezil with IC50 = 18 ± 0.2 nM, 29.9 ± 0.15 nM for 3f and donepezil, respectively. 3f was also found to effectively inhibit AChE activity in rat brain (AChE = 1.266 ng/mL) compared to tacrine (AChE = 1.137 ng/ml). An assessment of the ADMET properties revealed that compounds 3f, 3d, and 3i are drug-like and can penetrate blood–brain barrier. Findings presented here showcase highly potential cholinergic agents, with expected partial agonist activity towards glycine binding pocket of NMDAR which could lead to development and optimisation of novel nootropic drugs.An efficient method for synthesising NMDAR co-agonist Sunifiram (DM235), in addition to Sunifram-carbamate and anthranilamide hybrids, has been developed in high yields   Individuals with CI are usually in need for expensive nursing, safekeeping, and institutional care.,,N-methyl-D-aspartate receptor (NMDAR) is another approach in enhancing cognition,in vivo either by glycine itself or by D-serine.via the glycine site had developed like administration of glycine but this strategy is limited by the high activity of GlyT-1, so effort is moved to develop GlyT-1 inhibitors like Pfizer sarcosine analogue CP-802079  is a novel potent nootropic drug developed by Gualtieri research group in 2000 and is considered as new class of nootropic agents.The importance of Sunifiram “unsymmetric diacylated piperazines”  and it’sThis stimulated us to undertake the current study where we illustrated a new protecting-group-free synthetic route towards Sunifiram, and constructed novel Sunifiram analogues which are able to modulate NMDARs, and are equipped with the structural features that enable them to inhibit acetylcholinestrase enzyme thus increasing the concentration of acetyl choline by two mechanisms . CarbamaN-acylbenzotriazoles in high yields.N-acylbenzotriazoles (1a-k) by reacting carboxylic acids, namely, benzoic, propionic, nicotinic, anthranilc, p-aminobenzoic acid, and N-Boc aminoacids with 1 equivalent of 1H-benzotriazole and 1.4 equivalents. N,N′-Dicyclohexylcarbodiimide (DCC) in CH2Cl2 at 25 °C for 12 h  in n-butanol for 3 h to produce N-benzoylpiperazine (2a) in 76% yield.We have recently succeeded in monoacylation of aromatic and aliphatic symmetrical diamines using for 12 h .32–34 Su2a) with N-acylbenzotriazoles (1 b–k) in n-butanol for 1 h affords Sunifiram and various analogues in high yields in 80–90% yield -2,5-diphenyltetrazolium bromide (MTT) assay to select safe dose for evaluation of acetylcholine release.3c-j and 3a (Sunifiram) and subjected to MTT assay  with compound 3f (ACh = 176.1 pg/ml) being the most active even higher than the well-known cognitive enhancer Sunifiram (ACh = 144.3 pg/ml). This may be attributed to the ability of these compounds to directly enhance acetylcholine release from A549 cells and/or by inhibiting AChE, thereby result in increasing acetylcholine concentration as may be concluded from the lower level of free choline in case of 3f and 3d (221.5 pg/ml and 235.6 pg/ml) compared to control group or Sunifram-treated group with free choline levels of 255.7 pg/ml and 258.2 pg/ml, respectively.3i caused a 1.8-fold increase in acetylcholine concentration compared to the control. These results suggest that targets 3d, 3f, and 3i can be good lead compounds to develop novel cognitive enhancers. Other compounds  expressed enhanced ACh release with variable increments (1.3- to 1.5-fold) of normal ACh value. The high level of acetylcholine measured with carbamate compounds 3f and 3d may be attributed to a dual effect of these compounds as both AChE inhibitor and NMDAR co-agonist in contrast to the carbamate devoid compounds. So estimating the activity of the most potent compounds on AChE was also performed.The relative amount of ACh released in response to the target compounds 49 cells . Compounin vitro inhibitory activities against AChE based on Ellman’s method in comparison to donenpezil.3f possessing methionine side chain (IC50=0.018 ± 0.0002 µM), a value lower than that of the reference drug donepezil (IC50= 0.0299 ± 0.00015 µM) and close to that of rivastigmine (IC50= 0.0163 ± 0.00017 µM). Absence of 3f side chain (2-methylthioethyl group resulted in decreasing the potency of the targets to micromolar range (10.36–22.37 uM) (3f is important for potency and (ii) increasing the size of side chain at the α-carbon 2, ph) resulted in decreasing anti-AChE activity  . It was activity .3f was then subjected to ex-vivo experiment to assess its efficacy as AChE inhibitor.3f-(10 µg/kg) treated group and tacrine- (10 µg/kg) treated groups. AChE activity was determined according to modified Ellman assay method.The most active compound 3f can inhibit effectively AChE activity in rate brain with efficacy (AChE = 1.266 ng/mL) close to that of tacrine (AChE = 1.137 ng/ml). It could be concluded that:3f can cross BBBTarget 3f can inhibit AChE effectively in vivo and could be considered for further investigation as a nootropic agent capable of enhancing cognition in various diseases involving cognitive deficit such as Alzheimer’s disease and Parkinson’s disease.Target The results indicate that compound 3f and 3i was evaluated using Transformed Human Liver Epithelial-2 (THLE-2). Compounds 3f and 3i have been incubated with THLE-2 cells for 24 h and MTT assay was used to determine cell viability. Compound 3f was less cytotoxic with IC50 equal 40.58 ± 1.95 µM while compound 3i cause 50% decrease in cell viability at 26.979 ± 1.29 µM.Hepatotoxicity of the active compounds 3a-j were docked into the glycine binding pocket of the NMDA receptor ligand binding domain (LBD) to ascertain their chemical and physical feasibility towards the glycine binding pocket. The scoring functions incorporated in the molecular docking tools allow the evaluation of the binding affinity of each compound3i which exhibited the highest  binding affinities was selected for further in silico investigations. This top compound exhibited relatively similar binding affinity with Sunifiram as seen in Targets ,3c-j have LogP in the range 1.5–2.36, this value is optimal for BBB penetration as postulated by Hansch and Leo that found that BBB penetration is optimal when the LogP values are in the range of 1.5–2.7, while compound 3 b did not met this value (LogP = 0.94).3i  for molecular dynamics simulation relative to the native ligand glycine.Upon administration of a drug, the pharmacokinetics and the physicochemical properties of the drug