A hip fracture is a major adverse event for older individuals that has extremely high rates of mortality and morbidity, specifically functional decline. Thus, effective post–hip fracture rehabilitation is crucial to enable patients to regain function and improve their quality of life. Most post–hip fracture rehabilitation programs focus only on physical functioning, but rehabilitation goals related to the quality of life, social participation, and environmental issues are also crucial considerations. This study aimed to develop a core set of considerations based on the International Classification of Functioning, Disability, and Health (ICF) for use as a reference in designing comprehensive rehabilitation programs for patients with hip fractures. For this purpose, we recruited 20 experts from related fields working at a university hospital to complete a three-round Delphi-based questionnaire. Before beginning this process, a literature review related to ICF category selection was conducted. Next, a 5-point Likert scale was employed to rate the importance of each proposed category, and Spearman’s rank correlation coefficient and semi-interquartile range indices were analyzed to rate the consensus status. Categories for the ICF core set of considerations for post-hip-fracture rehabilitation were chosen on the basis of a high level of consensus and a mean score of ≥4.5 in the third Delphi-based questionnaire round. After selection, the ICF core set comprised 34 categories, namely 15 for bodily functions, 5 for bodily structures, 13 for activities and participation, and 1 for environmental factors. The proposed post-hip-fracture rehabilitation ICF core set can serve as a reference for developing effective rehabilitation strategies and goal setting by interdisciplinary teams. However, further feasibility evaluation is recommended for individualized rehabilitation program design. Hip fracture is a major adverse event for older individuals that has extremely high rates of mortality and morbidity, specifically functional decline ,2. FurthPreviously, the primary rehabilitation goal after a hip fracture was to minimize a patient’s hospitalization duration through multidisciplinary team intervention and then to continue with home rehabilitation after discharge. A recent study indicated that early discharge followed by geriatric interdisciplinary home rehabilitation resulted in similar recovery regarding independence in ADLs at 3 and 12 months of recovery compared with hospital geriatric care and rehabilitation . In addiThe World Health Organization developed the International Classification of Functioning, Disability, and Health (ICF) framework to provide a comprehensive and holistic description of the functioning and disability status of individual patients . This frThe Delphi-based consensus method was applied to determine the optimal ICF core categories for a post-hip-fracture rehabilitation program . To idenThe three rounds of the Delphi-based consensus questionnaire survey were conducted between 1 June and 31 August 2022 at a university hospital. Twenty multidisciplinary hip fracture care experts  were recruited for this hip fracture rehabilitation core set developmental study. All these experts had more than 5 years of clinical experience and the proportion of different experts was based on daily clinical practice hip fracture surgery and rehabilitation by multidisciplinary team intervention in a medical university hospital. These participants were informed of the study objectives, the consensus process methods, and the clinical scenario of hip fracture rehabilitation via email. After the participants had agreed to join this study, the questionnaire was sent to them. The questionnaire contained second-level ICF codes for potential post-hip-fracture rehabilitation-associated categories, and the content of these categories was presented in detail to facilitate the importance rating. The participants rated the importance of each of the selected categories by using a 5-point Likert-type scale . The questionnaire of three rounds were the same categories for rating scores and each category had an explanation of the content. The scores of each category given by all the participants were averaged, and the scores from the first and second rounds of the questionnaire were used as a reference during the second and third rounds, respectively, to provide information regarding previous scores and enable the participants to re-evaluate their scores for all the items on the basis of their previous scores and those of the other participants. After the three rounds of the questionnaire, the post-hip-fracture rehabilitation ICF core set was developed on the basis of an average Likert scale score of more than 4 points in the final round. The hallmark of this study is illustrated in To determine the most suitable ICF core set categories for post-hip-fracture rehabilitation, we conducted serial data analysis. Spearman’s rank correlation coefficient (rho) scores were calculated to compare the individual scores of the participants with the mean scores of all the participants for each ICF category in each round of the questionnaire. A rho value of more than 0.7 indicated strong agreement for a category between a participant and all the participants. Regarding the ICF core set, as long as the category scored more than 4.5 on the Likert scale in the third round of the Delphi-based consensus, it was considered suitable for the core set for post-hip-fracture rehabilitation. Data analyses were performed using SPSS , and a p-value of less than 0.05 was considered statistically significant.As mentioned, 20 experts from relevant fields completed all three rounds of the Delphi-based consensus questionnaire. In the first round, the mean (SD) Spearman’s rho value was 0.524. In the second round, it is 0.660, and it is 0.748 in the third round. The mean (SD) Likert scores of all categories in all three rounds are presented in A total of 34 categories scored more than 4.5 on the Likert scale in the third round of the questionnaire, and thus, these 34 categories were considered suitable for the ICF core set for post-hip-fracture rehabilitation. These categories were divided as follows: 15 for bodily functions, 5 for bodily structures, 13 for activities and participation, and 1 for environmental factors. The categories of Consciousness functions (b110), Muscle power functions (b730), Muscle endurance functions (b740), Gait pattern functions (b770), and Walking (d450) achieved the highest level of expert consensus  category was considered one of the most crucial categories in the post-hip-fracture rehabilitation core set; this finding is similar to that of a previous study, which noted that 40% of patients with hip fractures also have dementia . This fiThe Muscle power, Endurance, Gait pattern functions, and Walking categories were considered to be among the most crucial categories for inclusion in the ICF core set. These findings are in accordance with those of a previous study, which reviewed multiple hip fracture rehabilitation programs and concluded that the most frequently reported outcomes were associated with ambulation ability . AnotherBased on our ICF core set, the rehabilitation strategy can be focused on these categories. These categories can be included by different experts via a multidisciplinary team intervention. In the aspect of body functions, physiotherapists and occupational therapists can design the rehabilitation program via these core set categories. Similarly, the assessment of body structures can focus on the related dimension of ICF core set by clinical physicians and rehabilitation-related medical staff. The goal setting activities and participation in these