In addition, rehabilitation satisfaction was positively associated with reaching rehabilitation goals and perceived communication with healthcare workers. Digital interventions appeared effective in supporting mental health of psychosomatic rehabilitation patients’ post-rehabilitation. These findings support the inclusion of multidisciplinary and interdisciplinary digital and face-to-face treatment programs and call for more implementations of new technologies in a context of complexity to improve health and healthcare service.The need for new technologies in healthcare services has been stressed. However, little is known about the effectiveness of digital interventions integrated in psychosomatic rehabilitation processes. Data from 724 patients from psychosomatic rehabilitation clinics were analyzed with regard to the effectiveness of digital trainings indicated by a change in symptoms related to depression, anxiety, stress, and loneliness from pre– to post–rehabilitation. Rehabilitation satisfaction was examined in association with reaching rehabilitation goals and satisfaction with communication. A mixed repeated measures analyses of covariance, analyses of covariance, and hierarchical stepwise regression analyses were performed. Results indicated a superior effectiveness for the intervention group receiving all offered digital treatments in addition to the regular face-to-face rehabilitation program with regard to symptoms of depression (F (2674) = 3.93,  The effects of the coronavirus disease 2019 (COVID-19) on individuals’ health, especially on mental health and perceived well-being, are likely to be profound and long-lasting . TherefoFor individuals with a pre–existing mental health disorder, the lockdown measures have shown to be major stress factors and are associated with a deterioration of their mental health status due to changes in daily routine and social rhythms , reducedTo partially compensate for reduced access to support systems and discontinuation of psychotherapy, therapists have been more prone to offer digital psychotherapy sessions in addition to face-to-face sessions to guarantee the continuation of treatment as well as to protect and support the mental health of patients. The idea of blended psychotherapy as a combination of online treatment with face-to-face psychotherapy is rather a new research field and has received more attention during the COVID-19 pandemic ,11,12. BHowever, even though a few studies have examined the effectiveness of blended psychotherapy in outpatient settings , the effThe overarching aim of the rehabilitation system in Germany is to reintegrate and support social participation of patients but not to curate disorders. Patients admitted to rehabilitation clinics in Germany are generally treated on the basis of the biopsychosocial model . This isResearch has shown that blended therapy can be well integrated in the preparatory process before a rehabilitation stay , during Communication is the central element of psychotherapy and thus a central element of the psychosomatic rehabilitation process. Based on a patient’s verbal and nonverbal communication, psychotherapists are able to foster a diagnostic-therapeutic alliance with the patient ,24,25. FAn example of communication skills required in the healthcare context was proposed by Rider and Keefer . In the One theoretical model that describes the relationship between mental well-being and factors associated with well-being is the Compensatory Carry-Over Action Model (CCAM ). The CCThe psychiatrist or therapist fosters an early diagnostic-therapeutic alliance with the patient. S/he integrates information obtained through both the patient’s verbal and nonverbal communication and his or her own countertransference ,24. TherThe goal of the current study was to test the effectiveness of digital trainings provided to rehabilitation patients before and during their medical rehabilitation stay. In addition, as communication is a central element of (psycho)therapy, the present study aimed to evaluate the interrelation of communication with rehabilitation satisfaction and consequently with perceived rehabilitation success.With these research aims in mind and on basis of the theoretical background of the CCAM and previous findings, the following hypotheses were formulated. (1) Symptoms of depression, anxiety, perceived stress, and loneliness will decrease from pre– to post–rehabilitation. Additionally, we expected that (2) the intervention group who received all digital trainings will have a more substantial decrease of symptoms with regard to depression, anxiety, perceived stress, and loneliness. Further, we assumed that (3) the patients from the intervention group receiving all digital trainings will indicate a higher perceived rehabilitation success. We also predicted that (4) patients who perceived greater satisfaction with communication will be more satisfied with their rehabilitation process and will more likely indicate that they have achieved their rehabilitation goals.The present study was conducted at four psychosomatic rehabilitation clinics from the Dr. Becker clinic group in a longitudinal manner. Participants recruited to this study received regular treatment programs with regard to psychological and physical interventions. Those treatment programs included among others individual and group psychotherapy, physiotherapy, and occupational therapy.ClinicalTrials.gov Identifier: NCT04453475), which is supported by the Dr. Becker clinic group.Participants were recruited through the four participating clinics from the Dr. Becker clinic group. Before participation, patients were informed about the study in writing on the rehabilitation clinic group’s online portal. Thereby, it was guaranteed that only patients who had access to the digital portal with an individualized participant code could participate. Patients were invited to take part in a survey administered via the survey platform Unipark. Before participating in the survey, patients were asked to read the participation information and were asked to give informed consent. All data collected as part of this study were pseudonymized. Participants were not offered any form of compensation for participating in the study. The survey at the four psychosomatic clinics was administered between July 2020 and June 2021. Data collection was longitudinal with two measurement time points. Patients were invited to participate from six weeks before until the first day of rehabilitation (T1) as well as after their rehabilitation stay (T2). Participation after rehabilitation was possible for a maximum of 12 weeks post-rehabilitation. Reminders were sent out to the participants for the T2 survey after 1, 4, and 11 weeks post–rehabilitation. Ethical approval for the online survey concerning psychosomatic rehabilitation patients was given by the Ethics Committee at Jacobs University Bremen . The current study was conducted as part of the project “Anhand-COVID19-Offer to achieve treatment and rehabilitation goals in compliance with hygiene and social-distancing rules” (n = 1279 patients participated in the online survey at timepoint 1 (before rehabilitation stay). A total of 555 patients dropped out after the baseline assessment, leaving 724 participants who completed the survey at measurement timepoint 1 pre–rehabilitation as well as the survey at measurement timepoint 2 post–rehabilitation.In total, n = 193 (26.7%); an adjustment disorder (F43.2) with n = 159 (22.0%); and a major depressive disorder, single episode, moderate (F32.1) with n = 93 (12.8%). Patients’ ages ranged from 18 to above 60 years. Within the sample, 466 (64.4%) patients were female. Additionally, 155 (21.7%) patients had a secondary school diploma, 106 (14.8%) patients had a high school diploma, 319 (44.6%) patients had completed vocational training, and 135 (18.9%) patients indicated having a university degree.The most common three diagnoses that patients received, according to the International Classification of Disease-10 (ICD–10) manual, were as follows: a major depressive disorder, recurrent, moderate (F33.1) with As part of the incoming process and prior to the begin of the treatment stay, participants were asked to participate in a digital training on rehabilitation goals presented to patients in a digital PowerPoint presentation without face-to-face elements. Participation was on a voluntary basis. The digital training on rehabilitation goals could be accessed from home with a computer, laptop, tablet, or smartphone. This training was designed as a combination between psychoeducation and practical elements. Patients were educated on the importance of formulating goals and plans as well as on how to formulate those. After the educative element, participants were instructed to formulate their own plans for their rehabilitation treatment process. Further interactive tools such as digital exercise booklets supporting goal and plan formulation were provided to patients online. Patients were encouraged to make use of the supporting material after the training.As part of the rehabilitation process, participants diagnosed with a major depression were required to take part in the digital group training for depression. The digital group training was based on cognitive behavioral therapy (CBT) guidelines with evidence-based components of computerized cognitive behavioral therapy (eCBT) and internet-delivered cognitive behavior therapy (iCBT) interventions ,32,33. TThe informative digital training on legal rights for (severely) disabled was offered to all patients once during their rehabilitation stay in the form of a group session. Participation was mandatory irrespective of the ICD-10 diagnosis. The training consisted of a 20 min informative video and a subsequent 25 min face-to-face group session in which in-depth questions were discussed in accordance with the flipped classroom manner. Contents of the video and the group discussion included aspects of the law on severe disabilities, requirements for obtaining a degree of disability, and its consequences on everyday life.Hence, the study design was set up as follows: participants allocated to the control group received the care-as-usual rehabilitation program. Patients allocated to intervention group 1 (IG1) took part in the digital training on rehabilitation goals prior to the rehabilitation stay in addition to the care-as-usual rehabilitation program. As part of intervention group 2 (IG2), patients took part in the digital training on rehabilitation goals prior to the rehabilitation stay, the digital group therapy on depression, and the digital training on legal rights for (severely) disabled in addition to the regular care-as-usual rehabilitation treatment.To measure symptoms of depression and anxiety, the Patient Health Questionnaire-4 (PHQ-4) was used. The questionnaire was not used as a diagnostic tool as part of this study, but rather used as a measure of symptom intensity. The PHQ-4 is a composite measure with four items of the PHQ-2  and the As a measure of stress, the Perceived Stress Scale  was usePerceived loneliness was assessed by means of two items: ‘How often do you feel lonely?’ stemming from the Center for Epidemiologic Studies–Depression (CES–D) Scale  and ‘HowBefore and after rehabilitation, patients were asked to indicate whether they aimed to achieve eight possible rehabilitation goals on a scale from 1  to 4 (‘completely’) with a Cronbach’s alpha at T1 of 0.65 and at T2 of 0.89. Examples of possible rehabilitation goals included the reduction of mental health symptoms, an improvement of stress coping capabilities, improvement of cognitive abilities, ability to relax and rest, or the improvement of/return to past earning capacities. The items assessing rehabilitation goals were developed based on the provided content and the outcome aims of the digital trainings provided before and during the rehabilitation treatment.Perceived communication between rehabilitation patients and healthcare professionals  was examined from the perspective of rehabilitation patients through six items developed based Rider and Keefer’s interpersonal communication competencies with a Cronbach’s alpha of 0.88 .Post rehabilitation, patients were asked to indicate their satisfaction with medical rehabilitation with one item on a 6-point Likert scale from 1 (‘very dissatisfied’) to 6 (‘very satisfied’).For all analyses, SPSS Version 27 was used . The data were analyzed using 724 patients who were either allocated to the control group or to the intervention groups (IG1 to IG2). A randomization check was performed to confirm successful allocation to groups. Hence, the different groups (control group and two intervention groups) were compared for age, gender, educational status, symptoms of depression and anxiety, perceived stress, and perceived loneliness before rehabilitation. Therefore, a one-way ANOVA was used for continuous variables. Chi-squared tests were used for nominal variables. According to Tabachnick and Fidell , it has As the amount of missing data was below 5% for all items, no imputation of missing data was performed. Patients with missing data on the social-cognitive variables  were included for further analyses if they had at least one non-missing data point under the assumption of missing data (completely) at random.To evaluate significant changes in the symptom intensity with regard to depression, anxiety, stress, and loneliness, a 2 × 3 linear mixed-model repeated measures analysis of covariances (MMRM ANCOVA) was performed. In order to explain a significant time x intervention group interaction effect, the mean difference scores for the two time points were computed (before rehabilitation to after rehabilitation). Based on the mean difference scores, a series of analyses of covariances were performed to identify differences between the interventions.To determine the effect sizes of all measurements, partial eta squared and Cohen’s d values were computed. Based on the recommendation by Field , partialFurther, we performed several multivariate analyses of covariance (MANCOVA) to evaluate which treatment group was more likely to reach the proposed rehabilitation goals. Additionally, we investigated the association between the estimation of achieving rehabilitation goals and overall satisfaction with rehabilitation treatment by means of a stepwise hierarchical linear regression.With regard to the current study, n = 55 (7.6%) patients did not participate in any of the three digital trainings and were thus defined as the control group. Overall, 570 (78.7%) patients participated in the digital trainings on rehabilitation goals (intervention group 1—IG1), 80 (11.0%) patients participated in all three digital trainings (intervention group 2—IG2), and 19 patients (2.6%) were excluded from the analyses.2  = 1.60, p = 0.45), for age  = 11.84, p = 0.16), and for educational level  = 2.80, p = 0.83). In addition, there were no significant differences for symptoms of depression (F (2695) = 0.78, p = 0.46, ηp2 = 0.01), for symptoms of anxiety (F (2698) = 1.15, p = 0.32, ηp2 = 0.01), as well as for perceived stress (F (2695) = 1.61, p = 0.20, ηp2 = 0.01) and perceived loneliness (F (2686) = 1.22, p = 0.30, ηp2 = 0.01).There were no significant differences for gender , symptoms of anxiety (F (1678) = 6.80, p < 0.01, ηp2 = 0.01), and perceived stress (F (1672) = 17.63, p < 0.01, ηp2 = 0.03), as well as for perceived loneliness (F (1662) = 4.00, p < 0.05, ηp2 = 0.01).Results indicated a significant main effect across time, controlling for age and gender for symptoms of depression (F (1674) = 13.34, p < 0.05, ηp2 = 0.01) and symptoms of anxiety (F (2678) = 3.68, p < 0.05, ηp2 = 0.01). However, no significant interaction effect was found for perceived stress (F (2672) = 1.80, p = 0.17, ηp2 = 0.01) as well as for perceived loneliness (F (2662) = 2.69, p = 0.07, ηp2 = 0.01).A significant interaction between time x intervention, controlling for age, gender, and intervention group, emerged for symptoms of depression (F (2674) = 3.93, p = 0.56, ηp2 = 0.01), anxiety (F (2678) = 0.42, p = 0.66, ηp2 = 0.01), perceived stress (F (2672) = 0.832, p = 0.44, ηp2 = 0.01), and loneliness (F (2662) = 1.43, p = 0.24, ηp2 = 0.01). Reported effect sizes for the main effects of time and intervention as well as for the interaction effect of time x intervention were small for all outcome domains.No significant main effect for intervention, controlling for age and gender, was found for all four outcome domains: depression (F (2774) = 0.58, Overall, the average scores showed an improvement from pre–rehabilitation treatment to post-rehabilitation treatment with regard to symptoms of depression, symptoms of anxiety, and perceived stress across the control group and intervention groups see a–d. For p < 0.05, ηp2 = 0.02) and symptoms of anxiety (F (2638) = 4.19, p < 0.05, ηp2 = 0.02); however, significant differences were not found for perceived stress (F (2638) = 2.38, p = 0.09, ηp2 = 0.01) and perceived loneliness (F (2638) = 2.39, p = 0.09, ηp2 = 0.01).The results of the ANCOVA showed significant between–group differences with regard to the decrease in symptoms in the outcome domains from pre–rehabilitation to post–rehabilitation. Hence, significant differences were found for symptoms of depression (F (2638) = 4.50, diff = −0.74, p = 0.40) and a significant difference between IG1 and IG2  for symptoms of depression. With regard to symptoms of anxiety, Bonferroni’s post hoc test indicated a significant difference between IG1 and IG2 .Bonferroni’s post hoc test indicated a significant difference between the KG and IG2  and for group comparison purposes post-rehabilitation. From pre– to post–rehabilitation, Cohen’s d values indicated a medium effect for overall symptoms of depression (0.69) and symptoms of anxiety (0.69). From pre– to post–rehabilitation, the effect size of Cohen’s d values, taking the intervention and control group into consideration, for depression was significantly larger in the IG2 group (1.27) compared to the IG1 group (0.66) and to the control group (0.31). For symptoms of anxiety, Cohen’s d values were significantly larger in the IG2 group (1.08) than in the IG1 group (0.66).To evaluate the association between reaching rehabilitation goals post-rehabilitation and satisfaction with rehabilitation treatment, a stepwise hierarchical regression analysis was performed controlling for age, gender, and intervention group with rehabilitation goals as predictors . p < 0.01), improvement of physiological status , relaxation and resting , improvement of coping with stress and management of stress , and improvement of one’s own confidence . However, results showed a non-significant difference between treatment groups associated with the estimation of reaching rehabilitation goals post-rehabilitation  = 1.524, p = 0.08, ηp2 = 0.02). In addition, no significant differences regarding satisfaction with rehabilitation treatment were found between the intervention groups (F (2682) = 0.02, p = 0.98, ηp2 = 0.01; see Results underlined that patients who indicated to have achieved the following rehabilitation goals were also more satisfied with the overall rehabilitation treatment process: reduction of psychological symptoms , taking worries and fears seriously , and provision of sufficient information . The covariates of intervention group , age , and gender  were not significantly associated with the relationship between perceived communication and satisfaction with treatment  and with consideration of the ICD-10 diagnosis. Hence, complaints are, thus, translated by diagnostic tests into diagnoses, which are necessary and a prerequisite for the treatment process ,51. ConsWith regard to the effectiveness of different digital trainings offered during rehabilitation, results have highlighted that, for patients receiving different combinations of digital trainings  disabled), an average symptom reduction was found for depression, anxiety, and perceived stress. We found the same for participants who were part of the control group as well. However, perceived loneliness did not decrease for patients as part of the control group. Hence, the present results would suggest a beneficial effect of the rehabilitation setting especially with regard to depression, anxiety, and perceived stress. These findings are consistent with previous findings ,48,49.This was especially pronounced when comparing the patients allocated to the control group with patients from intervention group 1  as well as patients from intervention group 1 with participants from intervention group 2  disabled) concerning symptom reduction in depression. With regard to symptom reduction in anxiety, this effect was significant when comparing intervention group 1 with intervention group 2. Hence, intervention group 2 was shown to be significantly superior with regard to symptom reduction concerning depression and anxiety. Considering symptom reduction of perceived stress and perceived loneliness, intervention group 2 was shown to be on average superior to either intervention group 1 or the control group. These findings indicate that the interdisciplinary and multidisciplinary medical, psychosomatic rehabilitation program as a whole had a positive impact on mental health status, a finding that adds to previous research ,52. HoweDespite the promising results highlighted by the present study, it needs to be stressed that digital trainings as part of the psychosomatic rehabilitation process need to constantly be tailored, evaluated, adapted and modified to the needs of the patients, to current treatment guidelines, as well as to the newest scientific developments to ensure an effective care and treatment program as well as overall patient safety.So far, effect size benchmarks have only been postulated for regular face-to-face treatments but not for digital trainings as part of a medical, psychosomatic treatment process. Previous literature has defined effect size benchmarks for psychologically based treatment programs from pre– to post–treatment stay ,53. As pResults stressed that patients who perceived greater satisfaction with rehabilitation goals  also displayed greater satisfaction with the overall rehabilitation treatment process. Additionally, patients who were more satisfied with their treatment also perceived greater satisfaction with communication . However, no significant difference was found with regard to the intervention groups. These results are in line with previous literature on the potentials of telemedicine generally and especially in times of crisis like the COVID-19 pandemic .One of the main limitations of the current study is that we had no indication of the mental health status of psychosomatic rehabilitation patients before the outbreak of the COVID-19 pandemic. Hence, we cannot be certain whether the COVID-19 pandemic was associated with an aggravated symptom increase with respect to symptoms of depression, anxiety, stress, and loneliness, as shown by previous literature ,54. A fuIn addition, this study did not consider possible confounding correlations of physiological symptoms  with mental health. Another limitation that needs to be considered is that the digital trainings offered before and during the rehabilitation stay so far have not been validated or standardized, but rather align with the German regulations for rehabilitation treatments and were developed based on experiences by the rehabilitation clinics. Hence, with regard to future research, a standardized manual, such as the Curriculum Hannover for aftercare , should In addition, another limitation concerns the Cronbach’s alpha value of rehabilitation goals prior to the rehabilitation stay that has been found to be relatively low at 0.65  comparedAs countries are becoming more culturally diverse, further research should also be considered to replicate and validate the current findings in countries with different rehabilitation systems and with patients from different cultural backgrounds. With regard to the different cultural expression of emotions and acknowledgements of psychological disorders, as well as acceptance of psychotherapy as a form of treatment, the question remains whether different healthcare systems and psychosomatic rehabilitation treatments  would result in a similar outcome arises.The findings of this study suggest that medical, psychosomatic rehabilitation is effective in reducing symptoms related to mental health disorders. By providing new technologies, i.e., digital elements as part of the healthcare services and the treatment process, symptoms of depression, anxiety, perceived stress, and perceived loneliness could be reduced post-rehabilitation. This was especially the case with the multidisciplinary and interdisciplinary rehabilitation treatment program, i.e., a treatment program including digital rehabilitation goals, digital group therapy for depression, and a digital training on legal rights for the (severely) disabled. They were shown to be especially effective with regard to symptom reduction of depression and anxiety, which are the central goals of the medical, psychosomatic rehabilitation process. Furthermore, greater satisfaction with the rehabilitation process was associated with the perception of rehabilitation goals as well as with greater satisfaction with communication between patients and healthcare professionals.