influence their rate of absorption, distribution, metabolism, and excretion in human system.3i to the glycine binding pocket using a time-scale analyses of the trajectories generated by the molecular dynamics technique employed. The stability of the systems, mobility, compactness, and the fluctuations of the residues were estimated by computing the C‐α root-mean square deviation (RMSD), C‐α radius of gyration (RoG), and the C‐α root-mean-square fluctuation (RMSF). The C‐α RMSD measures the atomistic deviations as well as reflecting the stability and convergence of the systems. As observed in 3i system showed average RMSD values of 2.063 Å and 2.213 Å, respectively. Generally, the stability of the systems gives credence to the assumptions derived from the models. We further calculated the RMSF, which is predictive of the flexibility of the systems. As observed from the RMSF graph plots, 3i presents the less flexible domain with an average RMSF value of 13.081 Å, a value lesser that the unbound model and Sunifiram bound model which presented 15.595 Å and 14.127 Å, respectively. This suggests that the binding of 3i to the ligand binding domain induces a less flexible domain compared to Sunifiram. This could indicate that the binding of 3i further decreased structural flexibility reflective of distortions of backbone atoms and corroborated with the findings from RoG where in high RoG values indicate less compactness and high mobility of the C‐α atoms. RoG average figures of 19.315 Å, 19.448 Å, and 19.452 Å were presented for 3i, Apo, and Sunifiram, respectively. These as well indicate that the 3i induced high compactness and less residual mobility. Taken together, the ability of the compounds to induce a reduction in residual flexibility, compactness, and mobility could induce a signal to the membrane domain of the receptor that resultantly reliefs the TMD of magnesium thereby opening the channel for passage. It could therefore be inferred further that 3i with the most disruptive effect on the Ca atoms could be more potent and of therapeutic use making it a candidate for experimental validation.When chemical compounds bind to biological targets, they usually instigate changes in the primary structure of the biological target which resultantly disrupts the basal functionalities. We therefore investigated the structural changes that occur upon the binding of 3i were compared to the native co-agonist glycine to evaluate their differential effects on the ligand binding domain. As observed from 3i presented similar results as that of the native ligand. Both systems showed similar C‐α atoms deviation pattern during the period of simulation via their RMSD calculation with average RMDS values of 2.213 Å and 2.006 Å for 3i and glycine respectively. Relative to the unliganded LBD, both 3i and glycine are shown to decrease the stability of LBD. Also, RMSF and RoG estimations of both 3i and glycine reflect similarity of action on the ligand binding domain. For the residual fluctuations of the domain, both 3i and glycine presented lower figures compared to the unbound system, informative of the reductive effects of both ligands on the flexibility and mobility of the domain. Average RMSF figures of 13.081 Å and 14.398 Å were presented by 3i and glycine, respectively. The compactness of the domain as observed in the RoG figures of 19.315 Å and 19.305 Å for 3i and glycine, respectively, corroborates the reductive effect of both compounds on the mobility which ultimately increase compactness and rigidity of the domain. Presenting a further lower figure compared to the native ligand as observed in the case of 3i could imply more potency though this needs experimental validationFinally, the induced conformational and structural dynamics upon the binding of 3i-receptor complex and Sunifiran-receptor complex during the 250 ns simulation period and the evaluation of their binding energies (ΔG) involved in their complex formations. As shown in 3i and Sunifiram exhibited favourable free binding energies of −30.13 kcal/mol and −6.43 kcal/mol, respectively. Van der waals and electrostatic energies contributed immensely to their total binding energies which ultimately led to the stability of the compounds within glycine binding pocket.Probing further, we sought to ascertain the structural insights into the mechanistic binding and stability of the 3i complex. These results present higher residue energy contributions from the residues to the compound 3i relative to Sunifiram.These desirable binding energies of the compounds could explain their modulatory effects on the entire NMDA receptor. Intermolecular interactions between the compounds and the residues at the binding site facilitate the binding and stability of the compounds to the domain. As seen in in vitro results as can be seen from 3f is found to be the most potent in inhibiting AChE depending on that we focussed on it during simulation. The non-covalent binding mechanisms of new compounds were predicted using molecular docking simulation using rivastigmine and donepzil as reference. Molecular docking allowed for the prediction of the most suitable binding conformation of compound 3f which could favour its pocket stability, affinity, and inhibitory potential towards AChE.3f exhibited a docking of −8.8 kcal/mol. Relative to other synthesised analogues including the known AChE inhibitor donepezil ; 3f and rivastigmine  exhibited the lowest docking score towards AChE as shown in ,3f  allowed for the formation of strong binding pocket interactions that contributes towards its inhibitory potencyHowever, there are several crystal structures available for AChE, we have selected structures that contain carbamate derivatives as inhibitors as the synthesised analogues were based on carbamate for activity. Acetylcholinesterase(AChE) complexed with Ganstigmine was retrieved from the protein data bank (PDB) with ID: 2BAG.3f interactions revealed that at a peripheral anionic site (PAS) the –OC(CH3)3 moity is pointed towards TRP 279 forming π-sigma interaction also the –SCH3 moity participate in π–alkyl interaction with TRP 279 and PHE 290. TYR 70 and TYR 121 are close to show Van der Waals interaction with 3f  was also evident as seen in π–π stacked interaction between the phenyl ring of 3f and TRP84 and π-alkyl interaction between its piperazine part and PHE 330 and PHE 331. These interactions in addition to π–sulphur interactions with PHE288 and PHE331 