ICF core set categories can be applied for functional achievement after the rehabilitation program.In addition to the bodily functions and activities and participation categories, the bodily structures and environmental factors categories were also considered necessary as ICF core set categories. In addition to the hip region, other bodily structures related to movement were also included in the ICF core set, including those vital for maintaining balance and stabilization. In addition, health services, systems, and policies were considered environmental factors, indicating that post-hip-fracture rehabilitation policies and healthcare resources are crucial for effective functional restoration after a hip fracture. Based on the ICF core set, the healthcare system of the inpatient, post-acute care, and community interact with the functional restoration goals and directions. The ICF core set could provide information for effective healthcare resource use by the government and lessen the economic burden of hip fracture patients.This study also investigated the effectiveness of the post-hip-fracture rehabilitation ICF core set for comprehensive rehabilitation program design. Under the framework of the ICF, the proposed core set provides categories related to bodily functions, bodily structures, activities and participation, and environmental factors. On the basis of these categories, related experts can set goals for rehabilitation and intervention. Nevertheless, some limitations of the present study should be noted. First, perceptions of post-hip-fracture rehabilitation program designs based on the proposed ICF core set may differ among patients. In addition, experts  may focus on inpatient intervention for patients with hip fractures. To prevent this problem from occurring, the scenario of considering the rehabilitation status of a patient with a hip fracture was described in the questionnaire. Our goal for the proposed ICF core set is to enable patients with hip fractures to regain their preinjury level of functioning. Clear rehabilitation goals eliminate the problem of subjective interpretations of questionnaire ratings. Second, the experts who participated in this study were from multiple fields. The percentage of each field’s representation was based on our clinical experience of post-hip-fracture rehabilitation; however, other percentage divisions may be more appropriate in other countries or under other healthcare systems; for example, this study recruited no nurses or social workers, which is not to say that the inclusion of such professionals would not be beneficial in other contexts. Finally, whether the feasibility and validity of the proposed ICF core set for patients with hip fractures are applicable to other healthcare systems in other countries or whether experts in those different settings would yield different clinical feasibility ratings for the proposed core set are open questions. Thus, regarding clinical applications, further investigation into the clinical feasibility of the proposed ICF core set is recommended.To enable patients with hip fractures to regain their preinjury levels of physical functioning and minimize the socioeconomic burden of care, an effective post-hip-fracture rehabilitation strategy design is crucial, particularly for elderly people. The proposed ICF core set provides a multidimensional framework for rehabilitation program design. The categories of this core set can be considered rehabilitative components that are teachable to patients by experts from related fields and other medical professionals. Furthermore, the proposed ICF core set provides information regarding effective rehabilitation strategies, multidisciplinary team interventions, and goal setting for post-hip-fracture rehabilitation programs. Developing an individualized rehabilitation program under the framework of this ICF core set could be highly beneficial for patients with hip fractures.


Neuroendocrine prostate cancer (NEPC) is a multi-resistant variant of prostate cancer (PCa) that has become a major challenge in clinics. Understanding the neuroendocrine differentiation (NED) process at the molecular level is therefore critical to define therapeutic strategies that can prevent multi-drug resistance.Using RNA expression profiling and immunohistochemistry, we have identified and characterised a gene expression signature associated with the emergence of NED in a large PCa cohort, including 169 hormone-naïve PCa (HNPC) and 48 castration-resistance PCa (CRPC) patients. In vitro and preclinical in vivo NED models were used to explore the cellular mechanism and to characterise the effects of castration on PCa progression.We show for the first time that Neuropilin-1 (NRP1) is a key component of NED in PCa cells. NRP1 is upregulated in response to androgen deprivation therapies (ADT) and elicits cell survival through induction of the PKC pathway. Downmodulation of either NRP1 protein expression or PKC activation suppresses NED, prevents tumour evolution toward castration resistance and increases the efficacy of docetaxel-based chemotherapy in preclinical models in vivo.This study reveals the NRP1/PKC axis as a promising therapeutic target for the prevention of neuroendocrine castration-resistant variants of PCa and indicates NRP1 as an early transitional biomarker. The mortality associated with prostate cancer (PCa) is mainly due to its progression to therapeutic-resistant metastatic disease. Androgen deprivation therapies (ADT) have improved the management of the disease; however, the vast majority of tumours ultimately acquire resistance to ADT. In most cases, this is associated with genomic alterations affecting the androgen receptor (AR) axis and apoptotic pathways . Thus, rRB1 ).p = 0.00042; Fig. Examination of the Mondor dataset for AR-regulated genes using a signature previously defined by Hieronymus et al., 2006  see Tab revealedNRP1, NRP2, AURKA, EZH2, LAMB1, NLGN1 genes .We hypothesised that some overlapping members between a defined neurogenesis signature (GO:0022008) and the Mondor dataset might provide candidates for PCa transition to an NE phenotype. Examination of the Mondor dataset against this signature revealed upregulated expression of nes Fig. , severalWe decided to focus on NRP1 in NED because it has been linked to high Gleason score and ADT  and funcTo validate it as a potential candidate, we observed that NRP1 expression was upregulated in the CRPC Mondor dataset Fig. , downregwww.pradcellatlas.com, Epithelial dataset), we found that NRP1 and other neuronal genes from the neurogenesis signature comparison  analysis of 13 tumour biopsies from 12 PCa patients, including those with luminal, basal, or proliferative phenotypes  for detason Fig.  were simTo ask about NRP1 expression after ADT, we examined the SU2C-PCF dataset (208 CRPC mRNA samples across both Adeno-CRPC and NEPC (11%), . ClusterTo better focus on NE samples only, a cohort of 39 patients (37 mRNA samples) with high NEPC and low AR scores  and upregulation of NE markers such as CHGA, neuron-specific Enolase (NSE) and β-Tubulin III  , 36. We PRKCD was identified in the Mondor CRPC upregulated genes list . Upregulated phosphorylation was also observed in PKCα, PKCδ and PKCε proteins from LNCaP-NE compared to LNCaP cells  expressed in multiple cancers and during neuronal differentiation . Examinaurs Fig. . Examinaurs Fig.  confirmells Fig. . Finallylls Fig.  left. Aslls Fig.  right.We next examined the relationship between NRP1 and PKC isotypes in NED. Cross-correlation of NRP1 and PKCD in the Mondor dataset suggested that increased transcription was tightly coupled Fig. . FurtherTo ask which PKC isoform(s) might be requisite for the NE phenotype, siRNAs were