Rickettsia asembonensis is a flea-related Rickettsia with unknown pathogenicity to humans. We detected R. asembonensis DNA in 2 of 1,153 human blood samples in Zambia. Our findings suggest the possibility of R. asembonensis infection in humans despite its unknown pathogenicity. Rickettsia asembonensis is a fleaborne rickettsia closely related to Rickettsia felis and is thus referred to as an R. felis–like organism. R. asembonensis was first detected in cat fleas in Kenya and subsequently reported worldwide  from healthy volunteers from rural eastern (n = 200) and central (n = 200) provinces to assess rickettsia infection in healthy rural persons. The study was approved by the National Health Research Authority of Zambia through the Biomedical Research Ethics Committee (reference no. 007-10-18).We obtained 753 residual patient blood samples from hospitals in urban Lusaka (n = 519) and the Chongwe District (n = 234) of Zambia. Approximately half of the samples (303/753) were traceable to clinical records of patients. The common clinical conditions among these patients included fever, anemia, meningitis, septicemia, and sickle cell anemia (Appendix Table 1). In addition, we obtained dried blood spots on Whatman FTA classic cards  of Rickettsia. We subjected the positive samples to multiple-gene sequencing analysis targeting the 17-kDa common antigen (htrA), outer membrane protein A (OmpA), and outer membrane protein B (OmpB) genes using previously described primers (Appendix Table 2). We aligned the sequences using MAFFT  and performed phylogenetic analysis by the neighbor-joining method using MEGA7 (https://www.megasoftware.net). We determined the estimated Rickettsia bacterial burden in Rickettsia-positive blood samples by OmpA quantitative PCR by using published primers. We further testsed the gltA PCR-positive samples for malaria by nested PCR (Appendix Table 2).We extracted genomic DNA and subjected it to PCR screening that targeted the citrate synthase gene (R. asembonensis in 0.39% (2/519) samples from the urban Lusaka District by gltA PCR. The samples from the Chongwe District and the rural areas of the eastern and central provinces were all negative, although the possibility that dried blood spot samples from rural areas might have lower detection sensitivity cannot be ruled out. BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) of the gltA sequences obtained (GenBank accession nos. LC557154 and LC557155) showed 100% homology to R. asembonensis identified in cat fleas from human dwellings and domestic dogs in 3 countries: Senegal (GenBank accession no. JF966774), Kenya (accession no. JN315968), and Zambia (accession no. LC431490) (gltA with those detected in fleas from Peru (GenBank accession no. KY650697) and other regions in the Americas showed 99.8% similarity. Phylogenetic analysis of gltA confirmed the detected sequences’ closer relatedness by clustering with genes from cat fleas from sub-Saharan Africa, a distinct cluster from other regions . The obtained nucleotides are available in GenBank under accession nos. LC557154–61. Detection of genotypically similar R. asembonensis in persons and cat fleas in Zambia suggests possible human infection by R. asembonensis through cat flea bites. Nevertheless, the epidemiologic cycle and pathogenicity of R. asembonensis and other related R. felis–like organisms remain to be elucidated.We detected  regions . The OmpR. asembonensis was detected had a medical record of anemia and weight loss (Rickettsia infection with anemia, severe R. felis infection has been reported with severe anemia, possibly attributable to hemorrhage from vascular damage in rickettsial disease (R. asembonensis as the cause of these symptoms. The second R. asembonensis–positive sample from the patient identified as UTH_231 had limited clinical information, which did not allow for further interpretation. The 2 R. asembonensis–positive blood samples showed estimated DNA quantities of 890,000 copies/mL of blood from patient UTH_185 and 2,100,000 copies/mL of blood from patient UTH_231 (Rickettsia rickettsii estimated rickettsial burden (The patient identified as UTH_185 in whom ght loss . The mal UTH_231 . These rR. asembonensis of identical genotype in cat fleas and human blood in Zambia suggests possible transmission from cat fleas to humans. Given the worldwide distribution of R. asembonensis, further studies to elucidate its pathogenicity and epidemiologic cycle are warranted.In conclusion, detection of Rickettsia asembonensis in human blood collected in Zambia, 2019.Information related to the molecular detection and characterization of


The prevalence of allergic rhinitis (AR) is steadily rising in the Thai population, causing a major impact on the quality of life (QoL). Enhancing knowledge on common aeroallergens in the local setting helps in the appropriate prevention and management of AR. In this study, the demographic characteristics, clinical data, aeroallergen sensitization pattern, allergic symptoms, visual analog scale (VAS) score, and QoL are described. We evaluated the association between VAS, QoL, and severity of symptoms, except the aeroallergen sensitization pattern. We retrospectively reviewed the medical records of adult AR patients with a positive skin prick test (SPT) for at least one aeroallergen from January 2018 to May 2020. Standard descriptive and inferential statistics were used for analysis. A total of 366 patients were enrolled. Indoor aeroallergen sensitization and outdoor aeroallergen sensitization were observed in 32% and 7.9% of patients, respectively. Mono-sensitization was noted in 16.9% of patients, while poly-sensitization was noted in 83.1% of patients. Mites (65%) and sedge (39.3%) were the most common indoor and outdoor allergens. Nasal obstruction (74.6%), runny nose (63.7%), and nasal itchiness (61.5%) were the primary symptoms affecting the QoL. The association between VAS and symptom scores showed a trend of association with AR severity (Allergic Rhinitis and its Impact on Asthma [ARIA] classification) and VAS. AR has a significant effect on QoL in all domains of the validated generic  and specific  questionnaires. Mite and sedge remain the most common indoor and outdoor aeroallergens. The pattern of sensitization and number of aeroallergens were not associated with AR based on the ARIA guidelines. Meanwhile, symptoms of patients affected the QoL and VAS scores, which can be used as a quick and reliable tool for monitoring and stepping up or stepping down the treatment according to the next-generation guidelines. AR has a significant impact on the QoL of adult Thai patients. Allergic rhinitis (AR) is an immunoglobulin (Ig) E-mediated inflammation of the nasal mucosa induced after allergen exposure and has three cardinal symptoms: sneezing, nasal obstruction, and rhinorrhoea . AR may Allergic rhinitis represents a global health problem, affecting 10–20% of the population. The prevalence of AR is steadily rising in Thailand, with no signs of abating. According to a previous study, the prevalence of AR increased from 37.9 to 50.6% (Dermatophagoides pteronyssinus (Dp) remains the most common indoor aeroallergen, while sedge remains the most common outdoor aeroallergen among the Thai population (According to recently published 19-year 1998–2017) data from an ENT allergy clinic, Faculty of Medicine Siriraj Hospital, the mite pulation . This st–2017 datThis study was a retrospective chart review of adult patients diagnosed with AR by a positive SPT for at least one aeroallergen, conducted at the Rhinology and Allergy Unit, Department of Otorhinolaryngology, Faculty of Medicine Siriraj Hospital, Mahidol University, between January 2018 and May 2020. Patients who were unable to completely fill out the case record forms or QoL questionnaires were excluded from our analysis. The following demographic and clinical data were obtained: age, gender, presenting symptoms, duration of symptoms, SPT results for various aeroallergens, the score of each domain in SF-36 (generic QoL questionnaire), the score of each domain in the Rcq-36 (specific questionnaire), and classification and severity of AR based on the ARIA guidelines. The patients were asked to discontinue taking oral antihistamines for 7 days prior to the test day. The visual analog scale (VAS) was used to determine the severity of AR symptoms (0–10 cm), and a diary card was provided to record the patient's symptoms for the last 7 days prior to the SPT.Dp, American cockroach (Periplaneta americana), cats, dogs, molds , Bermuda grass (Cynodon dactylon), para grass (Brachiaria mutica), sedge (Cyperaceae), careless weed , and Kapok (Ceiba pentandra) were used in our center. Histamine and normal saline were used as positive and negative controls, respectively , which contains standardized allergen extracts and has proven allergenic potency, was used , 10. Allectively . All SPTValidated Thai versions of the SF-36  and Rcq-U-test for non-normally distributed data and using unpaired t-tests and ANOVA for normally distributed data. Clinical symptoms and the pattern of sensitization were evaluated for association with severity and QoL. The factors associated with the pattern of sensitization and ARIA classification with a p-value <0.05 using univariate analysis by stepwise method and multivariate analysis are shown as p-value and odds ratio (OR) with 95% CI. A p value < 0.05 was considered significant.Data processing and analysis were performed using PASW Statistics . The data were expressed as frequency and percentage for categorical data or mean ± SD for continuous data. Normally distributed continuous data were evaluated using the one-sample Kolmogorov-Smirnov test. The data were classified according to the ARIA guidelines. The differences in the type of aeroallergen and type of sensitization among the AR severity groups (ARIA classification) were determined by chi-square tests. The association between TNSS, VAS, and QoL scores in the SF-36 and Rcq-36 questionnaires and type of aeroallergen, pattern of sensitization, or ARIA classification was analyzed using the Kruskal-Wallis test; meanwhile, the association of TNSS, VAS, and pattern of sensitization was determined using the Mann-Whitney Si 296/2019).This study was approved by the Siriraj Hospital Institutional Review Board  patients, while mono-sensitization was observed in 62 (16.9%) patients. Sensitization to indoor aeroallergens was noted in 117 (32%) patients, while sensitization to outdoor allergens was noted in 29 (7.9%) patients. Sensitization to both the aeroallergens was observed in 220 (60.1%) patients. Nasal obstruction (74.6%), runny nose (63.7%), and nasal itchiness (61.5%) were the most common symptoms observed in AR patients in this study. These symptoms were frequently observed in patients with moderate-to-severe intermittent and persistent AR. Based on the ARIA classification, a majority of the patients had moderate-to-severe persistent AR (54%), followed by moderate-to-severe intermittent AR (22%), mild intermittent AR (14%), and mild persistent AR (10%) .Dp (65%) followed by house dust (60.1%), cockroach (41.2%), dog (35.4%), and cat (24%). Meanwhile, the outdoor aeroallergen was most frequently sensitized against sedge (39.3%) followed by para grass (31.4%), Bermuda grass (31%), careless weed (25.4%), Kapok (23.4%), Typha (16.9%), Cladosporium (11.1%), Penicillium (7.5%), and Aspergillus (6.4%)  .p = 0.54) and the type of sensitization (p = 0.49) (p< 0.05). TNSS is significantly associated with the type of sensitization (p < 0.05). The QoL in all domains was poorer as the severity of AR increases. Bodily pain, general health, and vitality were the most severely affected domains. The disease-specific QoL questionnaire (Rcq-36) also yielded poorer QoL in all domains, especially for rhinitis symptoms (54.5%), sleep (65.5%), and emotions (57.5%), with ANOVA yielding significant results  . Howevertization . An incrtization , 4. Fact9 . Howevtization . FiguresDp sensitization rate of 50.1% within a 12-year period (from January 2004 to December 2015) , while sedge remained the most common sensitizing outdoor aeroallergen (39.3%) reported in our study, which was conducted from January 2018 to May 2020. When compared to the 19-year (1998–2017) data from the same center , no signer 2015) , which eHouse dust mite is the most common sensitizing aeroallergen worldwide and causes sensitization in up to 90% of Asian atopic patients , 14. The2 concentration, temperature, rainfall, humidity, and wind speed and direction (Cynodon dactylon), para grass (Panicum purpurascens), sedge (Carex species), and careless weed (Amaranthus hybridus), is more commonly found in Bangkok than tree pollen , 64.5% were women, while only 35.5% were men. However, no significant gender discrepancies were observed in terms of mean age, mean duration of symptoms, and mean age of onset of AR. The female predominance might be attributed to the better health awareness among women, as suggested in a previous study . Bodily pain, general health, and vitality were the most affected domains in the SF-36 questionnaire. Meanwhile, rhinitis symptoms, sleep, and emotions were the most affected domains in the analysis of the disease-specific questionnaire (Rcq-36). The findings from our study reaffirm that AR continues to be significantly affecting the QoL of the Thai population. Adapting and advocating the next-generation ARIA guideline with the use of real-world evidence and an integrated care pathway in the Thai population may fill the gap and adequately address the needs of AR patients.A previous study in Thailand using the Thai version of the SF-36 questionnaire reported that AR patients had significantly impaired QoL scores compared with healthy individuals in all aspects, except the social functioning dimension. The same study also reported that the Rcq-36 questionnaire showed a higher correlation with the symptom scores compared with the SF-36 questionnaire, additionally including information on sleep and productivity (p = 0.49). There may be several factors that can have an effect on the clinical severity of AR due to the number of sensitizing aeroallergens. This should be addressed in future research. Similarly, our study also revealed that the type of aeroallergen and number of sensitizations had no association with VAS score and TNSS.Ciprandi and Cirillo  reportedOur study has some limitations. It was retrospective in nature and only used a 2-year dataset. Our findings would have been better supported if we had assessed the significance of the changes in VAS scores and QoL after the initiation of treatment. In addition, due to the prevailing coronavirus disease 2019 pandemic, only a few SPTs were performed from March to May 2020.Mite and sedge remain the most common sensitizing indoor and outdoor aeroallergens in adult Thai patients with AR. The VAS scores are significantly associated with AR severity. VAS can be used as a quick and reliable tool in adult Thai patients with AR to monitor and step-up or step-down treatment. AR has a significant impact on the QoL of adult Thai patients, and the severity of AR is not associated with the number and type of aeroallergen.