and several van der Waals interactions collectively anchor compound 3f within the AChE inhibitor binding pocket. Interestingly TRP279 and TRP84 were also participate in π–π stacked interaction with donepezil in addition to π–alkyl interaction was also observed with PHE330 and PHE331 in similar manner to 3f. Also, PHE331 involved in π–π interactions with rivastigmine like 3f , its ability to induce ACh release from A549 cells, its in vivo ability to lower AChE activity in rat brain makes it worthy of further investigation as a promising nootropic agent. It also showed molecular docking score = −1.7 kcal/mol when docked to glycine binding pocket of NMDA receptor compared to Sunifiram  and the anthranilamide hybrid 3i . Compounds 3i further bound preferentially to NMDA domain with high binding affinity interaction which enhanced its binding pocket stability and are promising leads as potent co-agonists that binds to the glycine binding pocket of NMDA receptor and expressed good AChE inhibitory activity with IC50 value of 10.36 ± 0.23 µM.In conclusion, novel Sunifiram-carbamate and Sunifram-anthranilamide hybrids were designed, synthesised, and evaluated for cholinergic activity. Introducing carbamate to the skeleton of synthesised targets enabled a good AChE inhibitory activity. Amongst all targets compound the Sunifram-carbamate hybrid 1H and 13 C NMR spectra, computational methodology, and pharmacological screening can be found via the “Supplementary Content” section of this article’s webpageFull experimental detail, 1H NMR (500 MHz) and 13 C NMR (125 MHz) spectra were recorded on JEOL a 500 MHz NMR Spectrometer and using DMSO-d6 and CDCl3 as solvents, at Faculty of Science, Mansoura University. Also Bruker 400 MHz NMR Spectrometer at Faculty of Pharmacy, Mansoura University was used. The chemical shift (δ) is reported in ppm, and coupling constants (J) are given in Hz. The HRMS was recorded on Q-TOF, 6530 (Agilent Technologies) at Faculty of pharmacy, Fayoum University. All reactions were monitored by TLC with visualisation by UV irradiation.Starting materials and solvents were purchased from common commercial sources and used without further purification. Melting points were determined on Fisher melting apparatus and are uncorrected. To the corresponding carboxylic acid (10 mmol), dissolved in dichloromethane (50 ml), benzotriazole , and dicyclohexylcarbodiimide  were added. The reaction mixture was left with stirring at 25 °C, overnight. Dicyclohexylurea was filtered, and dichloromethane was evaporated. The residue was crystallised from dichloromethane (20 ml) and hexane (30 ml). The product was filtered and dried under vacuum to give the desired compounds.H-benzo[d]triazol-1-yl)(phenyl)methanone (1a)., mp 111–112 °C (lit. 110–112 °C).1H NMR  δ 8.33–8.28 , 8.13–8.10 , 7.85–7.76 , 7.68–7.63 . 13 C NMR  δ 166.5 (C = O), 145.2 (C–N = N), 133.5 (Ar–C), 131.7 (Ar–C), 131.5 (Ar–C), 131.3 (Ar–C), 130.7 (Ar–C), 128.3 (Ar–C), 126.6 (Ar–C), 120.0 (Ar–C), 114.4 (Ar–C).H-benzo[d]triazol-1-yl)propan-1-one (1 b).1-, mp 78–80 °C ((lit. 80–82 °C)1H NMR  δ 8.28 , 8.10 , 7.64 , 7.49 , 3.45 , 1.40 .H-benzo[d]triazol-1-yl)ethan-1-one (1c).1-, mp 49–51 °C (lit. 49–51 °C).1H NMR  δ 8.22 , 7.78–7.74 , 7.61–7.57 , 2.94 .13C NMR  δ 169.6 (C = O), 145.4 (C–N = N), 130.5 (C–N), 130.4 (Ar–C), 126.1 (Ar–C), 119.8 (Ar–C), 113.8 (Ar–C), 23.0 (CH3).H-benzo[d]triazol-1-yl)-2-oxoethyl)carbamate (1d).tert-Butyl  mp 139–141 °C ((lit. 140 °C).H-benzo[d]triazol-1-yl)-1-oxopropan-2-yl)carbamate (1e).tert-Butyl (S)-, mp 68–70 °C ((lit. 68–69 °C).1H NMR  δ 8.28 , 8.23 , 7.80 , 7.63 , 7.45 , 5.48–5.41 , 1.51 , 1.38 3). 13 C NMR  δ 173.3 (–CO–N), 156.0 (–OCO–), 145.8 (=N–C=), 131.5 (Ar–H), 131.1 (Ar–H), 127.1 (Ar–H), 120.6 (Ar–H), 114.4 (Ar–H), 79.1 (–C(CH3)3), 50.2 (–CH–), 28.6 (–C(CH33)), 17.1 (–CH–CH3).H-benzo[d]triazol-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (1f).tert-Butyl (S)-, mp 119–120 ((lit. 120–121).H-benzo[d]triazol-1-yl)-4-(methylthio)-1-oxobutan-2-yl)carbamate (1 g).tert-Butyl (S)-. 1H NMR  δ 8.23–8.09 , 7.87 , 7.64–7.50 , 7.38 , 7.28 , 5.83–5.73 , 2.71–2.56 , 2.15–2.06 , 1.43 3CH).13C NMR  δ 175.4 (CON), 171.8 (COO), 145.9 (NC=), 138.7 (Ar–C), 131.1 (Ar–C), 125.9 (Ar–C), 120.3 (Ar–C), 114.3 (Ar–C), 80.7 (C(CH3)3), 53.8 (CH), 32.2 (CH2CH), 30.0 (2CHSCH3), 28.3 ((CH3)3), 15.3 (SCH3).H-benzo[d]triazol-1-yl)-1-oxo-3-phenylpropan-2-yl)carbamate (1 h).tert-butyl (S)-, mp 142–144 °C ((lit. 144–145 °C).H-benzo[d]triazol-1-yl)methanone (1i).(4-Aminophenyl), mp 178–180 °C (lit. 178–180 °C).1H NMR  δ 8.21 , 7.96 , 7.74 , 7.58 , 6.71 , 6.51 . 13 C NMR  δ 164.6 (C = O), 155.0 (C–NH2), 144.9 (C–N = N), 134.6 (Ar–C), 132.2 (Ar–C), 130.0 (Ar–C), 126.0 (Ar–C), 119.7 (Ar–C), 115.9 (Ar–C), 114.3 (Ar–C), 112.6 (Ar–C).H-benzo[d]triazol-1-yl)methanone (1j).(2-Aminophenyl), mp 130–132 °C ((lit. 132–133 °C).H-Benzo[d]triazol-1-yl)(pyridin-3-yl)methanone (1k)., mp 101–102 °C (lit. 101–102 °C).1H NMR  δ 9.22 , 8.90 , 8.50–8.48 , 8.33 , 7.86 , 7.71–7.66 . 13 C NMR  δ 165.3 (C = O), 153.3 (CH–N), 151.3 (N = CH–C–C = O), 145.2 (C–N = N), 138.8 (Ar–C), 131.4 (Ar–C), 130.9 (Ar–C), 128.0 (Ar–C), 126.8 (Ar–C), 123.3 (Ar–C), 120.1 (Ar–C), 114.3 (Ar–C).3n-butanol. To the dissolved solution, 1-benzoylbenzotriazole (1 A)  was added. The mixture was stirred at 25 °C for 3 h. The reaction mixture was filtered and the n-butanol was removed under reduced pressure. The residue was dissolved in 2 cm3 methanol and loaded on a silica gel column. A mixture of hexane—ethylacetate—methanol (2:3:5) was used for elution of the pure 1-benzoylpiperazine. which was dried under reduced pressure.In a round bottom flask, piperazine  were dissolved in 10 cm1-Benzoylpiperazine (2a). Oily, yield 1.38 g (73%). 1H NMR  δ 7.42–7.37 , 3.57 2H–), 2.87 2H–), 1.24 .n-butanol (5 ml) followed by addition of the corresponding N-acylbenzotriazole 1 b-k (1 mmol). The mixture was heated at 60 °C for 1 h. Upon completion of the reaction (monitored by TLC), the organic solvent was evaporated. The semisolid was dissolved in ethyl acetate (20 ml) and was washed with saturated Na2CO3 , water  and brine . The organic layer was dried over anhydrous sodium sulphate. Hexane (20 ml) was added to the filtrate, and then the solid obtained was dried under vacuum to give the target compounds 3a-j.In a round bottom flask, 1-benzoylpiperazine 2 A  was added to 1–(4-Benzoylpiperazin-1-yl)propan-1-one (3a).1H NMR  δ 7.46–7.41 , 3.62–3.45 , 2.32 , 0.99 . 