used to target PKCα, PKCδ or PKCε in LNCaP-NE cells, and NE profile markers examined by western blot. Knockdown of PKCδ resulted in a significant reduction of CHGA and SYP, suggesting a reversal of the NE phenotype Fig. .We also observed co-expression of PKCα and PKCδ and downstream target BCL2 in our SU2C/PFC NEPC NRP1 + population Fig. , confirmTogether, these results suggest that PKC expression is linked to NRP1 and that PKC might be a significant contributor to the NE phenotype.Based on the findings that PKC expression and activation are upregulated early in NED and could drive NE transdifferentiation Fig. , we postWe then evaluated the combined effect of castration and Enzastaurin on tumour growth in vivo using LNCaP ectopic xenograft model. The LNCaP model is commonly used in vitro and in vivo to model the response to ADT of PCa. Xenografted male mice were castrated by surgery to block androgen synthesis and promote an apparent LNCaP-NE phenotype as defined by drug resistance in injected LNCaP tumours. In response to castration, tumour growth in control mice remained low for two weeks, after which time tumour cells proliferated despite castration, and developed androgen-independent tumours Fig. . Enzasta50 from 1 to 4 nM  have failed to reverse NE phenotype (data not shown), suggesting that these pathways may not have a direct impact on NRP1-induced NED.Because of its limited molecular characterisation, there is no standard treatment for patients with NEPC. Although multiple pathways, including PI3K/AKT and MAPK, likely converge to drive the emergence of NE phenotype, no studies so far have demonstrated clear clinical benefits from targeting these pathways . In our Our results show that NRP1 upregulation correlates with PKC activation as determined by increased phosphorylation during ADT and that NRP1 co-immunoprecipitates with PKC, suggesting a functional link. Further, both NRP1 overexpression and knockdown studies support the notion that NRP1 upregulation during ADT is directly required for PKC activation. Work in unrelated fields has shown that NRP1 directly associates with VEGFRs for VEGF-mediated PKC induction during angiogenesis  We have Microarray data from the patient cohort (Mondor dataset) showed that PKCα and PKCδ were most highly expressed in human CRPC, and our in vitro results and examination of a scRNAseq database  confirmePKC pathway activation in response to androgen deprivation has been shown to promote resistance to AR-targeted therapy . We founFor decades, taxane-based chemotherapies have been the main treatment for metastatic CRPC. Although it prolongs overall survival for some patients, many do not respond to treatment, while others invariably develop resistance. We have previously reported that CRPC-NE cells are resistant to multiple cytotoxic agents . In our Numerous cellular pathways involving apoptosis, signalling components, drug efflux pumps and tubulin are implicated in the development of chemoresistance . Both NRWhile several aspects of therapy-resistant treatment-induced NEPC have been explored, how to therapeutically target these aggressive metastatic NE subsets remains a clinical challenge . We propOur work reveals several novel findings with implications for patients with CRPC and drug-resistant NE disease. These findings support a real promising clinical value of the NRP1/PKC-targeted axis in the treatment and prevention of therapy-resistant treatment-induced NED. NRP1 would provide an excellent biomarker of PCa progression and particularly early diagnosis of NE disease.Supplementary methodsaj-checklistNC3Rs ARRIVE Guidelines ChecklistSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure 7Supplementary Table-S1Supplementary Table-S2Supplementary Table-S3Supplementary Table-S4Supplementary Table-S5Supplementary Table-S6Supplementary Table-S7


Corona virus infectious pandemic makes outdoors rehabilitation a potential hazard. Patient education to perform simple home-based exercises seems to be an interesting and sometimes a mandatory option. This study provides a comparison between the conventional and home-based virtual rehabilitation after surgical repair of medial meniscus root tears.In this prospective study, all patients who underwent medial meniscus posterior root repair with a modified trans-tibial pull-out technique from March 2019 to March 2021 were evaluated. Those who underwent surgery after December 2019 were trained to perform self-rehabilitation. The rest had undergone outdoors specialized rehabilitation according to a unified protocol and these were used as a historical control group. All patients were followed up for a minimum of 2 year after surgery. Final Lysholm scores were utilized to compare functional outcomes after considering the effect of age, body mass index and time from surgery by multivariate linear regression analysis.p < 0.05), except the “Using cane or crutches” item (p = 0.065). Nevertheless, the final Lysholm knee score improvement was higher in patients who performed outdoors specialized rehabilitation and in patients with shorter time-to-surgery.Forty-three consecutive patients with medial meniscal root tears were studied. Thirty-nine (90.7%) were women and 4 (9.3%) were men. The mean age of participants was 53.2 ± 8.1 years. The total Lysholm knee score, and all its items were significantly improved in both groups at a two-year follow-up (Regardless of age and gender, home-based rehabilitation after meniscal root repair with the modified trans-tibial pull-out technique improved the patients’ function at a two-year follow-up. Nonetheless, this effect was still significantly lower than that of the outdoors specialized rehabilitation. Future work is required to clarify basic protocols for home-based tele-rehabilitation programs and determine clinical, radiological and functional results.Level IV, therapeutic, historically controlled study. Meniscal roots convert the axial load into hoop stress and distribute the pressure symmetrically in the articular surface . Root inThe effects of corona virus infectious disease COVID-19) pandemic on orthopedics cannot be overlooked –19. It h pandemicIn the current study, patients with medial meniscal posterior root tear (MPRT) underwent surgical repair of the tear with a modified trans-tibial pull-out technique. We sought out to determine (1) if these patients can experience significant improvement in function with home-based self-rehabilitation, and (2) if there is a significant difference in functional outcomes between the patients who are forced to perform home-based self-rehabilitation and those who have access to specialized physical therapy.This retrospective cohort studies all patients who underwent surgical repair of the MPRT from March 2019 till March 2020 in a tertiary knee center. The study protocol was reviewed and approved by the local ethics committee. The procedure was described for all patients and informed written consents were obtained. Two separate fellowship trained knee surgeons were involved who used the same surgical technique for root repair. Baseline patient characteristics and the time interval from the acute onset/exacerbation of knee pain to the surgery  were recorded. The severity of knee osteoarthritis prior to and after surgery was assessed based on Kellgren- Lawrence (K-L) classification . Patient mm Drill (Arthrex) was inserted through the reamed canal, to (1) confirm the tunnel’s position in the anatomical footprint with arthroscopy and (2) perform minimally invasive inside-out reaming of the tibial tunnel. Both ends of the loop and post constructs were retrieved through the tunnel. Tension was applied to the thread ends in 30˚ of knee flexion, and then they were fixed on the tibial cortex