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Siriraj Institutional Review Board. Certificate of Approval No. Si 296/2019. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.PK: data collection, methodology, formal analysis, project administration, designed and wrote the first draft of the manuscript, and writing—review and editing. BP: data curation, statistical analysis, visualization, and writing—review and editing. KT: data collection and curation. PT: conceptualization, methodology, project administration, supervision, and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dissemination of antibiotic resistance (AR) in marine environments is a global concern with a propensity to affect public health and many ecosystems worldwide. We evaluated the use of sea turtles as sentinel species for monitoring AR in marine environments. In this field, antibiotic-resistant bacteria have been commonly identified by using standard culture and sensitivity tests, leading to an overrepresentation of specific, culturable bacterial classes in the available literature. AR was detected against all major antibiotic classes, but the highest cumulative global frequency of resistance in all represented geographical sites was against the beta-lactam class by a two-fold difference compared to all other antibiotics. Wastewater facilities and turtle rehabilitation centres were associated with higher incidences of multidrug-resistant bacteria (MDRB) accounting for an average of 58% and 49% of resistant isolates, respectively. Furthermore, a relatively similar prevalence of MDRB was seen in all studied locations. These data suggest that anthropogenically driven selection pressures for the development of AR in sea turtles and marine environments are relatively similar worldwide. There is a need, however, to establish direct demonstrable associations between AR in sea turtles in their respective marine environments with wastewater facilities and other anthropogenic activities worldwide. Staphylococcus aureus were isolated in healthy edible marine fish in South Africa [Vibrio spp. and Salmonella spp. in humans following the consumption of sea turtle meat and eggs. Despite the existence of these specific examples pertaining to the threat of AR in the marine environment, our comprehensive understanding of ecological and anthropologic factors that contribute to the development and dissemination of AR in such environments is lacking. To study and elucidate factors that contribute to AR in otherwise very diverse and complex marine ecosystems, sentinel animal species whose health is considered to reflect the wellbeing of these ecosystems should be used. Sea turtles are considered a good sentinel species for monitoring the health of marine ecosystems; they have been used in many toxicological and trophic ecology studies [Antibiotics have provided effective and safe treatments for bacterial infections and are an essential tool in modern medicine . Unfortuh Africa . Similarh Africa  reported studies ,10. This studies ,12,13. F studies ,13,14. HMany of the antibiotic compounds used in medicine and agriculture are naturally occurring, and therefore, co-existing bacteria have evolved mechanisms to survive exposure to antibiotics or other environmental threats . This me6 cells/mL), sediment on the seafloor (estimated at 108 cells/mL), and on or within macroscopic organisms such as turtles, fish, and sea mammals [Marine ecosystems are home to the largest and most diverse communities of flora and fauna, presenting different habitats that cover 75% of the earth’s surface and harbour over 90% of all life on earth, including bacteria . Marine- mammals ,19,20. F mammals .The dissemination of antibiotics and resistance-causing genes into marine environments is a major global concern, as it allows for the selection of antibiotic-resistant bacteria (ARB) within these highly concentrated and diverse bacterial populations . ResearcRun-off from the agricultural industry that may not be directly associated with WWTPs is another important source of antibiotics, bacteria, and ARGs in marine environments. There is extensive antibiotic use in the agricultural industry of many countries not to just treat infections but also as growth promoters to pre-emptively prevent infections. This standard agricultural practice of long-term utilisation of broad-spectrum antibiotics can provide the perfect environment for cultivating ARB and propagating ARGs ,28,29. TRelevant literature was gathered systematically from three bibliographic databases: Medline (Ovid), Scopus, and Web of Science. The search terms used were: “Antibiotic Resistance Genes” or “Antibiotic Resistance”; and turtle or Testudines. This allowed for the identification of older documents analysing antibiotics by culture to obtain resistance identification, as well as newer documents identifying AR genes through metagenomic methods. Eligibility was not limited to publication type, including journal articles, reviews, and case studies. The eligibility criteria did not include language limitations, and coincidentally, all articles found to fit the eligibility criteria were publications in English. The exclusion criteria included papers that were not focused on the study of (or previous study of) AR in marine turtles. All used databases were searched from time of inception to December 2020. To allow for a critical review of the literature, subsequent citations from the results of the previously found articles were also appraised for suitability and inclusion in this review. Quality appraisal was ensured by using the CASP tool checklist relevant to each piece of literature, which allowed for the evaluation of the quality of each publication . The litp < 0.05 was considered significant.All relevant data were extracted from text, figures, and tables. Data were then stratified according to predetermined variables including turtle species involved, anatomical sampling sites on turtles, the most prevalent bacterial isolates, classification of antibiotics against which AR was tested, geographical locations, and the potential source of AR. Quantitative data on AR were presented as mean percentages of observed AR in confirmed bacterial isolates. Where applicable, statistical comparisons between groups were done using the Kruskal–Wallis test, and Fifteen published studies focusing on AR, ARB, and ARG isolated in sea turtle species were identified . Three oThe geographical distribution of marine turtles is worldwide with the exception of the Arctic and Antarctic waters . Their hThe coastal areas of the Mediterranean and Arabian seas are shared by 21 cities and 6 nations representing Western Europe, the Middle East, and North Africa . These cOn the east coast of the Pacific Ocean of Australia, AR was detected in sea turtle rehabilitation centres as well as wild sea turtles in surrounding areas such as the Cleveland Bay but no definitive and specific sources or causes of AR were identified ,38. It iChelonia mydas) represented the majority (~75%) of the publications (Dermochelys coriacea), which feeds almost exclusively on jellyfish, may accumulate antibiotics, ARGs, and ARB from any area in which the food source has travelled. Thus, turtle species depending on a more protein-rich diet may not represent AR profiles within their immediate environment, but possibly other geographical regions. In contrast, more mature green sea turtles primarily feeding on seagrasses and algae in times of non-migration are likely to provide more accurate and site-specific information on the bioaccumulation of antibiotics, ARGs, and ARB due to the stationary nature of seagrass and algae within near-shore environments. Certainly, the ability of seagrass to bioaccumulate other chemical compounds and pollutants has been documented previously [In all documented peer-reviewed studies on the use of turtles to monitor AR in marine environments herein, green turtles  were represented by multiple studies at different geographical sites . IrrespeWhile not all antibiotics were tested for AR in all reported studies included in this review, there was no apparent difference in reported frequencies of resistance to commonly used, first-line drugs such as ampicillin, which was tested at multiple sites. Moreover, a relatively similar incidence of MDRB across all major antibiotic classes was observed in all represented geographical locations in the currently available studies . The anaOn average, WWTPs and turtle rehabilitation centres accounted for 58% and 49% of MDRB isolates from sea turtles in all published studies. This result is not surprising because the rehabilitation of sick or injured turtles for purposes of conservation is usually associated with marked broad-spectrum antibiotic usage, as methods for rapid diagnosis for wild animals are sorely lacking. Antibiotics classified as quinolones , beta-lactams (ampicillin and amoxicillin-clavulanic acid), and tetracyclines (doxycycline) showed the highest frequency of resistance that potentially originated from WWTPs and turtle rehabilitation centres. This directly correlates with the increased usage of these antibiotic classes at rehabilitation centres because they have a broad spectrum of activity and are safe to use in reptiles. These arguments on the observed AR patterns are supported by the fact that there was no resistance against aminoglycosides (gentamicin and streptomycin) when tested for at turtle hospitals or rehabilitation centres. Coincidentally, aminoglycosides are severely nephrotoxic in reptiles, and as such, the application of this class to treat infections in turtles is very limited. In contrast, chloramphenicol is not a drug of primary choice in treating sick turtles, but it showed a relatively high frequency of resistance. This may be attributable to generalized non-specific mechanisms of resistance such as the increased expression of efflux pumps by bacteria.tetA), sulphonamides (sulII), beta-lactams (blaTEM and blaCTXM), and quinolones (qnrS), were also isolated in wild sea turtles [int1 gene, encoding for the mobile element class 1 integron has also been identified in bacteria isolates from sea turtles [The AR pattern associated with WWTPs or other unidentified sources was more diverse, affecting all the major antibiotic classes. It is plausible that this is because WWTPs are points of convergence for a diverse mix of antibiotics, ARGs, MDRB, and other pollutants that may select for AR. However, specific sources and mechanisms contributing to observed AR patterns in turtles in marine environments are not known. It is reasonable to assume, however, that similar pools of ARGs and means of AR acquisition seen in other ecosystems are involved here as well. For instance, the respective genes encoding for some of the reported resistance patterns against tetracyclines ( turtles . In addi turtles . This suThis review evaluates the use of sea turtles as a sentinel species for monitoring as well as reporting incidences, dissemination, and distribution patterns of AR in marine environments. The extensive geographical distribution and relatively long lifespan exhibited by sea turtles allow for their utilisation as a sentinel species for AR surveillance in marine environments. However, the majority of studies originated from the Mediterranean and Arabian Seas, suggesting a bias in previous research towards these geographical areas, likely due to the accessibility of turtles in these regions during mating and migration. To date, five marine turtle species including green, loggerhead, leatherback, olive ridley, and hawksbill have been used in studies on monitoring AR in marine environments. While there were no statistically significant differences in reported frequencies of AR when data were stratified by turtle species or anatomical origin of the tested specimen, it can be hypothesised that turtle species with higher protein-rich diets may contain AR elements that are representative of their mobile food sources. Conversely, species that rely on more stationary food sources may provide a more accurate representation of their immediate feeding