13 C NMR  δ 171.6 (CH3CH2CO–), 169.3(Ph–CO), 135.7 (=C–CO–), 129.7 (Ar–C), 128.5(Ar–C), 127.0(Ar–C), 47.0 (=C–CON(C2H–2)–),), 44.8 (–CH2CON(C2–)2H–), 25.6 (–CH2CH3), 9.3(–CH2CH3).–(4-Benzoylpiperazin-1-yl)ethan-1-one (3 b). White microcrystal, yield 0.20 g (86%) mp 94–96 °C (lit. 94–95 °C).1H NMR  δ 7.48–7.43 , 3.46–3.38 2(CH2)2N–), 2.03 . 13 C NMR  δ 169.7 (CO), 169.0 (CO), 136.2 (–CO–C=), 130.1 (Ar–C), 128.9 (Ar–C), 127.5 (Ar–C), 45.8 ((=C–CO–N(2)2CH–), 41.4 (=C–CO–N(CH2)22)2(CHN–), 21.7 (CH3).1tert-Butyl (2–(4-benzoylpiperazin-1-yl)-2-oxoethyl)carbamate (3c). White microcrystal, yield 0.316 g (91%) mp 173–175 °C; 1H NMR  δ 7.48–7.44 , 6.81 , 6.43 , 3.82 , 3.45 2(CH2)2N–), 1.40 3C–). 13 C NMR  δ 169.7(=C–CO–), 168.1 (–CH2CO–), 156.2 (–OCO–), 136.1 (=C–CO), 130.2 (Ar–C), 128.9 (Ar–C), 127.5 (Ar–C), 78.4 ((CH3)3C–), 44.2 (=C–CO–N(2)2CH–), 42.2(–2CH–CO), 41.8 (–CH2–CO–N(2)2CH–)), 28.7 ((3)3CHC–). HRMS (ESI): m/z cald for C18H25N3O4 [M + H–C(CH3)3OCO] + 248.1394, found 248.13909tert-Butyl (S)-(1–(4-benzoylpiperazin-1-yl)-1-oxopropan-2-yl)carbamate (3d). White microcrystal, yield 0.33 g (91%) mp 70–72 °C. 1H NMR  δ 7.50 , 7.08–7.0 , 6.69 , 4.51–4.48 , 3.53 2CH–)), 3.41 2CH–), 1.43 3(CHC–), 1.19 . 13 C NMR  δ 171.4 (–CHCO–), 170.8 (=C–CO–), 155.4 (OCO), 136.1 (–CO–C=), 130.1 (Ar–C), 128.9 (Ar–C), 127.5 (Ar–C), 78.5 –C(CH3)3, 46.3 (–CH–), 45.0 (=C–CO–N(2)2CH–), 41.9(–2)2(CHN–CO–CH–), 28.7 (–C(3)3CH), 18.1 (–CH3CH). HRMS (ESI): m/z cald for C19H27N3O4 [M + H] + 362.2074, found 362.20796.tert-Butyl (S)-(1–(4-benzoylpiperazin-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (3e). White microcrystal, yield 0.36 g (92%) mp 75–77 °C. 1H NMR  δ 7.21 ), 6.66 , 6.51 , 6.27 , 3.95 , 3.45–3.26 2CH–), 3.12–3.03 2CHN–CO–CH–), 1.72–1.67 2), 1.13 3(CH), 0.60 2CH). 13 C NMR  δ 170.8 (–CHCO), 170.2 (=C–CO), 156.0 (OCO), 136.1 (=C–CO–), 130.1 (Ar–C), 128.9 (Ar–C), 127.5 (Ar–C), 78.5 (–C3)3(CH), 55.5 (–NH–CH–), 45.4 (=C–CO– N(2)2CH–), 42.3 (–(2)2CHN–CO–CH–), 30.2 (–CH(CH3)2), 28.6 (–C(3)3CH), 19.9 (–CH(3)2CH). HRMS (ESI): m/z cald for C21H31N3O4 [M + H] + 390.2387, found 390.23950.tert-Butyl (S)-(1-(4-benzoylpiperazin-1-yl)-4-(methylthio)-1-oxobutan-2-yl)carbamate (3f). White microcrystal, yield 0.39 g (93%), mp 146–148 °C. 1H NMR  δ 7.46 ), 7.15–7.02 , 6.75 , 4.52 , 3.52 2(CH–), 3.38 2CHN–CO–CH–), 2.47 , 2.05 , 1.83–1.77 , 1.39 3CH). 13 C NMR  δ 170.6 (CO), 170.1 (CO), 155.8 (–CO–O–), 136.1 (=C–CO), 130.1 (Ar–C), 128.9 (Ar–C), 127.5 (Ar–C), 78.7 (–C(CH3)3, 49.7 (–CH–), 45.0(=C–CO-N(2)2CH–), 42.0 (=C–CO–N(CH2)22)2(CHN–)), 31.6 (–S–CH22CH–), 30.2 (–S–CH2–), 28.6 (–C3)3(CH), 15.1 (–S3CH). HRMS (ESI): m/z cald for C21H31N3O4S [M + H] + 422.2108, found 422.21218.tert-Butyl (S)-(1–(4-benzoylpiperazin-1-yl)-1-oxo-3-phenylpropan-2-yl)carbamate (3 g). White microcrystal, yield 0.39 g (89%) mp 123–125 °C. 1H NMR  δ 7.47 , 7.25–7.12 , 4.57 , 3.41 2(CH2)2N–), 3.28–3.25 , 2.81–2.76 , 1.33 3). 13 C NMR  δ 170.6 (CO), 170.5(CO), 155.5 (OCO), 138.1 (Ar–C), 138.0 (Ar-C), 129.9 (Ar–C), 129.8 (Ar–C), 128.9 (Ar-C), 128.6 (Ar–C), 127. 5 (Ar–C), 126.8 (Ar–C), 78.6 (–C(CH3)3, 51.7 (–CH–), 45.0 (=C–CO–N(2)2CH–), 41.9 (–CH–CO–N(2)2CH–)), 37.8 (–CH2), 28.6 ((3)3CHC–). HRMS (ESI): m/z cald for C25H31N3O4 [M + H]+ 438.2387, found 438.23948.(4-(4-Aminobenzoyl)piperazin-1-yl)(phenyl)methanone (3 h). Buff microcrystal, yield 0.26 g (84%) mp 195–197 °C. 1H NMR  δ 7.47–7.43 , 7.17 ), 6.57 ), 5.58 , 3.66–3.54 2(CH2)2N–). 13 C NMR  δ 170.6 (CO), 169.7 (CO), 151.2 (=C–NH2), 136.2 (Ar–C), 130.1 (Ar–C), 129.9 (Ar–C), 128.9 (Ar–C), 125.0 (Ar–C), 121.9 (Ar–C), 113.1 (Ar–C), 45.1 (Aliph–C), 42.2 (Aliph–C). HRMS (ESI): m/z cald for C18H20N3O2 [M + H]+ 310.1577, found 310.1591.4-(2-Aminobenzoyl)piperazin-1-yl)(phenyl)methanone (3i). Pale yellow microcrystal, yield 0.27 g (87%) mp 170–172 °C. 1H NMR  δ 7.45–7.41 , 7.09 , 7.01–6.99 , 6.71 , 6.56 , 5.21 , 3.66–3.45 . 13 C NMR  δ 169.7 (CO), 169.4 (CO), 146.3 (=C–NH2), 136.1 (Ar–C), 130.6 (Ar–C), 130.1 (Ar–C), 128.9 (Ar–C), 128.3 (Ar–C), 127.5 (Ar–C), 119.5 (Ar–C), 119.4 (Ar–C), 116.0 (Ar–C), 47.4 (Aliph–C), 42.0 (Aliph–C). HRMS (ESI): m/z cald for C18H20N3O2 [M + H]+ 310.1577, found 310.1590.((4-benzoylpiperazin-1-yl)(pyridin-3-yl)methanone (3j). Buff microcrystal, yield 0.24 g (81%) mp 145–148 °C. 1H NMR  δ 8.66 , 7.88 , 7.47 , 3.69 2CHN–CO–ph), 3.51 2CHN–nicotinoyl). 13 C NMR  δ 169.8 (CO), 167.5 (CO), 151.1 (Ar–C), 148.2 (Ar–C), 136.0 (Ar–C), 135.4 (Ar–C), 131.9 (Ar–C), 130.2 (Ar–C), 128.9 (Ar–C), 127.5 (Ar–C), 124.0 (Ar–C), 47.4 (–(2)2CHN–CO–ph), 42.2 (–(2)2CHN-nicotinoyl). HRMS (ESI): m/z cald for C17H18N3O2 ([M + H+]) 296.1421, found 296.1438.Male Sprague-Dawley rats  were used in the present study. At our laboratory, the animals were provided from Department of Pharmacology, the Faculty of Veterinary Medicine, Zagazig University, Egypt. Animals were housed under standard conditions  in a group of five rats with free access to food and water. The number of animals used and their suffering were minimised as possible as we could. The experimenters and data-processing persons were blind to the treatment of rats. All animal experiments were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committees at Zagazig University (ZU-IACUS), Egypt.3f- (10 µg/kg) treated group and tacrine- (10 µg/kg) treated groups. The rats in the normal control group received an equivalent volume of 5% DMSO in saline (vehicle). All the rats were treated with a single intraperitoneal injection of the respective drugs and sacrificed 30 min later. The brains were quickly removed on an ice-cold plate. These tissues were homogenised in a 10-fold volume of cold 10 mM phosphate buffer (pH 7.4). The homogenates were centrifuged at 3500 rpm for 10 min, and the supernatants were employed as sources of enzyme in AChE assay. All the above steps were carried out at 4 °C. AChE activity was determined according to a modified Ellman assay method.The rats were randomly divided into three groups of five animals each: normal control group, ,3f via experimental methods, it was then docked into the Ganstigmine binding pocket of AChE. The grid box coordinates for molecular docking included; centre: X = –3.90, Y = 65.61 and Z = 63.99 and dimensions: X = 22.33, Y = 23.89 and Z = 22.28. To view the molecular interactions of the docked complex, we employed Discovery Studio Visualiser.Acetylcholinesterase (AChE) complexed with Ganstigmine was retrieved from the protein data bank (PDB) with ID: 2BAG.Click here for additional data file.