around a screw-washer construct. We tend to over-reduce the meniscus by tensioning the root to the point that at least 5 mm of the root enters the tunnel before final fixation.The loop-post construct technique, which was introduced in 2020, as a modification of the standard trans-tibial pull-out method of repairing meniscal root tears was used . After pth week. Passive range of motion exercises to reach 90 degrees of flexion during the first six weeks after the operation included supine wall slides and hanging the leg from the bed. Patellar mobilization exercises were started and performed by the therapist or the patient himself. Open chain quadriceps exercises were performed immediately after surgery during hospitalization period under supervision. Furthermore, partial weight-bearing exercises  started during the first two weeks, with the brace locked in extension. Full weight-bearing was permitted after six weeks.Rehabilitation protocols were adjusted from Mueller et al. meniscus root rehabilitation concepts . The knePostoperative rehabilitation was performed either as an outdoors specialized rehabilitation or by the patients themselves as a home-based self-rehabilitation due to the force of isolation after the COVID-19 outbreak. Self- rehabilitation at home included training of the patients to perform straight leg raising, range of motion and patellar mobilization exercises. Patient education was performed by the surgeon and the physical therapist before discharge from the hospital. After being discharged, virtual education and virtual follow-up with the physical therapist using social media, mostly Whatsapp  helped to ensure proper adherence to the instructions and patient progressions during this process were supervised by both the surgeon and the physical therapist. The virtual postoperative follow ups were scheduled weekly until 6 weeks or until achievement of 90’ flexion and full weight bearing; whichever happened sooner. After which the patient was followed virtually at 3 months postoperative.Meanwhile, outdoors specialized rehabilitation was performed by a trained physical therapist with a uniform protocol including range of motion exercises, vastus medialis strengthening, patellar mobilization, open-chain quadriceps isometric exercises, hamstring stretching and pain reduction modalities.Statistical analysis was applied by R programming language (version 3.3.1 for Mac OS) with deducer graphical user interface (GUI) package, and the results were visualized by GraphPad Prism (version 8.2.1 for Mac OS). Quantitative and qualitative variables were described using mean ± standard deviation (SD) or median and frequency (percentage), respectively. The primary objective was to compare the baseline and the two-year post-surgical total Lysholm knee score and its domains. The effect of demographic, clinical variables and type of rehabilitation on Lysholm Knee Score change (Δ LKS) was analyzed using the Wilcoxon rank-sum test. Comparing means for normally distributed variables was performed by paired t test. Correlations were test by the Mann–Whitney test. A p-value < 0.05 was considered as statistically significant. Multivariate linear regression analysis was performed to detect the effect of age, BMI, time from surgery and type of rehabilitation on final LKS.p = 0.065) , leaving 43 patients who participated in this study. Patient characteristics and clinical outcomes are provided separately Tables  and 2. A+rehabilitation = 20 ± 9.13 vs, Δ LKS -rehabilitation = 12.55 ± 5.01; p = 0.012)  = -0.7264, -0.2026; p = 0.002). Patient’s age  and sex , on the other hand, did not show any significant correlation with final Lysholm knee scores.The increase in the final Lysholm knee score  was higher in patients in the non-COVID era (Δ LKS P = 0.008).Multivariate analysis Table  showed tDuring the last 15 years, techniques for surgical repair of MPRT have been developed to restore joint biomechanics and joint contact pressures, to prevent the joint from early osteoarthritis. Trans-tibial pull-out repair is one of the most common procedures for meniscal root repair . The traIncluded patients were mostly females at their mid-fifties. Patients with a stable knee and no sign of severe osteoarthritis or major malalignment were scheduled for surgical repair of the MPRT. This demographic pattern and surgical indications are in consistency with previous reports , 29. ConNo correlation was seen between demographic characteristics such as age or gender and functional outcomes after root repair. This finding is interesting since we expected to see variable results of self-rehabilitation between different age/sex groups, due to different levels of compliance. Frankly this study involved a limited age range, which might have masked this effect. Besides, previous studies such as Laprade et al.  reportedTime interval from the onset/ exacerbation of knee pain to surgery was significantly correlated with clinical outcomes. As a result, assigning meniscal root repair methods as soon as possible amongst eligible patients might improve the outcomes of the surgery, a notion which has been stated in the previous reports .All patient undergoing root repair in our centers were started on a specialized rehabilitation program with a unified protocol in the non-COVID era. Outbreak of the SARS-COV-2 infection in December 2019 and isolation protocols prevented the patients from participating in such programs. Most patients were afraid to perform outdoors rehabilitation and were unable to afford home-based private physical therapy either. Therefore, we were forced to train the patients to perform simple rehabilitation tasks at home.Our results show significant improvement in patient reported outcomes in both non-COVID and COVID era patients. Fortunately, no case of limited knee range of motion was encountered in neither group. Still and all, those who had access to specialized physical therapy (the non-COVID era group) experienced significantly better outcomes. While no study was found in the literature after the COVID-19 outbreak to take this matter into consideration, some previous reports have considered a comparison between restricted and accelerated rehabilitation –35. VandBefore jumping into any conclusions, one must consider some serious limitations of this study. We did not use visual analogue scale for pain, however we reported Lysholm scores which show the level of pain patients encounter during everyday activities. Lack of MRI evaluation and a follow-up of two years are two important limitations of this study. The COVID-19 outbreak has only begun since two to three years ago; therefore, follow-up period could not be any longer and our sample size is relatively small. Even so, we felt compelled to share our concerns and results. Indeed, this seems to be a global on-going problem which might deeply affect not only our routine clinical practice, but also our rehabilitation protocols and postoperative care. Due to a lack of control subjects, we compared the results with historical controls who had completed their postoperative rehabilitation before the start of the pandemic. Another limitation, is the modification of the standard trans-tibial pull-out technique that we used to repair root tears . No biomIn summary, the results reveal that regardless of age and gender, patients can reach significant functional improvements even with home-based simple rehabilitation tasks after arthroscopic repair of MPRT. Nonetheless, better outcomes were associated with postoperative specialized rehabilitation programs and earlier surgery. Future work is required to clarify basic protocols for home-based tele-rehabilitation programs and determine clinical, radiological and functional results.