grounds. In monitoring the prevalence of AR in marine environments using sea turtles, the majority of studies utilised adult animals capable of, or during active, migration. This means that identified microbiomes and associated AR profiles may only indicate the existence of AR in respective marine environments, without a clear indication of specific geographical sources of the problem. The sub-adult class of green sea turtles may be a more effective animal model for assessing AR in specific geographical locations and thus recommend that future AR surveillance studies should use the non-migratory juvenile stage of green sea turtles.Furthermore, the true magnitude of bioavailable antibiotics and ARB in entire microbiomes in marine turtles has remained unknown. Herein, we show that the bulk of knowledge on surveillance of AR in marine environments using turtles has relied on culture and antibiotic sensitivity tests with the identification of phenotypic AR traits. These data are not likely to represent the entire resistomes for all ARB in tested microbiomes due to the existence of bacteria that is not culturable under ordinary laboratory conditions. In addition, our knowledge of the prevalence of specific ARGs and other mechanistic determinants of the phenotypic AR exhibited by sea turtles in marine environments is very limited. There is therefore a need to determine if ARGs and ARB profiles in sea turtles in marine environments are associated with similar profiles in specific anthropogenic AR “hot spots” such as WWTPs. Despite all these gaps in knowledge, analyses in this review show that there is a predominance in resistance to the beta-lactam class of antibiotics and this reflects the extensive usage of this class of antibiotics globally. Nonetheless, MDRB and resistance against less commonly used antibiotics including quinolones and aminoglycosides was also detected in sea turtles. This presents a serious threat to environmental and public health. Future studies should concentrate on the accurate determination of point sources, specific causes, and mechanisms, as well as prevalence and distribution patterns of AR in marine environments. The acquisition of this knowledge will be critical in designing effective mitigation strategies.


Augmented reality (AR) is a rapidly expanding technology; it comprises the generation of new images from digital information in the real physical environment of a person, which simulates an environment where the artificial and real are mixed. The use of AR in physiotherapy has shown benefits in certain areas of patient health. However, these benefits have not been studied as a whole.This study aims to ascertain the current scientific evidence on AR therapy as a complement to physiotherapy and to determine the areas in which it has been used the most and which variables and methods have been most effective.augmented reality, physiotherapy, physical therapy, exercise therapy, rehabilitation, physical medicine, fitness, and occupational therapy. The methodological quality was evaluated using the PEDro scale and the Scottish Intercollegiate Guidelines Network scale to determine the degree of recommendation. The Cochrane Collaboration tool was used to evaluate the risk of bias.A systematic review registered in PROSPERO  was conducted following PRISMA  recommendations. The search was conducted from July to August 2021 in the PubMed, PEDro, Web of Science, Scopus, and Cochrane Library scientific databases using the keywords z=1.65; P=.10) and the Timed Up and Go test .In total, 11 articles were included in the systematic review. Of the 11 articles, 4 (36%) contributed information to the meta-analysis. Overall, 64% (7/11) obtained a good level of evidence, and most had a B degree of recommendation of evidence. A total of 308 participants were analyzed. Favorable results were found for the Berg Balance Scale (standardized mean change 0.473, 95% CI −0.0877 to 1.0338; AR, in combination with conventional therapy, has been used for the treatment of balance and fall prevention in geriatrics, lower and upper limb functionality in stroke, pain in phantom pain syndrome, and turning in place in patients with Parkinson disease with freezing of gait. AR is effective for the improvement of balance; however, given the small size of the samples and the high heterogeneity of the studies, the results were not conclusive. Future studies using larger sample sizes and with greater homogeneity in terms of the devices used and the frequency and intensity of the interventions are needed.PROSPERO International Prospective Register of Systematic Reviews CRD42020180766; https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=180766 New technologies are rapidly emerging in our society to streamline, optimize, and perfect some of the activities we perform in our day-to-day lives . Among tAR is currently being used in different fields such as advertising , psycholAR technologies have significant advantages: they provide new experiences to patients during physiotherapy sessions, increasing engagement and improving physical outcomes ; they caOf the few investigations that have been conducted on AR, most were performed on healthy people with the aim of determining strategies that could later be used in the clinic . InteresRecently, a protocol for interactive AR-based telerehabilitation in patients with adhesive capsulitis was published  and anotIn the given context and taking into account all the advantages that the use of this kind of tool could have in physiotherapy, this review aims to determine how progress has been made in this regard, with the objective of ascertaining the current scientific evidence on AR therapy as a complement to physiotherapy, determining in which areas it has been used the most and which variables and methods have been most effective.A systematic review and meta-analysis was conducted and registered in PROSPERO  using the PRISMA  guidelines .augmented reality, physiotherapy, physical therapy, exercise therapy, rehabilitation, physical medicine, fitness, and occupational therapy were used, combining them by means of the Boolean operators AND and OR in the different searches in English or Spanish.A search of scientific evidence published from 2011 to August 2021 was conducted between July and August 2021 in the following scientific databases: PubMed, PEDro, Web of Science, Scopus, and Cochrane Library. In addition, gray literature  and AR conference proceedings were searched. The keywords The criterion that was taken into account for selecting the articles was clinical trials published in indexed scientific databases. The selected intervention was AR used with patients aged >18 years with some pathology of the musculoskeletal system of neurological or physical origin that was subsidiary to improvement in any physical measure analyzed in an objective and standardized way. Duplicate studies, qualitative trials, case reports, single-subject studies, reviews, meta-analyses, studies conducted on healthy individuals, and studies using VR were excluded.After performing the search, potentially relevant articles were identified after reading the title and abstract and eliminating duplicates. All studies identified in the searches were assessed for inclusion by 2 independent reviewers (MJVG and GGM). Any disagreements were resolved through discussions to reach a consensus. The following information was extracted from each included article: authors, year of publication, study population, type of intervention, number of participants, mean age, frequency of sessions per week, time of each session, total duration of the intervention, outcome measures, measurement instrument, and results obtained.To assess the quality of the trials used for the review, we used the PEDro scale , which cThe risk of bias was calculated for each study using the Cochrane Collaboration tool , referriThe effect size measure was the difference in the standardized mean change with raw standardization (SMCR) between the intervention (AR) and control groups ,43 for 2The entire selection process during the corresponding phases is detailed in The sample size was variable, with the largest sample (75 patients) being in the study by Rothgangel et al  and the The AR systems used varied widely: projectors connected to computers with webcams where images were shown , virtualThe intervention time ranged from 20 minutes  to 2 houThe most widely used measurement scale in the selected studies was the BBS, used both for stroke ,59 and iThe results found regarding the interventions conducted in the field of physiotherapy were diverse in the different plots. In stroke, intensive and repetitive task-oriented exercises were used for upper limb functionality , posturaThe results of the methodological quality assessment can be found in The results of the risk of bias can be observed in z=1.65; P=.10) for the BBS and −1.211  for the TUG, both differences favoring the intervention group, although the null hypothesis cannot be rejected. The forest plot  was present among the studies, with estimated variance components of 0.148  and 3.098 . No identifiability problems for the variance components were found  of studies were selected to evaluate the differences in mean changes in BBS and TUG scores. The power for detecting differences was low because of the reduced number of studies and small sample sizes. The data used for the meta-analysis are shown in re found . The 2 ore found  seems toARkanoidAR, which improved and corrected movement with the use of auditory, textual, or imaging feedback.In this systematic review and meta-analysis of clinical trials, we wanted to determine the use of AR in conjunction with conventional therapy in the different fields of physiotherapy. In our study, favorable results were obtained in balance and gait ,59, uppeThe aspects related to AR interventions and their positive results are as follows. Regarding their use in patients with amputations, the 2 articles found conflicting results on AR in phantom limb pain. In the trial by Ortiz-Catalán et al , pain waIn patients who had a stroke, we found improvement in the functionality of the upper limb , with hiRegarding geriatrics, favorable results were found in lower limb strength, balance, muscle mass, physical performance, exercise self-efficacy, and fall prevention. It is in this area that we have seen greater consistency in the findings. In this sense, for older adults who normally depend on visual information to achieve balance, AR training could effectively improve proprioception of the lower limbs, favoring static balance. It would be even better if the used system provides visual feedback .There are other areas within physiotherapy where AR could be used to improve these parameters, such as Parkinson disease, where VR has been used to improve balance . ExperimIt was possible to conduct the meta-analysis by taking into account the BBS and TUG. The BBS comprises 14 items where the patient is asked to perform several specific tasks to check their balance. Total scores range from 0  to 56  . IndividAdvantageous aspects of AR use have also been described -56,59. HDisplays used in AR can be classified into the following categories: head-worn, handheld, and projective . In our There are some obstacles that limit the generalized use of AR, such as technological and user interface limitations . Other nHowever, it should be considered that AR has certain advantages that VR does not have. For example, VR cannot recognize the real dangers that can cause injury, whereas, in AR, the patient is aware of the possible risks . In addiFurthermore, the benefits of AR in the use of telerehabilitation demonstrate its effectiveness in the remote monitoring of the patient and can even modify according to their progress, providing high-quality attention with reduced costs ,76. ThusThis study may serve as an aid in clinical practice through the use of AR systems. It may also serve as a preliminary step toward further research with a more homogeneous methodology and the ability to experiment with these technological systems in other areas of physiotherapy where pain, functionality, balance, and fall prevention may be an objective to be pursued.In terms of the limitations found, we must mention the limited number of studies with low quality and the wide variety of AR interventions with respect to the system used, number of sessions, and frequency and duration of the treatment sessions. There was no homogeneity with respect to the instruments used to measure the variables studied or the variables themselves. Similarly, the need for authors to use the same measuring instruments stands out as, in some cases, it was not possible to compare studies statistically because different versions of the same scale or different units of measurement were used.After 10 years of the review by Hussain Al-Issa  and withAccording to the results obtained, we can say that AR, in combination with conventional therapy, has been used for physical performance, treatment of balance and prevention of falls in geriatrics, functionality of the lower limb and upper limb in stroke, and pain in phantom pain syndrome. However, no positive results were obtained with turning and timing in the freezing of gait in Parkinson disease. Owing to the diversity of the interventions and the variables measured, no consensus can be reached on the best AR system in each area studied, although the most commonly used were the level 3 projectives.Future clinical trials are needed using larger sample sizes and with greater homogeneity in terms of the devices used and the frequency and intensity of the interventions.