Background: Although the COVID-19 pandemic led to a series of governmental policies and regulations around the world, the effect of these policies on access to and provision of rehabilitation services has not been examined, especially in low and middle- income countries. Aims: The aim of this study was to investigate the impact of governmental policies and procedures on the number of patients who accessed rehabilitation services in the public sector in Jordan during the pandemic and to examine the combined effect of sociodemographic factors (age and gender) and the governmental procedures on this number of patients. Methods: A retrospective cohort study was conducted based on records of 32,503 patients who visited the rehabilitation center between January 2020 and February 2021. Interrupted time-series analysis was conducted with three periods and by age and gender. Results: The number of patients who visited the rehabilitation clinics decreased significantly between January 2020 and May 2020 due to government-imposed policies, then increased significantly until peaking in September 2020 (p = 0.0002). Thereafter, the number of patients decreased between October 2020 and February 2021 as a result of the second wave of the COVID-19 pandemic (p = 0.02). The numbers of male and female patients did not differ (p > 0.05). There were more patients aged 20 years and older attending rehabilitation clinics than younger patients during the first strict lock down and the following reduction of restriction procedures periods (p < 0.05). Conclusions: The COVID-19 public measures in Jordan reduced access to rehabilitation services. New approaches to building resilience and access to rehabilitation during public health emergencies are needed. A further examination of strategies and new approaches to building resilience and increasing access to rehabilitation during public health emergencies is warranted. In March 2020, the outbreak of coronavirus disease COVID-19) caused the sixth public health emergency of international concern declared by the World Health Organization (WHO). In many countries where rehabilitation services are recognized as an essential part of quality healthcare systems, the COVID-19 pandemic led to increased demands for acute care rehabilitation, with a greater number of rehabilitation professionals being reassigned to emergency rooms and joining the acute care teams in working with hospitalized patients with COVID-19. Although several studies include descriptions of the impact of COVID-19 on rehabilitation services and activities in Europe 9 caused , studiesDecisions to shift, transform, delay, or discontinue rehabilitation services are complex and have likely impacted the quality of life of many patients who needed rehabilitation . WorldwiAfter the first case of COVID-19 was diagnosed in early March 2020, the Jordanian government imposed a policy of extensive contact tracing followed by quarantine or hospital isolation of symptomatic cases. This was supported by a strict countrywide lockdown, nightly curfew, and border closure on 14 March 2020. Afterward, a Royal Decree was issued approving the implementation of the National Defense Law on 17 March 2020. As of 11 April 2021, there had been 662,395 confirmed cases and 7708 confirmed deaths in Jordan. Two spikes were observed in November 2020 [However, rehabilitation services in Jordan were deemed nonessential during the emergency response. Since its recognition in the 1970s, rehabilitation services have been provided within a traditional medical model . RehabilIt is known that the role of rehabilitation extends to the post-acute phase, during which time patients need services that aim to improve gas exchanges, reduce dyspnea, improve muscle functioning, reduce disability, and optimize function ,10. ThisIn line with the WHO initiative “Rehabilitation 2030: A Call for Action”, the Cochrane Rehabilitation and the World Health Organization Rehabilitation Programme has published the Rehabilitation Research Framework for COVID-19 patients (CRRF) . The CRRThe study was conducted and results were reported following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) collaboration guidelines . The stuThe Ministry of Health (MoH) is considered the largest provider of rehabilitation services in Jordan, which comprises three zones: the northern, central, and southern parts of Jordan. Rehabilitation services are provided by rehabilitation teams in 21 public hospitals and centers. There are no recognized acute care rehabilitation services in the hospitals of the MoH, but a rehabilitation team, including a physiatrist, physiotherapist, and other members as needed, provides rehabilitation assessment and intervention based on regular visits to different wards and in response to consultations with other specialties. The Rehabilitation Medicine Department at Al-Bashir hospital is the largest and most comprehensive provider of rehabilitation services in Jordan. As of 2021, the Al-Bashir Hospital included eight Physical Medicine and Rehabilitation Specialists in the MoH hospitals, 45 physiotherapists, 3 occupational therapists, 1 speech and language pathologist, and 12 prosthetics and orthotics among the total staff in the MoH hospitals. Since its establishment in 1965, the rehabilitation services at Al-Bashir hospital have been expanded to provide a comprehensive range of such services, including physiotherapy, occupational therapy, speech and language therapy, orthosis, and prosthesis, as well as physiatrists’ procedural interventions through the clinic for botulinum toxin injection and spasticity treatment, a stroke clinic, a nerve conduction study, a pain management clinic, and a joint clinic for neurosurgery and pediatrics.In this retrospective cohort study, we included patients who received rehabilitation services in the largest public hospital in Jordan between January 2020 and February 2021. The medical records of 32,503 patients were reviewed. Patients’ ranged from 1 month old infants to elderly people more than 60 years of age . Around 22% of the patients were younger than 20 years of age and 78% were between 20 years and 60+ years. The records were extracted from the national electronic medical system (HAKEEM).The records of all patients who utilized the rehabilitation services at Al-Basheer hospital were extracted from the national electronic medical system (HAKEEM). HAKEEM is linked with patients’ national identification numbers and all the information related to the patients’ healthcare in the public sector was accessed through this electronic database. Retrieved data included the numbers of patients who visited the rehabilitation clinics grouped by age and gender. The data extraction process for this study was initiated by a request from the head of the Physical Medicine Department to have the required unidentified data exported to Excel files by an information technology employee who had no connection to the research team. The researchers did not have access to the full database or patients’ records in HAKEEM. Therefore, the data were anonymous and had no personal identifying information linked to patients’ identities, ensuring the integrity of the data. The study was approved by the Ministry of Health national information board, exempting the study from obtaining patients’ consent.The independent variables included in this study were three time periods selected based on the governmental policies imposed and participants’ age and gender at the time of their visit to the rehabilitation center. The dependent variable was the number of patients who visited the rehabilitation clinics between January 2020 and February 2021. In this study, the number of patients who visited the rehabilitation clinics during each time period were examined.p-value, and standardized coefficients are reported for the differences between the three periods of interest.Data were entered into a Microsoft Excel database, where line graphs were used to observe the trends in the number of patients who visited the rehabilitation clinics between January 2020 and February 2021 (dependent variable). To achieve the first aim of the study, we used an interrupted time-series analysis to assess the effects of the governmental lockdown procedures on the number of patients who visited the rehabilitation center in the largest public hospital in Jordan during three time periods: (1) period 1 represented the first wave of the pandemic and the first strict lockdown, which extended from January 2020 until May 2020; (2) period 2 represented the reduction in restriction procedures and extended from June 2020 until September 2020; and (3) period 3 represented the pandemic’s second wave and the second period of strict lockdown and extended from October 2020 until February 2021. Interrupted time-series analysis enabled us to examine changes in the trend lines by calculating the differences between the slopes of the three time periods. The slopes, To achieve the second aim of the study the combined effect of sociodemographic factors (age and gender) and the governmental procedures on the number of patients who visited the rehabilitation center from January 2020 to February 2021 was examined. An interrupted time-series analysis was also used to assess the effects of the governmental lockdown procedures on the number of patients who visited the rehabilitation center as grouped by gender  and age group (<20 years vs. ≥20 years) during each time period.p < 0.05. Analyses were conducted using both Excel and SPSS version 25 software.To achieve the third aim of the study, descriptive analysis and line graphs were utilized to describe the services provided in the rehabilitation department during the three time periods of the study. The significance level was set at The number of patients who visited the rehabilitation clinics decreased sharply between January 2020 and May 2020 (period 1). Only 78 patients accessed the rehabilitation clinics in May 2020 compared to 3394 patients in January 2020 see . Afterwat (5) = 10.18, p = 0.0002) and during periods 2 and 3  = −3.12, p = 0.02).t (4) = −6.80, p = 0.0024), whereas no significant differences were found between the number of male and female patients