Acute allograft rejection (AR) following renal transplantation contributes to chronic rejection and allograft dysfunction. The current diagnosis of AR remains dependent on renal allograft biopsy which cannot immediately detect renal allograft injury in the presence of AR. In this study, sensitive biomarkers for AR diagnosis were investigated and developed to protect renal function.We analyzed pre- and postoperative data from five databases combined with our own data to identify the key differently expressed genes (DEGs). Furthermore, we performed a bioinformatics analysis to determine the immune characteristics of DEGs. The expression of key DEGs was further confirmed using the real-time quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and immunohistochemical (IHC) staining in patients with AR. ROC curves analysis was used to estimate the performance of key DEGs in the early diagnosis of AR.We identified glutamic-oxaloacetic transaminase 2 (GOT2) and syntaxin binding protein 3 (STXBP3) as key DEGs. The higher expression of STXBP3 and GOT2 in patients with AR was confirmed using RT-qPCR, ELISA, and IHC staining. ROC curve analysis also showed favorable values of STXBP3 and GOT2 for the diagnosis of early stage AR.STXBP3 and GOT2 could reflect the immunological status of patients with AR and have strong potential for the diagnosis of early-stage AR. An increasing number of novel plasma and urinary biomarkers have been explored for monitoring AR –27, inclIn this study, RNA-seq analysis of peripheral blood samples obtained before and after surgery was used to detect the dynamic changes in the pre- and postoperative DEGs. The combination of five online gene expression microarray datasets and our center gene expression mRNA-Seq analysis was utilized to filter out pre- and postoperative DEGs. Bioinformatics analysis and the xCell website were used to further screen the genes associated with immune response and map these selected genes to the corresponding immune cells. Then, the expression of selected genes at transcriptional and translational levels was determined using real-time quantitative real-time PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and immunohistochemical (IHC) staining. Two promising genes were identified, namelySTXBP3 and GOT2, through continuous screening of the key gene set. Subsequently, ROC curve analysis was applied to validate the AR early diagnostic value of STXBP3 and GOT2 in the serum of patients before undergoing kidney transplantation. We demonstrated that STXBP3 and GOT2 are sensitive biomarkers for early AR diagnosis.2 Materials and methods2.1 Data collectionThe study recruited 42 patients who underwent on renal transplantation between 2018-01-01 and 2019-01-31 due to ESRD in the Kidney Disease Center of the First Affiliated Hospital, College of Medicine, Zhejiang University, which included 23 patients with AR episodes and 19 patients without AR episodes (NAR). The inclusion criteria were ESRD patients aged>18 years who had received conventional treatment for at least 3 months. Exclusion criteria included acute kidney injury, active inflammatory or malignant disease, pregnancy, and inability to provide informed consent. Kidney biopsies were performed for all patients. The Banff criteria were used to define acute rejection based on kidney biopsy data. Peripheral blood mononuclear cells (PBMCs) from allograft recipients were stored at -80°C until used. Informed consent was obtained from all patients enrolled in this study.2.2 RNA sequencinghttps://intro2r.com/citing-r.html) was used to conduct log2 conversion, quantile normalization, and experimental batch correction on the read counts in comparison with the transcription levels among the samples  was used to obtain the total RNA (1000 ng) of PBMCs. The concentration and purity of the extracted RNA were determined using a NanoDrop2000 spectrophotometer . RNA integrity was confirmed using the 2100 Bioanalyzer . Samples with an RNA integrity number (RIN) > 8 were screened for RNA sequencing. mRNA-sequencing was performed using Illumina HiSeq X-ten according to the manufacturers' protocols. The ComBat R package ( samples . The fra samples . Genes w2.3 Identification of DEGs involved in acute rejectionwww.ncbi.nlm.nih.gov/geo/) with accession numbers GSE112927, GSE120396, GSE120649, GSE131179 and GSE145503. Additionally, we included mRNA-Seq-based gene expression data from our cohort of 42 patients in this study. The six data sets included two pretransplant  and four post-transplant  datasets.The study design is illustrated in The six datasets included in this study were generated using different sequencing platforms such as Illumina HiSeq - 4000, 2000, 2500, 2500 and NextSeq 500. DEG analysis was performed using LIMMA R package .http://bioinformatics.psb.ugent.be/webtools/Venn/) were used to represent the intersecting DEGs among the six datasets (https://xcell.ucsf.edu/)  . FinallyBioJupies was used to generate heatmap, and perform enrichment analysis of STXBP3, GOT2, and MAP4K5. Statistical significance was set at p < 0.05 .2.4 DEG validation using RT-qPCR, ELISA, and IHC stainingThe expression of 15 DEGs (kinesin family member 3B (KIF3B); fumarate hydratase (FH); eukaryotic translation initiation factor 4 gamma 1 (EIF4G1); SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 1 (SMARCD1); integrin subunit alpha L (ITGAL); heterogeneous nuclear ribonucleoprotein U like 1 (HNRNPUL1); mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5); elongator acetyltransferase complex subunit 3 (ELP3); advillin (AVIL); heterogeneous nuclear ribonucleoprotein L (HNRNPL); pre-mRNA processing factor 19 (PRPF19); glutamic-oxaloacetic transaminase 2 (GOT2); syntaxin binding protein 3 (STXBP3); chloride intracellular channel 3 (CLIC3); and protein phosphatase, Mg2+/Mn2+ dependent 1G (PPM1G)) were found to significant when RT-qPCR was performed. RNA was first extracted from each sample using QIAamp MinElute Virus Spin kits followed by quantification using a NanoDrop2000 spectrophotometer and BioAnalyzer. cDNA was synthesized from 10 μL of RNA using the PrimeScriptTM RT reagent kit . The subsequent RT–qPCR screening of the enrolled subjects for the 15 markers was performed using the primers listed in 2.5 Statistical analysisStatistical analyses were performed using R version 4.0.4, SPSS version 26 (IBM), and GraphPad Prism 8.4.2. The AR diagnostic values for STXBP3, GOT2, and MAP4K5 were assessed using area under the ROC curve (AUC) analysis. Statistical significance was set at p < 0.05.3 Results3.1 Identification of key DEGsAdetailed flow chart of the selection process for key DEGs is shown in 3.2 Clinical and demographic characteristics of patients with ARp=0.042) and GFR was decreased (p=0.019) in the AR group. No statistically significant differences in other