An estimated 30,000 men in the United States will die of metastatic prostate cancer (PCa) each year due to the development of therapy resistance, most notably resistance to second-generation antiandrogen enzalutamide. The vast majority of PCa is driven by the androgen receptor (AR). Enzalutamide is an AR antagonist, which extends patient survival and is widely used in the clinic for the treatment of castration-resistant prostate cancer (CRPC); however, many patients will have primary or develop acquired resistance and continue to progress. Characterization of the molecular mechanisms of enzalutamide resistance provides insight into potentially efficacious therapies for enzalutamide-resistant CRPC (ER-CRPC). Understanding these mechanisms is critical for the identification of biomarkers predictive of therapy resistance and the development of therapeutic strategies to target ER-CRPC. Despite the introduction of seven new Food and Drug Administration-approved therapeutic agents since 2007, metastatic PCa is incurable. The mainstay of treatment for metastatic PCa remains androgen deprivation therapy (ADT), via pharmacological or surgical castration. The majority of patients will initially respond to ADT; however, a significant proportion becomes therapy-resistant and develops castration-resistant prostate cancer (CRPC),4.Nearly 30,000 men in the United States will die of metastatic prostate cancer (PCa) in 2019,6. However, primary or acquired resistance to these agents is common with cancer recurrence and progression. Multiple mechanisms of enzalutamide resistance have been defined and offer insights into further therapeutic strategies against enzalutamide-resistant CRPC (ER-CRPC)-9. In this review, we present a critical evaluation of the defined molecular mechanisms, potential biomarkers, and therapeutic options for ER-CRPC.The androgen receptor (AR) is a steroid hormone nuclear receptor that drives the vast majority of PCa. CRPC remains dependent on AR signaling and second-generation antiandrogens, such as the AR antagonist enzalutamide or the androgen synthesis inhibitor abiraterone, extend the overall survival of metastatic CRPC patients by a median of 4-5 months,11. Thus, AR antagonists, like enzalutamide, apalutamide, or darolutamide, may be effectively unable to antagonize all AR proteins in this context. Furthermore, since CRPC and ER-CRPC tumors may synthesize their own androgens, sustained or enhanced AR signaling is noted, even under castrate-level serum androgen conditions. Thus, AR amplification and increased AR signaling are robust mechanisms of resistance to first- and second-generation antiandrogens,11,13-16.For receptor-ligand interactions, the relative concentrations of receptors, agonists, and antagonists in a cell can dictate whether antagonists will be efficacious in inhibiting their target. Stoichiometric ratios of AR, enzalutamide, and androgens determine whether AR is bound to agonist or antagonist, and whether AR signaling is active. Sequencing of CRPC and ER-CRPC patient tumors demonstrates that 70% of patients have significant AR pathway alterations, with the vast majority containing AR gene amplifications-16. Similarly, resistance to enzalutamide has been modeled to be driven by an AR F876L point mutation. This mutation has been infrequently identified in ER-CRPC samples and confers agonistic properties to both enzalutamide and apalutamide-19. Importantly, since these mutations are specific for each antiandrogen, other antiandrogens can have activity against AR. For example, CRPC cells with T877A and W741C mutations are resistant to flutamide and bicalutamide, respectively, but are sensitive to enzalutamide and apalutamide. While AR mutations can confer resistance to enzalutamide, the low observed prevalence of this mutation in ER-CRPC patients does not support a significant clinical role for this mutation in enzalutamide resistance.AR point mutations that convert first-generation antiandrogens, such as flutamide and bicalutamide, from antagonists to agonists have been well-characterized. These mutations are in the ligand binding domain (LBD), and include T877A and W741C point mutations, which mediate resistance to flutamide and bicalutamide, respectively-24. AR variants primarily appear after the selection pressure of castration and more frequently after enzalutamide and abiraterone treatment-22,24-31. The most common AR-V is AR-V7 (AR3), which contains exons 1, 2, 3, and a cryptic exon 3b but lacks the LBD,26,27. AR-Vs can be constitutively active, and detection of AR-Vs in CRPC correlate with poor survival, progression, and therapy resistance-22,24-31. Assays to detect AR-Vs in circulating tumor cells are commercially available and predict therapy response to enzalutamide and abiraterone. AR-Vs can substitute for the full-length AR (AR-FL) and can bind to canonical androgen responsive elements (AREs) on the chromatin, heterodimerize with AR-FL, and drive transcription from AREs-34. AR-Vs have been shown to be capable of independently driving transcription, cell proliferation, and DNA repair in CRPC. However, AR-Vs are rarely seen without significant amplification of AR-FL, and its activity may be dependent on full-length AR,32,36.There are a large number of known AR splice variants (AR-Vs)In vitro studies with knockdown of variants in CRPC cell lines that express both AR-FL and AR-Vs indicate that AR-V expression confers a distinct growth advantage, when treated with antiandrogens-34,37. Treatment with niclosamide inhibits AR-V7 recruitment to AR target genes, reduces AR-V7 protein levels in a proteasome-dependent manner, and re-sensitizes ER-CRPC cells to enzalutamide. Induction of AR-Vs through NF-κB2/p52 can also enhance enzalutamide resistance-41. While these data suggest that AR-Vs can contribute to ER-CRPC growth and can mediate resistance to enzalutamide, they do not definitively establish that AR-Vs drive resistance to enzalutamide.Importantly, the ability of AR-Vs to independently drive ER-CRPC has not been proven. . Ongoing clinical trials with AR degraders will also likely target AR-FL and AR-Vs, and are unlikely to establish AR-Vs as independent molecular drivers of ER-CRPC.The true contribution of AR-Vs in driving resistance can only be clearly established from drugs that specifically target AR-Vs and not AR-FL; however, no such agent has been developed. Since AR-FL and AR-Vs share a common N-terminal domain (NTD), compounds designed to target the NTD will target both AR-FL and AR-Vs. For example, ESSA Pharma’s EPI-506, which was designed to target the AR NTD, could target both AR-Vs and AR-FL, but demonstrated no activity against enzalutamide- and abiraterone-resistant CRPC patients in Phase I clinical trials-48. Modulating the expression of these cofactors can enhance or attenuate AR transcriptional activity. For example, alterations in expression of coactivators SRC-1, SRC-2, SRC-3, CBP/p300, and FOXA1 and co-repressor ZBTB16 are frequently seen CRPC and ER-CRPC patients and are associated with worse prognosis,11,49-53. Since enzalutamide can modulate the recruitment of AR cofactors, altering the expression of co-activators or co-repressors in CPRC can potentially bypass the inhibitory effects of enzalutamide and confer resistance,54,55.Upon binding androgens and activation, AR alters its protein interactome, translocates to the nucleus, and regulates a pro-proliferative transcriptional program. AR interacts with a number of co-activators to enhance transcription, like members of the p160 steroid receptor coactivator (SRC) family, histone acetyltransferase CBP/p300, and pioneer factor forkhead box A1 (FOXA1), and co-repressors to inhibit transcription, such as zinc finger and BTB domain containing 16 (ZBTB16),57. Upon binding its ligand, GR transcriptionally activates a stress response program, which enhances the expression of anti-inflammatory genes and suppresses pro-inflammatory genes-60. Overexpression of GR was noted in ER-CRPC and mediates enzalutamide resistance, where it may bypass the need for AR signaling. Primary GR-dependent enzalutamide resistance may be observed in