during periods 2 and 3. This indicated that females accessed rehabilitation services more during the early months of the pandemic and up to the first strict lockdown in the country when the numbers dropped both for males and females. However, without controlling for socioeconomic variables, it is impossible to confirm that this difference is gender-related.t (4) = −16.87, p = 0.0001) and period 2  = 3.98, p = 0.0073), whereas no significant difference was found during period 3. It has been observed that less patients under the age of 20 visited the governmental rehabilitation centers both during the strict lockdown as well as after the process of social distancing was eased by the government.In response to the research gaps identified in the Rehabilitation Research Framework for COVID-19 patients , this stAt the mesosystem level, the descending trend in the number of patients receiving rehabilitation services in the public health sector in Jordan indicated limited access to public outpatient rehabilitation services during the pandemic. This is consistent with findings from an international study that included 38 countries that showed that up to two million patients did not have access to various rehabilitation services in different settings  during the COVID-19 pandemic . Prvu BeAt the macrosystem level, demand for rehabilitation services in different settings in Jordan is expected to increase dramatically in the future among patients who did not receive rehabilitation services during the lockdown and post-COVID-19 patients. The overall declining trend in the number of patients accessing rehabilitation services was found to be similar for all patients, regardless of their age. There was, however, a greater number of adult and elderly patients visiting governmental rehabilitation services during the pandemic than younger patients. This can be attributed to the increased need for rehabilitation services among older people during the pandemic. Nevertheless, considering that many young people in Jordan who receive rehabilitation services in the public sector have disabilities , furtherThe pandemic revealed a need for a paradigm shift in the provision of services for children with disabilities all around the world. Based on expert opinion, investigators in two studies recommended that special attention be given to the needs of children with cerebral palsy and autism spectrum disorders and their families during the COVID-19 pandemic; in particular, during periods of quarantine ,17. ChilInterestingly, a high number of patients were referred by physiatrists to electro- and hydrotherapy clinics in this study. After the lockdown was eased in August 2020, the highest numbers of patients were referred to the Botox and cerebral palsy clinics. This reflects a traditional medical model of services, in which the focus of rehabilitation services is on medical management and modalities rather than functional training and exercises . EvidencThe limitations of this study were mainly due to the retrospective cohort design and the utilization of secondary data, which limited our ability to examine many variables that could have affected the provision of rehabilitation services during the pandemic. For example, we were not able to assess the clinical implications of the reduction in the number of patients receiving rehabilitation during the COVID-19 outbreak due to the lack of clinical outcomes documented in the HAKEEM system. We could not differentiate between regular patients and new patients who accessed rehabilitation during the study timeframe. In addition, since we were unable to access socioeconomic variables in the HAKEEM system, we were not able to control for factors such as income, education, and residency when examining the change in the number of patients who visited the rehabilitation clinics.Following the COVID-19 pandemic, demand for rehabilitation services is expected to increase, which will require an increase in supply through health planning and adjustments to the financing of healthcare. It is therefore possible to adopt the following recommendations to help Jordan’s health systems implement resilient rehabilitation services: (1) incorporating acute care rehabilitation services into national health care plans as an essential component of quality care, (2) establishing policies to ensure continuity of rehabilitation during periods of limited access, such as policies regulating telerehabilitation, home programs, and in-home virtual reality services, (3) requiring rehabilitation professionals to attend continuing education courses that address the acute care competencies of the rehabilitation workforce, (4) providing services to people with disabilities through client-centered models, and (5) promoting public-private partnerships among healthcare sectors in order to meet the in-creased demand for rehabilitation services.The COVID-19 pandemic public measures in Jordan reduced access to rehabilitation services. A further examination of strategies and new approaches to building resilience and in-creasing access to rehabilitation during public health emergencies is warranted. As part of efforts to limit major disruptions to Jordan’s health care system, current rehabilitation practice models, such as client-centered care models, may be considered. Through these models, patients can actively participate in their rehabilitation process in addition to im-proving their ability to access and navigate the rehabilitation process.


Although the use of Augmented Reality (AR) in language learning has increased over the last two decades, there is still little research on the preparation of pre-service teachers as AR content creators. This paper focuses on analyzing the digital competence and attitudes of teacher candidates to integrate AR in the foreign language classroom. For this purpose, eighty-five college students were assigned into different teams to create their own AR-based projects which aimed at teaching English and content to young learners. The teacher candidates employed several software development kits (SDKs) to construct collaborative AR projects in a five-week period, including discursive and illustrative representations of the learning content. In this research based on a mixed method, quantitative and qualitative data were gathered trough AR project presentations and surveys encompassing two validated scales, the Technological Pedagogical Content Knowledge (TPACK) framework and the Augmented Reality Applications Attitudes Scale (ARAAS). The statistical data and qualitative findings revealed that the participants lacked practical knowledge on AR content creation and implementation in Education. The major problems were related to the TPK  intersection since participants had been previously trained in AR technology just as recipients and not as content creators and educators.The online version contains supplementary material available at 10.1007/s10639-022-11123-3. The use of Augmented Reality (AR) has increased over the last two decades thanks to the widespread of portable electronic devices, such as tablets and smartphones, and the emergence and availability of free or low-cost AR tools. The adoption of AR has been particularly prolific in digital gaming and education, which has resulted in a rich body of literature in both areas as illustrated in systematic reviews . Originally, Azuma  describeThe three types of AR technology commonly referred to are marker-based, markerless and location-based, although some authors reduce them to two: vision-based (marker-based and markerless) and location-based. All of them have been integrated with different levels of success in several educational contexts , an umbrella term used to describe modern practices in bilingual education aimed at learning associatively a content subject and a foreign language. Although the CLIL foundations can be traced back to previous models of language immersion programs in Canada and the USA, this approach has become popular in some European countries over the last two decades. After its inception in the early 1990s Marsh, , this meIn fact, AR technology can be very useful in CLIL since it combines language and content learning. However, studies on AR adoption in this area remain very scarce to date. Expósito et al.  reportedDifferent studies have underlined the need for better training in AR technology among current and future educators. Some authors have even identified certain predictors for teacher readiness and willingness to use AR such as prior knowledge  and any existing correlation with their technological and pedagogical skills (TPACK).The objectives of this paper are three:What knowledge do teacher candidates have about the use of AR in education?What technological and pedagogical skills do teacher candidates have to integrate effectively AR in the CLIL classroom?What are the teacher candidates’ attitudes toward the adoption of AR after developing their own collaborative AR projects?These objectives are related with the following research questions:https://bit.ly/3PpTiRV.The research was based on a convenience sampling method. Initially, there were 92 undergraduate students enrolled in the Applied Linguistics class taught by the authors in a medium-sized university in Spain. The students were officially divided into two groups of approximately 45 participants that would meet twice a week during two-hour class sessions. Seven students dropped the class during the first two weeks and were excluded from the research. The remaining 85 students completed all the tasks and were randomly arranged in small teams (4–5) in order to create collaborative AR projects that aimed at teaching English to children in a CLIL classroom. This digital project was part of their evaluation. Their English language level corresponded to a B2 or higher according to the CEFR framework, and they all lacked any previous training in AR content creation or implementation. The last unit in the subject was devoted to CLIL as a dual-focused educational approach and it covered different aspects such as its origins and aims, principles and strategies, types and models, benefits and challenges, and modern practices. The ages of all participants