clinical indicators were observed between the two groups.Primary patient demographics and clinical features are shown in 3.3 RNA-Seq and DEG analysisIn our mRNA-Seq dataset, 975 genes were identified to be differentially expressed in AR compared to NAR patients based on the selection criteria of log2|FC| > 1.5 and p < 0.05 for the identification of DEGs. A total of 776 upregulated and 199 downregulated genes were identified among these 975 genes. The same method was used to identify 3084 DEGs in GES112927, 1306 DEGs in GES120396, 1205 DEGs in GES 120649, 3384 DEGs in GES 131179 and 1352 DEGs in GES 145503. The specific biological functions or pathways which these DEGs affect or are involved in are presented in the GO and pathway enrichment analyses. These genes were enriched in inflammatory and immune-related signaling pathways , B cells, and T cells and enhanced in inflammatory fibroblasts in patients with ulcerative colitis (UC) . STXBP3 The application of STXBP3 and GOT2, with two clinical indics donor age and eGFR, may facilitate the evaluation of AR risk before kidney transplantation. Monitoring the dynamics change in AR-related genes helped predict thepostoperative risks of AR episodes.Our study has several novel aspects that offer clues for diagnosing AR early. The dynamic trends of DEGs were compared before renal transplantation and during the AR episode, dynamical monitoring of the expression changes of AR-related genes in the peripheral blood is expected to distinguishAR episodes and prompt interventions in a timely manner. The subsequent bioinformatics analysis and xCell online tool correlated gene expression profiles and functions with specific types of immune cells; therefore, critical immune cell populations of the AR process were identified, which plays a similar role to that of single-cell/single-nuclei sequencing. Additionally, the data in our study for the screening of DEGs related to AR came from multiple centers which makes these results relatively objective. However, this study had several limitations. First, we had a few samples available for clinical validation owing to the limited accessibility of the positive specimens. The precise mechanisms of STXBP3 and GOT2 have not been explored in acute renal transplant rejection. Therefore, adequate clinical cases should be further evaluated to validate the results in this study.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Kidney Disease Center of the First Affiliated Hospital, College of Medicine, Zhejiang University. The patients/participants provided their written informed consent to participate in this study.QY performed the data curation and wrote the manuscript. WX collected the clinical data and analyzed the data. HJ designed the study and experiments. CW and YW conducted the experiments. YC and QZ interpreted the results. DC and JC supervised the manuscripts and experiments. All authors contributed to the article and approved the submitted version.This work was funded by NSFC81970651, U21A20350, Sino-German CENTER GZ1572, and NSF81802085.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.


Anion exchanger 1  is a major membrane protein of red blood cells and plays a key role in acid-base homeostasis, urine acidification, red blood cell shape regulation, and removal of carbon dioxide during respiration. Though structures of the transmembrane domain (TMD) of three SLC4 transporters, including AE1, have been resolved previously in their outward-facing (OF) state, no mammalian SLC4 structure has been reported in the inward-facing (IF) conformation. Here we present the cryoEM structures of full-length bovine AE1 with its TMD captured in both IF and OF conformations. Remarkably, both IF-IF homodimers and IF-OF heterodimers were detected. The IF structures feature downward movement in the core domain with significant unexpected elongation of TM11. Molecular modeling and structure guided mutagenesis confirmed the functional significance of residues involved in TM11 elongation. Our data provide direct evidence for an elevator-like mechanism of ion transport by an SLC4 family member. CryoEM structures and computational modeling of bovine anion exchanger 1 in both inward and outward facing conformations reveal an elevator-like mechanism of ion transport by a solute carrier ion transport family 4 member. AE1 (SLC4A1) constitutes ~30% of the membrane mass of the red blood cell, amounting to ~106 AE1 molecules per cell2. Carbon dioxide generated by metabolic processes in the tissues diffuses into the red blood cells where it reacts with water to form HCO3‾ and protons in a reversible process catalyzed by carbonic anhydrase II2. As the HCO3‾ concentration in the erythrocyte increases, AE1 mediates the electroneutral exchange of Cl− for HCO3− at the extremely fast exchange rate of ~50,000 ions per second3. Thus, ~90% of the CO2 is taken from the tissues to the lungs as the more soluble form, HCO3−. In the lungs, the process is reversed and CO2 is exhaled4. AE1 also plays an essential role in the acidification of the urine in the collecting duct5. Inherited mutations in AE1 cause anemia with red cell morphological abnormalities as well as distal renal tubular acidosis6.AE1, a member of the solute carrier ion transport family 4 (SLC4), mediates the exchange of HCO2. The CD plays an important role in cytoskeletal attachment and binding to various proteins, e.g., ankyrin, band 4.2 and band 4.1 proteins, glycolytic enzymes, hemoglobin, deoxyhemoglobin, hemichromes, p72syk protein tyrosine kinase, glycophorin A, adducin and integrin-linked kinase, and is proposed to play a key role in mediating red blood cell elasticity8. The TMD, which mediates anion exchange10, has a 7 + 7 inverted repeat fold and is subdivided into core and gate domains similar to the human electrogenic sodium bicarbonate cotransporter 1 11, the rat sodium dependent chloride carbonate exchanger 12, the plant boron transporter Bor113 homologous to the mammalian H+/NH3 transporter SLC4A11, the fungus purine symporter UapA14, the bacterial uracil transporter UraA15, the bacterial proton-coupled fumarate symporter SLC26Dg16, and the murine Cl− transporter Slc26a917. The crystal structures of the human AE1 CD expressed in bacteria, and the TMD in the OF conformation purified from human erythrocyte membranes, as well as low-resolution negative stain EM structures of full-length mammalian AE1 were previously reported9. Preliminary cryoEM full-length human AE1 structures in apo-, and holo-forms with substrates/inhibitors reported TMD structures solved in the OF conformation18. The structures of the membrane domain of other mammalian SLC4 transporters, NBCe111 and NDCBE12, were also resolved only in the OF state. Thus far, no 7 + 7 inverted repeat fold transporter has been resolved in both IF and OF state precluding