a subset of CRPC tumors that have increased basal expression of GR. Acquired GR-dependent enzalutamide resistance entails a de-repression mechanism, whereby AR normally inhibits GR expression, and enzalutamide enables enhanced GR expression by blocking AR signaling. Interestingly, chromatin immunoprecipitation-sequencing (ChIP-seq) studies in enzalutamide-resistant cells identified GR binding over 50% of AR binding sites on the chromatin, with the strongest AR-regulated genes also being regulated by GR. Thus, overexpressed GR functionally substitutes for AR. Therapeutic targeting of GR in ER-CRPC has been proposed, with at least two companies, ORIC Pharmaceuticals and Corcept Therapeutics, developing GR antagonists.Like AR, the glucocorticoid receptor (GR) is a steroid hormone nuclear receptor, which binds DNA as a homodimer in an inverted repeat fashion-65. Acute enzalutamide treatment induces EMT through a number of mechanisms, including increasing TGF-β1 expression and STAT3 activation, as well as Snail induction-68. Metformin blocks enzalutamide-induced EMT and improves PCa sensitivity to enzalutamide. In addition, autocrine IL-6 can facilitate CRPC growth and confers enzalutamide resistance mediated by STAT3 activation. Furthermore, AR directly represses Snail transcription, and acute enzalutamide treatment enhances Snail expression and EMT. Importantly, in models of chronic enzalutamide treatment, enzalutamide resistance can be mediated by Snail induction of both AR and AR-V7 expression, leading to increased AR signaling,70. Whether in an AR-dependent or -independent manner, programs that enable cell transitions in response to selective pressures are important mechanisms of resistance to enzalutamide.The epithelial-mesenchymal transition (EMT) is a process by which epithelial cells become more mesenchymal, a state characterized by increased invasive capacity, apoptotic resistance, and enhanced motility and metastatic potentialMost molecular mechanisms identified in ER-CRPC have focused on AR transcriptional regulation and maintenance of AR signaling, despite AR inhibition. Evaluations of the downstream programs that confer enzalutamide resistance often culminate in metabolic alterations, as the metabolic state governs whether cells will resist stress and proliferate.. Intracrine androgen synthesis has recently been shown to confer enzalutamide resistance through upregulation of steroid synthesis genes, such as aldo-keto reductase family 1 member C3 (AKR1C3),30,71-75. AKR1C3 catalyzes the conversion of androstenedione and 5 α-androstanedione to testosterone and DHT, respectively, and is enriched in acquired and de novo ER-CRPC,76,77. Increased AKR1C3 levels results in upregulated intracrine androgen synthesis and confers resistance to enzalutamide.Increased androgen synthesis can overwhelm the ability of enzalutamide to block AR signaling. PCa cells that are capable of stimulating hypoxia-induced survival programs through the upregulation of hypoxia response genes, such as glucose-6-phosphate isomerase (GPI), are clonally selected to become AR-independent and resistant to enzalutamide. Under normoxic and normal androgenic conditions, AR enhances glycolysis and the pentose phosphate pathway (PPP),80. Under hypoxic and normal androgenic conditions, the PPP is slightly upregulated, AR inhibits GPI, and glycolysis is inhibited. However, under hypoxic and castrate conditions, the PPP is inhibited, GPI is upregulated, and glycolysis is stimulated. PCa cells that are thus able to redirect glucose away from the PPP and toward glycolysis are able to evade stress and proliferate normally. Glycolytic inhibitors, such as 2-deoxyglucose, may be useful in this context; however, toxicity is a concern due to lack of selectivity. Selective glycolytic inhibitors in development may be efficacious against some forms of ER-CRPC. Of note, GPI is preferentially overexpressed in neuroendocrine prostate cancer (NEPC) tumors, attesting to the importance of metabolic rewiring in driving neuroendocrine disease.An AR-independent mechanism of enzalutamide resistance involves hypoxia and the metabolic consequences of hypoxia-induced programs driven by hypoxia-inducible factor (HIF). In CRPC cells, loss of protein kinase C (PKC) λ/ι allows cells to transition from a luminal, AR-dependent phenotype to a basal, AR-independent phenotype through enhanced one-carbon metabolism and resulting epigenetic changes. This upregulation in one-carbon metabolism is dependent on mammalian target of rapamycin 1 (mTORC1) and cyclic AMP-dependent transcription factor 4 (ATF4) and culminates in an increase in S-adenosylmethionine (SAM), which supports epigenetic reprogramming (DNA methylation). Enhancer of zeste homolog 2 (EZH2) inhibition has been shown to reverse NEPC to a more AR-dependent state sensitive to antiandrogens, which demonstrates the potential efficacy of this strategy and further indicates the importance of epigenetic changes for the development of NEPC,82. Additionally, DNA methylation inhibitors, such as decitabine, may be efficacious in targeting neuroendocrine disease. mTORC1 inhibitors, such as everolimus, may also show benefits in NEPC patients with a PKCλ/ι deficiency.Increased reliance on serine and one-carbon (1C) metabolism promotes enzalutamide resistance in NEPC. With enzalutamide treatment, cells become AR-independent and therefore enzalutamide-resistant. In PCa, lineage plasticity is a state characterized by significant epigenetic changes, decreased AR signaling, and an increased expression of neuroendocrine and stem cell markers-84.Lineage plasticity is a mechanism through which cells can acquire characteristics of a lineage that no longer requires a certain drug target .The proposed mechanism involves increasing cell plasticity, which confers resistance through lineage switching to an AR-independent, basal-like cell. Similarly, loss of p53 and Rb1 creates a stem cell-like epigenetic environment due to derepression of EZH2 (and SOX2), which allows for adaptation to selective pressures, such as enzalutamide.Enzalutamide resistance can develop from loss of tumor suppressors tumor protein p53 (TP53) and retinoblastoma 1 (Rb1) and a downstream SRY-box 2 (SOX2)-driven shift. BRN2 expression is inhibited by AR, is required for the expression of neuroendocrine markers, and expressed in NEPC. Enzalutamide derepresses AR inhibition of BRN2 in CRPC and enables BRN2-driven transdifferentiation into enzalutamide-resistant NEPC. Furthermore, BRN2 regulates SOX2, and these proteins directly interact at the enhancers of neuronal genes and cooperate to drive a neuroendocrine phenotype. Targeting BRN2 remains an attractive option for preventing lineage plasticity and the development of AR-independent PCa.Regulators of Sox2, like POU-domain transcription factor BRN2 (POU3F2), drive the emergence of NEPC and enzalutamide resistance-87. N-Myc and EZH2 cooperate to drive transdifferentiation into NEPC and enzalutamide resistance. N-Myc differentially regulates the DNA damage response in a context-dependent manner. Upregulation of N-Myc inhibits ataxia-telangiesctasia mutated (ATM), which allows PCa to become CRPC. In CRPC, overexpression of N-Myc with EZH2 blocks ATM inhibition, leading to ATM upregulation and the development of enzalutamide-resistant NEPC. Given the dependence of NEPC on epigenetic reprogramming and EZH2 in particular, targeting EZH2 may be an effective therapeutic option. While some EZH2 inhibitors have failed clinical trials [NCT01897571], other agents, such as Constellation Pharmaceuticals’ CPI-1205 and Daiichi-Sankyo’s DS-3201b, may offer hope for selected patients with NEPC .N-Myc overexpression is found in 5% of primary PCa patients, 20% of CRPC patients, and roughly 40% of NEPC patientsDiscovering the molecular underpinnings of enzalutamide resistance has led to a greater understanding of the factors that drive progression and the heterogeneity that belies ER-CRPC. Ongoing studies will enable the identification of biomarkers predictive of therapy resistance and the development of targeted therapies to overcome therapeutic resistance.