were aged between 22 and 30, 82% were female and 18% were male students. Written informed consent was obtained from all participants, including the use of data analysis and images of their class projects for scientific purposes. The research procedure followed the instructions provided by the Ethical Review Board of the University of Alicante available on its website The research design was based on a mixed method in order to collect both quantitative and qualitative data Riazi, . The expIn a later stage, the teams developed their AR collaborative projects following the instructions previously provided: the projects needed to include a minimum of ten multimedia activities, which should be meaningful, organized in a sequential manner and specifically designed to learn content and language about the topic they had selected. In the final stage, the different teams shared their projects and explained the topic, target students, multimedia activities and learning goals to the rest of the class, and participated in a semi structured discussion about the benefits and limitations of AR in the CLIL classroom after their experience as content creators. Then, the teacher candidates were post-tested using the TPACK framework, which was partly adapted to our research objectives . BesidesIn relation to the TPACK framework, CK in this experiment refers to the knowledge on CLIL concepts and principles. PK is related to the knowledge on the different pedagogical methods and practices in the CLIL classroom and TK is associated with the knowledge on general technology and AR, both for content creation (use of SDKs) and implementation . These main TPACK sections and intersections are summarized in Table The research participants created 17 AR projects using different SDKs, the three most popular being Zapworks, Aumentaty and Roar. Each team had to select a specific topic and an educational level (elementary or secondary education) for the CLIL classroom. The AR projects needed to include both discursive (theory) and illustrative (practice) representations through different images or objects (triggers) and multimedia content (overlays). The content should cover various language skills and be properly organized as a sequence of meaningful activities  in order to guide the students through the learning process. Therefore, the projects should be clearly based on a lesson plan and carefully designed taking into account the learning goals, both at the content and language level. Appendix According to the pre-test results, only 4.7% of the teacher candidates had been previous exposed to AR technology in other subjects and none of them had ever used SDKs for AR content creation. Prior experience outside of the classroom stemmed mostly from video games, as 33% of the participants had played Pokémon GO, followed at a distance by Resident Evil, Eyepet, Invizimals, Star Wars Battlefront and Harry Potter Wizards Unite. Concerning AR and VR visualization tools, the most popular were smartphones (74.1%), smart glasses (43.5%), tablets (29.4%) and head-mounted displays (22.4%).In this experiment, participants received a training session on AR concepts and tools but they required an average of 5–10 hours outside of the classroom to build up their projects. As shown in Table Regarding the TPACK results in the post-test, the data analysis provided similar means in the three main sections with small differences, as summarized in Table Compared to the scores obtained in the three main sections the intersections yielded lower results, which is consistent with previous studies on TPACK and EFL among in-service teachers Tseng, . The meaHowever, the intersection which provided the lowest outcome  was the Technological Pedagogical Knowledge (TPK), i.e. knowledge on how to use different pedagogical methods and approaches for AR integration in the CLIL classroom. In line with previous research conducted with in-service teachers, this result evidenced that pre-service teachers equally require better training in digitally-oriented pedagogical methods because the lack of this knowledge ‘poses a challenge to many practicing EFL teachers when it comes to dealing with presenting instructional materials via the employment of technology in EFL classrooms’  evidenced the moderate position adopted by the teacher candidates about their preparation to use different pedagogical methods and integrate AR technology in the CLIL classroom.The participants’ attitudes toward AR were measured in the post-test through the Augmented Reality Applications Attitude Scale (ARAAS), based on Küçük et al.  and DíazAll four dimensions yielded positive results, which indicates that the teacher candidates clearly support AR adoption for different reasons: the feeling of reality (#3 M = 3.86) and the practical view about the subject content (#9 M = 3.53) in the ‘relevance’ dimension; increased teacher-student interest (#11 M = 3.91 & #16 M = 3.72), enjoyment (#12 M = 3.79) and motivation (#14 M = 3.31), in the ‘satisfaction’ dimension; improved quality of teaching (#17 M = 3.60), collaborative learning (#18 M = 3.66), enhanced cognitive (#19 M = 3.79) and instrumental skills , in the ‘reliability’ dimension. As pointed out in previous research , correlational and linear regression analyses were implemented. Also, key assumptions of the linear regression model  were checked and confirmed using the approach recommended by Baños et al. .First, bivariate correlations were performed to explore relationships between the ARAAS factors, the participants’ previous experience and knowledge on AR technology, their satisfaction with and perceived difficulty of the SDK, and the seven different types of knowledge in the TPACK framework, which were computed as variables. Several statistically significant correlations were found as shown in Table Such correlation analyses led up to multivariate linear regressions, which aimed to look into the predictive strength of (i) students’ reported experience in the use of AR tools, (ii) students’ reported knowledge on AR tools, and (iii) any or all of the ARAAS subdimensions  on any of the different types of knowledge in the TPACK framework. As shown in Table A thematic analysis was used for qualitative data obtained through class observation and semi-structured debates. The debates were carried out after the presentations in the last stage and included exploratory questions related to the participants’ level of satisfaction with the AR projects and perceptions about the benefits and limitations of integrating AR in the CLIL classroom. Since all lessons were held online due to Covid-19 restrictions, student presentations and debates were digitally recorded and later transcribed verbatim. Then, the two authors inductively codified the data into several patterns of meaning to identify the main themes. Table However, two major limitations were pinpointed by the teacher candidates. Technologically, some participants mentioned certain constraints experienced with the SDK they selected, such as lack of image sensitivity, cloud storage capacity, limited number of scans in the free version, lack of compatibility between different operating systems, limited re-editing options and short-term availability (S24 and S37). These problems are closely related to the fact that AR tools are mostly business-oriented, and although some can be used for educational purposes their free versions have limited functionalities. Pedagogically, the teacher candidates manifested their previous lack of knowledge regarding AR creation and implementation in a real classroom as they had not been trained in technology-oriented pedagogies. Therefore, they ignored how to integrate AR effectively in a CLIL classroom, not for replacing but supplementing other pedagogical materials and to provide the learners with a more lifelike experience. In this sense, the participants advocated for a better training in modern pedagogies where technology is used in a transformative manner (S9). They also expressed some concern about the isolating factor this emerging technology may have among students if used for a prolonged time in the classroom.Considering the first research question about teacher candidates’ prior AR knowledge, the results evidenced that their experience is mostly based on video games and that their exposure to this emerging technology is limited to the role of AR recipients but not as content creators. This confirms previous studies  and a positive attitude toward AR technology can also be predictors of the digital confidence in AR implementation among future educators. This may contradict previous results Okumuş,  in whichAlthough the implementation of AR technology in Education is in its infancy, this paper has evidenced the need for better pedagogical training among pre-service teachers, particularly in the CLIL classroom which is aimed at learning language and content associatively. In this experiment, the teacher candidates used AR to create their own projects through different SDKs and were post-tested on their self-perceived knowledge of and attitudes toward AR through the TPACK framework and the ARAAS scale. Three main conclusions can be drawn from this research.First, the TPACK results demonstrated the participants’ higher self-perceived knowledge in the three main sections  compared to the intersections, which can be explained by the fact that the more specific the knowledge becomes the higher the need of adopting a holistic approach. In other words, isolated knowledge about a technology and isolated knowledge about pedagogy are not sufficient to effectively integrate new technologies in the classroom as pointed out by Mishra and Koehler , since tSecond, the results of the ARAAS scale revealed the participants’ positive attitudes in all the scale dimensions: usage reliability, satisfaction and relevance. Consistent with previous works