the analysis of their underlying transport mechanisms. Given that AE1, one of the fastest ion exchangers19, is of key importance in mammalian biology and general studies of secondary transport by proteins, understanding of its transport mechanism is a priority and requires resolution of its IF structure.Mammalian erythrocyte AE1 is a ~100 kD integral transmembrane glycoprotein consisting of an N-terminal cytoplasmic domain  and a C-terminal trans-membrane domain Here, by single-particle cryoEM, we determined the structures of the TMD of bovine AE1 at near-atomic resolution and captured both IF and OF conformations. Heterodimers (OF–IF) of bovine AE1 were detected in addition to homodimers. The IF state features an elongated TM11, which incorporates residues from intracellular loop 5 (IL5) between TMs 10 and 11, and partially unfolded TM10. The most significant conformational change occurs in the core domain, which combines a downward movement with small lateral displacement and a slight rotation respective to the gate domain in the OF to IF transition. The data supports an elevator-like transport mechanism combined with rotational movement of the ion coordination site and reorganization of IL5 between β-hairpin and α-helical form as it incorporates into TM11. Computational modeling demonstrates that the IF cavity is well hydrated and that anions tend to accumulate at the interface between the two monomers at the intracellular side of AE1 from where they can access laterally the IF cavity.8, was ~98% purity and predominantly dimeric  of bovine AE1 . The TMD can be separated into gate  and core domain  31 calculations of bAE1 monomers in IF and OF state and multiple 1 µs MD simulations of the symmetric (IF–IF) and asymmetric (IF–OF) dimers, embedded in a POPC bilayer and in the presence of a 0.75 M NaCl + 0.75 M NaHCO3 solution. Figure 27. In the IF state, the anions from the intracellular solution accumulate at the entry of the permeation cavity  transported by AE1, we evaluated ion densities from 1 µs MD simulations of the bAE1 dimers. The bicarbonate densities in the asymmetric (IF–OF) dimers (cyan iso-surface) are mapped in Fig. − maps indicate Cl− presence in the same areas  in agreement with the SILCS maps , R760C and S762C (R778C and S780C in bovine AE1) mutants demonstrated a greater impairment of transport (<10% activity). Human AE1 mutations at residues R760, and S762 have been previously associated with hereditary stomatocytosis and spherocytosis1. The location of these residues in the TM11/IL5 scaffold of bovine AE1 is shown in Fig. Mutagenesis studies of several residues of human AE1 from TM11 and IL5, the protein area involved in TM11 elongation and β-hairpin to α-helix transition, demonstrate the functional importance of this region Fig. . Althoug33. Transporters with the same fold as AE1 have been resolved as both monomeric and dimeric 34 or solely dimeric14. The available SLC4 structures  have been resolved as dimers12. SLC26Dg is dimeric in proteoliposomes but monomeric in the presence of maltoside detergents16. We have previously hypothesized that the increased interaction of two gate domains in the dimeric structures might confer a greater transporter stability in the plane of the membrane during the transport cycle11.Our cryoEM data demonstrated that AE1 is a homodimer confirming previous biochemical crosslinking and hydrodynamic studies36. The resolved structures demonstrate the existence of asymmetric AE1 dimers with one IF and one OF monomer. In this way, AE1 resembles the prokaryotic glutamate transporter homolog GltPh that has three monomers, which can adopt different conformations37.Our data is an additional example of the ability of cryoEM to sort particles from a heterogenous dataset8. This hypothesis is supported by the near atomic resolution data obtained in this study showing large changes in the position of the CD relative to the TMD and the recent characterization of AE1 complexes with ankyrin and protein 4.2 by cryoEM39.We have previously demonstrated that there is a flexible linker between CD and TMD in the AE1 monomer that may play a significant role in the red blood cell elasticity, providing a mechanism to connect the cytoskeleton (given that AE1 CD binds various cytoplasmic proteins) with the red blood cell membrane40. Ions diffuse to the protein center aided by the presence of a number of charged residues lining the cavities , are blocked by TM3 in the IF bovine AE1 and occluded Bor1 structures and are opened only when TM3 moves upwards and away with respect to the gate (as seen in the OF bovine AE1 structure). The IF cavity and the access to the N-termini of TM3 and TM10 is blocked by TM10 in the OF bovine AE1 and (to some extent) in the occluded Bor1 state. TM10 must move downwards and away with respect to the gate (as seen in the IF bovine AE1 structure) for the IF cavity to open. The elevator-like motion of the core allows the protein center, where key binding residues such as R748 and E699 (R740 and E681 respectively in human AE1) are located, to be consecutively exposed to the extracellular and intracellular solutions , which does not indicate complex gating rearrangement in this domain, are typically observed in elevator transporters44. The vertical shift of the core ion coordinating residues during the AE1 OF-IF transition is ~ 5 Å, which is similar to the coordination site shift observed in some elevator transporters44. The more modest protein reorganization during the elevator-like motion of the core might be advantageous in contributing to the observed high rate of transport by AE1 .Comparison of the OF and IF conformations reveals significant changes in the position and shape of the core region Figs.  and 9 in45, activation of the bacterial thermosensor DesK that involves elongation of its transmembrane helix46, and T cell receptor major histocompatibility complexes (MHC) interaction that involves MHC alpha helices conformational changes47. Folding/unfolding dynamics of α-helixes plays an important role in conformational transitions of proteins. More specifically, the α-helix to β-hairpin transformation has been observed and characterized in various peptides related to neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington’s disease as well as prion diseases like Creutzfeldt-Jacob’s disease and other spongiform encephalopathies49. Experimental and computational studies of model polypeptides, such as polyalanine, polylysine, and polyglutamate have demonstrated the thermodynamic underpinnings of this transition and its dependence on environmental factors, such as temperature, pH, and salt concentrations51. In the context of the SLC4 family, a switch between two meta-stable states (α-helix and β-hairpin) of the IL5 might facilitate and accelerate the observed OF to IF