To validate the experimental conditions used, we assessed the activity of various AEDs typically used in the fight against DS, including combination therapy. Overall, our results are highly consistent with the treatment algorithm proposed by the updated current practice in the clinical management of DS. Our results show that (+)-FFA, (−)-FFA and (+)-norFFA displayed significant antiepileptic effects in the preclinical model, and thus can be considered as compounds actively contributing to the clinical efficacy of FFA. In case of (−)-norFFA, the results were less conclusive. We also investigated the uptake kinetics of the enantiomers of FFA and norFFA in larval zebrafish heads. The data show that the total uptake of each compound increased in a time-dependent fashion. A somewhat similar uptake was observed for the (+)-norFFA and (−)-norFFA, implying that the levo/dextrotation of the structure did not dramatically affect the uptake. Significantly, when comparing (+)-FFA with the less lipophilic (+)-norFFA, the data clearly show that the nor-metabolite of FFA is taken up less than the parent compound.Dravet syndrome (DS) is a rare genetic encephalopathy that is characterized by severe seizures and highly resistant to commonly used antiepileptic drugs (AEDs). In 2020, FDA has approved fenfluramine (FFA) for treatment of seizures associated with DS. However, the clinically used FFA is a racemic mixture (i.e. (±)-FFA), that is substantially metabolized to norfenfluramine (norFFA), and it is presently not known whether the efficacy of FFA is due to a single enantiomer of FFA, or to both, and whether the norFFA enantiomers also contribute significantly. In this study, the antiepileptic activity of enantiomers of FFA (i.e. (+)-FFA and (−)-FFA) and norFFA (i.e. (+)-norFFA and (−)-norFFA) was explored using the zebrafish  SCN1A which encodes for an α (pore-forming) subunit of the brain voltage gated sodium channel type-1 (NaV1.1), occurs in a large majority of patients  was a gift from Prof. Berten Ceulemans . The enantiomers of FFA and norfenfluramine (norFFA) were provided by Zogenix International . Compounds were dissolved in dimethylsulfoxide (DMSO), and diluted in embryo medium to achieve a final DMSO concentration of 0.1% w/v. Embryo medium with 0.1% w/v DMSO served as a vehicle control (VHC).To evaluate the maximum tolerated concentration (MTC) of the individual compounds, a dozen of WT zebrafish larvae (6 dpf) were individually incubated in single wells of a 96-well plate, and treated with two-fold serial dilutions of the compounds. After 22 h incubation under standard conditions , larvae were individually examined for their touch response, posture, edema, signs of necrosis, morphology, heartbeat rate, and swim bladder condition under the microscope. The MTC was defined as the highest concentration at which a compound did not exert any sign of toxicity in any of the larvae used.scn1Lab−/− mutants) (6 dpf) were individually positioned in single wells of a 96-well plate, and incubated in 100 μL VHC or VHC supplemented with compound or combined compounds for 22 h at 28 °C on a 14/10 h light/dark cycle. Then, the plates were placed immediately in an enclosed tracking device , followed by a 30 min chamber habituation and 10 min recording. Locomotor activity over the total tracking period of 10 min was quantified by ZebraLab software  using the lardist parameter  and 150 bkg (background) as a threshold, as reported previously by our group [WT and homozygous zebrafish larvae  (6 dpf) were treated as described above. After incubation, larvae were immobilized in 2% low-melting-point agarose (Invitrogen) at room temperature (RT) and the epileptiform activities were measured by noninvasive local field potential (LFP) recording from the skin above the optic tectum (midbrain). The single glass electrode filled with artificial cerebrospinal fluid (ACSF)  was positioned on the skin above the optic tectum. Each recording lasted for 10 min. Epileptiform activity was quantified by using Clampfit 10.2 software , as reported previously by our group [WT and homozygous zebrafish larvae  (6 dpf) were treated as described above. After incubation with compounds, larvae were washed and euthanized by exposure to cold Milli-Q water. Then, the heads of larvae were carefully separated under the microscope, and five heads were transferred collectively into one 1.5 mL Eppendorf tube with acid-washed glass beads  and 275 µL extraction medium . Next, the samples were homogenized by 10 min of ultrasonication  at 4 °C. The overall ultrasonication process encompassed 10 cycles of 30 s with pauses of 30 s in-between with high energy input [WT and homozygous zebrafish larvae (gy input . After tdp = 1.9 μm) column was utilized for the chromatographic separations at 40 °C. Samples were injected in a volume of 1 μL and the flow rate was set to 0.2 mL/min. The mobile phase consisted of 10 mM ammonium acetate in H2O: acetonitrile 75:25 (v/v) and separations were carried out isocratically. The elution time of (−)- and (+)-FFA was 4.1 min, while the elution time of (−)- and (+)-norFFA was 2.6 min. The LC instrument was hyphenated to a mass spectrometer (MS) with a triple quadrupole detector  and equipped with an electrospray ionization source. The MS/MS analysis was conducted in multiple reaction mode (MRM) in positive mode. The MS settings were optimized by direct infusion of compound standards diluted in methanol. The optimal MS parameters are summarized in Table Analyses were performed using an Infinity 1200 LC system  equipped with an autosampler, binary pump, and a thermostated column oven compartment. An YMC-Triart C18  were 0.993, 0.998, 0.992, and 0.990 for (+)-FFA, (−)-FFA, (+)-norFFA and (−)-norFFA, respectively.Quantification was performed through the use of a calibration curve created for each compound separately in blank matrix, which was derived from a pooled set of blank fish heads (n = 40). Calibrators consisted of 90 µL of blank matrix spiked with 10 µL of the corresponding standard dissolved in MeOH to obtain a concentration range varying between 0.025 μM to 7.5 μM for (+)-FFA, 0.02 μM to 5 μM for (−)-FFA, 0.05 μM to 10 μM for (+)-norFFA, and 0.02 μM to 10 μM for (−)-norFFA. Ranges consisted of at least 5 concentrations, each of which was analyzed in replicate (n = 5). A weighed least squares (WLS) regression model with 1/The mean head weight (± S.D.) of a 6 dpf and 7 dpf zebrafish larva was 192 ± 10 µg and 196 ± 13 µg, respectively, as measured by weighing three batches of 50 fresh heads after removing excess water with filter paper. The final uptake was calculated according to the method reported by Copmans et al. , and exp*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.The locomotor activity data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison tests. Electrographic brain activity data were analyzed by Kruskal–Wallis testing with Dunn’s multiple comparisons. All test were performed in GraphPad Prism 8 software . Significance was calculated only when compound treatment decreased the seizure activity. Significance levels: scn1Lab−/− mutant model and the experimental conditions used in this study, we first tested a series of antiepileptic drugs (AEDs) proposed by different treatment algorithms for DS [To validate the zebrafish s for DS , 22, 23,s for DS , 24.Various concentrations of the AEDs were examined for their adverse effects, allowing a maximum tolerated concentration (MTC) to be determined. MTC is defined as the highest concentration at which the compound did not exert any sign of toxicity in any of the larvae tested. MTC values were determined as 1 mM for VPA, 50 µM for ( ±)-FFA, 200 µM for TPM, 50 µM for STP, 6.25 µM for CBD, 100 µM for CLB, 10 mM for LEV, 50 µM for CBZ, 100 µM for PHT, and 100 µM for LTG. By using these maximal concentrations for all further investigations, we sought to reduce the risk of false positive results to a minimum, which is particularly critical for locomotor activity measurements.scn1Lab−/− mutant model. As shown in Fig. Next, the effect of each of these AEDs as a single treatment on locomotor and brain activities was evaluated by behavioral and electrophysiological assays using the zebrafish scn1Lab−/−mutants. In the clinic, however, VPA shows dose-limiting side-effects after prolonged use like fatigue, hair loss and hyperammonemia amongst others [Of interest, VPA rarely provides adequate seizure control in DS patients, so that it requires the addition of others AEDs as second-line therapies , whereast others . Possiblscn1Lab−/− mutants, but also dramatically attenuated the frequency of epileptiform events -FFA -FFA are typically combined in the clinic with VPA for second line treatment , we furtvae Fig. .Fig. 3BeSignificantly, the combination of VPA (250 µM) and STP (50 µM) turned out to effectively alter the locomotor activity and decrease the epileptiform discharges of the mutant larvae, whereas the single treatments were not active Fig. A–C.Furthermore, investigating the combination outcome of VPA (250 µM) and (±)-FFA, the concentration of the latter compound was reduced from 50 µM (MTC) to 3.13 µM. This concentration continued to induce a significant effect on the locomotor activity but not on the epileptiform brain discharges of the mutant larvae  was combined with CBD (6.25 µM), the VPA + CBD-treatment showed a clear effect on the locomotor and LFP results obtained with the mutant larvae as compared to the single treatments that were not active Fig. G-I.scn1Lab−/−mutant model. Noticeably, CLB was previously proven to be ineffective in the DS zebrafish mutant model [scn1Lab−/−mutant model is yet to be investigated into more detail, for instance by exploring its additional activity in combination with VPA and STP, as suggested by Cross et al. [As far as alternatives for second line treatment are concerned , i.e. TPnt model . Althougnt model . Whethers et al. .scn1Lab−/− mutants in this study, as shown before [LEV has been categorized as a third-line drug for DS by the North American consensus panel , but is n before .In order to investigate the uptake kinetics of the enantiomers of FFA and norFFA in larval zebrafish heads, we immersed larvae in solutions of the individual compounds at their MTC for 30 min, 4 h and 22 h. Next, the compounds present in extracts of the heads were quantified by LC-MS analysis. The data depicted in Fig. A somewhat similar uptake was observed for the (+)-norFFA and (−)-norFFA enantiomers, implying that the levo/dextrotation of the structure did not dramatically affect the uptake. Conversely, a direct comparison between (+)-FFA and its (−)-enantiomer is hard to draw as different immersion concentrations were used due to the different MTCs of the respective compounds.scn1Lab−/− mutants, they were first examined by a behavioral assay, at a wide range of concentrations. As illustrated in Fig. As uptake of compounds in larval heads was maximal after 22 h, we proceeded to use this prolonged incubation condition to explore the pharmacological activity of the FFA and norFFA enantiomers, as performed previously , 19. To Subsequently, we examined the effect of the compounds on the epileptiform discharges of the mutant larvae by recording local field potentials (LFP) of the brain. Representative traces of brain activity during the recordings are shown in Fig. scn1Lab−/− mutant zebrafish DS model was previously performed, using a shorter incubation protocol than present in this study [Overall, our results mirror well the order of multiple treatment options proposed by the updated current practice in management of DS . Notablyis study , 28. HowSurprisingly, a substantial increase in concentrations could still be observed in the 4–22 h time window. To the best of our knowledge, this study is the first to quantify the uptake of any drug compound in heads of zebrafish larvae as a function of time, and consequently it is not possible to conclude whether FFA and norFFA are actually unique in this respect. However, a certain parallel is apparent with the clinical condition, where accumulation of (+)-FFA and (+)-norFFA in humans typically follows a step-wise increase to steady state concentrations after long-term repetitive drug administration .Other studies reported the uptake of compounds in heads and brain of larval zebrafish after 1 h incubations , 30. Of scn1Lab−/− mutants [50 values ranging from 52 to 287 nM. Other effects of the enantiomers relate to potent serotonin uptake inhibition and agonistic effects on 5-HT2 subtype receptors  brain levels are involved in epileptogenesis and/or seizure propagation , 33, and mutants . Signifi5-HT2A-R , therebyThe data available therefore show that the enantiomers of FFA and norFFA possess somewhat different pharmacological potencies on a subset of receptors that have been implicated in their anti-epileptic activity. However, in view of the larval head concentrations of the individual compounds found in this study, one would not anticipate any major difference in outcome for both locomotor and LFP read-outs. Moreover, as the pharmacological fingerprint of (−)-norFFA is not substantially different from the one of (−)-FFA, it is rather surprising that the former compound exerted a less conclusive inhibitory activity in the DS zebrafish model. Evidently, the slightly conflicting results might be explained by enantiomers’ relative affinities for zebrafish proteins differing to those for their mammalian counterparts. However, the mechanism-of-action of the antiepileptic effect of FFA is multi-dimensional, also involving σ1-receptors and possibly other targets, further complicating the interpretation of the finding. Clearly more investigations are needed to better understand the relationship between the pharmacology of the compounds and their respective antiepileptic activities against DS.scn1Lab−/− mutant model by using a combined treatment of AEDs, further supporting the application of the zebrafish-based model as a rapid screening platform to find precision medicine for DS, and possibly for other difficult-to-treat epilepsies. In addition, our results show that (+)-FFA, (−)-FFA and (+)-norFFA displayed significant antiepileptic effects in the preclinical model, and thus can be considered as compounds actively contributing to the clinical efficacy of FFA. In case of (−)-norFFA, the results were less conclusive. Whether this inconsistency is related to a different pharmacological fingerprint is presently unexplored and warrants further investigation.Taken together, our study is the first to validate the