Augmented reality (AR) is an interactive technology that uses persuasive digital data and real-world surroundings to expand the user's reality, wherein objects are produced by various computer applications. It constitutes a novel advancement in medical care, education, and training.The aim of this work was to assess how effective AR is in training medical students when compared to other educational methods in terms of skills, knowledge, confidence, performance time, and satisfaction.We performed a meta-analysis on the effectiveness of AR in medical training that was constructed by using the Cochrane methodology. A web-based literature search was performed by using the Cochrane Library, Web of Science, PubMed, and Embase databases to find studies that recorded the effect of AR in medical training up to April 2021. The quality of the selected studies was assessed by following the Cochrane criteria for risk of bias evaluations.I2=99.9%; P<.001), confidence , and satisfaction  more than what occurs under control conditions. Further, AR did not have any effect on the participants’ knowledge  and skills . The meta-regression plot shows that there has been an increase in the number of articles discussing AR over the years and that there is no publication bias in the studies used for the meta-analysis.In total, 13 studies with a total of 654 participants were included in the meta-analysis. The findings showed that using AR in training can improve participants' performance time (The findings of this work suggest that AR can effectively improve performance time, satisfaction, and confidence in medical training but is not very effective in areas such as knowledge and skill. Therefore, more AR technologies should be implemented in the field of medical training and education. However, to confirm these findings, more meticulous research with more participants is needed. Augmented reality (AR) is an interactive technology that uses persuasive digital data and real-world surroundings to expand the user's reality, wherein objects are produced by various computer applications ,2. Its uThere are many applications of AR in medical settings. Kamphuis et al , for exaIn their work, Eckert et al  providedThe literature shows some discrepancies in the effectiveness of AR in medical training. To date, most of the meta-analysis research on the efficiency of VR and virtual patients in medical education has been carried out by Chen et al  and KonoRQ1: What are the characteristics of studies using AR in medical training?RQ2: What kind of AR interventions were used to assist with medical training?RQ3: Does the use AR have an effect on medical training when compared to other methods?Methods section discusses the approaches used to obtain specific publications as well as the constraints and eligibility requirements that were used [The ere used . Followiere used . A checkThe meta-analysis included studies that (1) were published between 2013 and April 2021, (2) were written in English, and (3) primarily focused on AR for learning and training. Further, we excluded studies that (1) used other virtual platforms, such as VR and virtual patients, and (2) had no experimental data. However, based on the PICO  framework, our final analysis incorporated various trials, including those with randomized controlled and mixed designs. Criteria and descriptionsPopulationSex Age (>15 years)Target group InterventionAll types of augmented reality technology used in training or learning.ComparisonAugmented reality, nonaugmented reality, and conventional methods OutcomesKnowledgeConfidenceSkillsPerformance timeSatisfactionWe conducted a web-based literature search in the Cochrane Library, PubMed, Embase, Web of Science and Google Scholar databases from February to April 2021. We also manually checked the reference lists of the eligible studies to identify and acquire additional relevant publications. We used the following set of keywords, terms, and logical operators: “augmented reality” OR “AR” OR “mixed reality” OR “patient simulation” AND “medical training” OR “medical education” OR “health professions training.” Some of the search strategies and terms used are presented in Data were placed into an extraction spreadsheet by using Microsoft Excel 2019. From each of the studies, information was extracted based on the study characteristics, designs, and participants. These data included the names of authors, the years of the studies, the locations of the studies, sample sizes, participant types, study designs, and interventions and outcomes. EndNote X9 was also used to obtain some publication data, such as titles, publishers, URLs, digital object identifiers, page numbers, issue numbers, and volume numbers. The process of data extraction was initially performed by 2 authors (HA and A Alwadain) and was then validated by another 2 authors (WNWA and LFC).The quality of the studies was assessed by 2 authors (AB and A Alghail). We followed the criteria of the Cochrane Handbook for Systematic Reviews of Interventions  in the rmetacont package on R was used to transform the data on the sample sizes, SDs, and means of the control and AR groups. Data on the sample sizes, means, and SDs of only the AR groups were transformed by using the metamean package. Only 1 study [metan command. The statistical heterogeneity among the selected studies was measured by using I2 in each analysis and a 5% significance level. The random effect model was selected for the analysis carried out because the true effect sizes underlying all studies were stochastic.RevMan 5.4 (The Cochrane Collaboration) software was used to perform the risk of bias assessment; different scales were used by different authors to measure the outcomes; therefore, raw data could not be compared directly. Thus, the R 4.0.2  software was used to transform raw data, so that STATA 14.0 (StataCorp LLC) could be used to run the analysis directly. The  1 study  recordedFrom our initial search of 5 potential databases, we found a total of 1832 records. We identified a total of 1839 records, including relevant reference lists (n=7). Duplicate records  were removed. Following that, a total of 866 of the 1839 (47.1%) studies were screened based on their titles, abstracts, and keywords, and 798 were excluded after applying the eligibility criteria. A total of 68 studies were downloaded and evaluated via full-text screening, of which 55 were excluded for various reasons . FinallyAs presented in In recent years, the use of AR in the medical field has attracted the attention of academics and researchers, as evidenced by the increasing number of publications devoted to AR. This is demonstrated by the fact that 8 of the 13 studies were conducted between 2019 and 2021. The characteristics of the selected studies, including the outcome measures, interventions, and types of participants, are shown in The most significant issue with meta-analyses is the possibility of bias and error arising from combining multiple studies, especially unrelated studies. Creating a funnel plot is one of the key methods used for testing common publication bias. The graphics in the funnel are obvious scatter plots for the effect size approximated in each selected study versus the unit of sample size reported in the studies . If therAs can be seen in As shown in A meta-analysis was performed by using the data set in Z=0.130; P=.90).Only 6 of the 13 selected studies recorded knowledge as scores ,30,32,33Z=1.668; P=.10). There is high heterogeneity among the studies.A total of 4 studies used skill as the outcome measurement ,27,28,33P value was significant according to the plot  between the AR and control groups in terms of confidence. Participants' confidence was improved by AR when compared to that of participants in control conditions. It was found that AR training had a greater effect on participants’ confidence.There was a statistical difference . Of the 7 studies that recorded satisfaction as an outcome, 4 [Between the control and AR groups, there was a significant difference in participants’ satisfaction (tcome, 4 ,32-34 inA meta-analysis of performance time was also carried out. The findings suggested that AR was more effective than other training methods at reducing performance time. However, we found that AR was more efficient than other methods in enhancing performance time. The observed heterogeneity could have been due to the various research designs and settings used in the selected studies, such as surgical projects, AR devices, and control group training methods. One study  on the eOne of the main strengths of this work is being among the first meta-analyses to be conducted on the effectiveness of AR in medical training, which presents a significant contribution to the advancement of AR in the medical field. Furthermore, our work involved an assessment of 13 trials with a total of 654 participants from various countries.Our work has some limitations as well. The first is that we only included articles that focused on AR as an intervention, which made it difficult to find a large number of studies. Second, some of the studies included in the review omitted information about allocation concealment, sequence generation, and blinding methods. Finally, the 13 trials we evaluated used different teaching and training methods for their control groups, which could lead to significant heterogeneity.The effectiveness of AR training methods in the medical field was assessed in this work. The meta-analysis included 13 trials with a total of 654 participants that were completed between 2013 and 2021. In all trials, AR training was used as an intervention in AR groups, while conventional methods were used for control groups. Based on our findings, medical students' performance time, confidence, and satisfaction can be improved by using AR training and education methods. However, there was no statistical difference between the skills and knowledge that participants gained by undergoing AR training and those gained via conventional training methods. The use of AR should therefore be adopted in medical department training because of its significant effect on the performance time of participants. In general, AR should be used to expand knowledge and as a supplement to other simulation approaches to enhance clinical practice quality and safety. It is very noticeable that there are considerable gaps in the literature regarding the use of AR technology in medical training, and a limited number of studies exist, suggesting that further research efforts in this area are needed.