transition, with unusual TM11 elongation. In the available structures of SLC4 proteins in the OF state 12, IL5 is fully resolved only in NDCBE. In all structures, although antiparallel β-hairpin conformation is not explicitly assigned due to the somewhat disordered organization of this region  or stomatocytosis 12. AE1 is bidirectional (HCO3− import coupled with Cl− export or Cl− import coupled with HCO3− export), depending on the existing ion gradients. Both substrate ions likely bind at the same residues at the protein center as suggested by previous SILCS and MD simulations27. In the OF state, the binding of the substrate ion at the ion coordination site allows the transformation of the OF to the IF state, which includes the elongation of TM11 and the rearrangement of the β-hairpin of IL5. In the IF conformation, the elongated α-helical TM11 is unstable due to the incorporation of charged and polar residues. In addition, the potential accumulation of anions at the dimeric interface allows for their swift lateral entry into the IF cavity. This likely accelerates the IF to OF transition, during which the α-helical structure of TM11 quickly unfolds and reforms into a β-hairpin with IL5, once the required substrate ion binds to the IF state   or folds into an α-helix leading to elongation of TM11 (in the IF state). The transported anions permeate vertically from the extracellular solution into the OF cavity in the OF state and accumulate at the dimeric interface at the intracellular side from where they can access laterally the IF cavity in the IF state. The modest protein reorganization during transport, the oscillation between two meta-stable states of IL5–TM11 and the easy anion access into the IF and OF cavities of AE1 may explain the observed high transport rates of this protein.8. AE1 was extracted from the ghosts with dodecyl maltoside and purified using our method8 except a 5 ml HiTrap ANX FF column  was used instead of a 3.5 cm DE-52 DEAE column (Whatman).Bovine AE1 was purified from red blood cell ghosts isolated from defibrinated bovine blood (Quad Five) as we previously reportedg for 5 min). Amphipol containing protein was further purified on a Superose 6 column in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl. The peak corresponding to dimeric AE1 was used for cryoEM analysis.For preparation of samples for cryoEM, AE1 was mixed with amphipol PMAL-C8 (Affymetrix) at 1:3 (w/w) dilution with gentle agitation overnight at 4 °C. Detergent was removed with Bio-Beads SM-2 (Bio-Rad) incubated with samples for 1 h at 4 °C, and the beads were subsequently removed by centrifugation at 2000 27. The cells were bathed initially in the absence of Na+ in a Cl‾-containing bicarbonate-containing solution: 115 mM tetramethylammonium chloride, 2.5 mM K2HPO4, 1 mM CaCl2, 1 mM MgCl2, 24 mM tetramethylammonium bicarbonate, 5% CO2, pH 7.4, and 30 µM 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). After a steady state, AE1 mediated transport was induced by switching to the following Cl−-free solution: 115 mM tetramethylammonium hydroxide, 115 mM gluconic acid lactone, 2.5 mM K2HPO4, 7.5 mM calcium gluconate, 1 mM magnesium gluconate, 24 mM tetramethylammonium bicarbonate, 5% CO2, pH 7.4, and 30 µM 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). The total cell buffer capacity (intrinsic (βi) plus bicarbonate (27. The rate of change of pHi (dpHi dt-1) was measured in the initial 10–15 s after a bath solution switch and converted to the rate of change of [H+in] (d[H+in] dt-1). The H+ flux (mM s−1) for each construct was calculated as (βi + βHCO3) × (d[H+in]·dt-1).HEK293 cells were transfected with wt-human AE1 cloned into a pcDNA3.1(+) expression vector, the empty vector, and specific human AE1 TM11 mutant constructs. Twenty-four hours later, the transport assays were performed. Twenty-four hour following transient transfection, pHi was measured with the various constructs in HEK293 cells grown on PEI coated coverslips as describedg, 4 °C). The supernatant containing >90% of the plasma membrane protein fraction was collected and incubated on a rotating shaker (4 °C for 4 h) with streptavidin-agarose resin (50 μl) (Thermo Fisher Scientific). To elute the bound proteins, the resin was pelleted and washed with the lysis buffer (60 °C for 5 min) with 2 × SDS buffer containing 2% 2-mercaptoethanol .Plasma membrane proteins were labeled and pulled down with Sulfo-NHS-SS-biotin. In this protocol the cells were washed with PBS  24 h following transfection with various constructs. The cells were incubated  with 1.1 mM sulfo-NHS-SS-biotin (Thermo Fisher Scientific). The reaction was then stopped using 50 mM Tris buffer at 4 °C . The cells were collected and washed with PBS, and lysed on ice in 150 mM NaCl, 0.5% sodium deoxycholate (Thermo Fisher Scientific), 1% (vol/vol) Igepal (Sigma-Aldrich), 10 mM Tris·HCl, 5 mM EDTA (Sigma-Aldrich), pH 7.5, with protease inhibitors (Roche Life Sciences). The insoluble material was pelleted over 10-min , 1% (vol/vol) Igepal (Sigma-Aldrich), 10 mM Tris·HCl, 5 mM EDTA (Sigma-Aldrich), pH 7.5). The human AE1 constructs were pulled down using the AE1 2-M anti-human monoclonal antibody (Alpha Diagnostics) .The sample proteins were resolved using 7.5% polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. The expression levels of the pulled-down biotinylated proteins and whole cell lysates were determined by probing the blots with the mouse monoclonal AE1 2-M antibody  in TBSTM buffer (0.1% (vol/vol) Tween 20; 137 mM NaCl, 20 mM Tris, pH 7.5), containing 5% (wt/vol) nonfat milk). After 1 h incubation (room temperature), the blots were washed with TBST and then probed with Peroxidase AffiniPure Donkey Anti-Mouse IgG (H + L)  at 1:10,000 dilution in TBSTM buffer and incubated at room temperature for 1 h. The blots were washed with TBST and signals were detected with ECL Western Blotting Detection Reagent .−1) was applied to a glow-discharged EM grid covered with a thin layer of carbon film. After 10-s incubation, the grid was stained with 0.8% uranyl formate. For cryoEM, 3 μl of bovine AE1 (~0.4 mg ml−1) was applied to a glow-discharged Quantifoil 300-mesh R1.2/1.3 grid. The grid was blotted with filter paper to remove excess sample and flash-frozen in liquid ethane with FEI Vitrobot Mark IV.For electron microscopy of negatively stained protein, 2 µl of bovine AE1  were acquired with a Gatan K2 Summit direct electron detection camera operated in the super-resolution mode  at a calibrated magnification of 36,764 and defocus values ranging from −1.4 to −3.2 μm. A GIF Quantum LS Imaging Filter (Gatan) was installed between the electron microscope and the K2 camera with the energy filter (slit) set to 20 eV. The dose rate on the camera was set to ~8 e− pixel−1 s−1 and the total exposure time was 12 s fractionated into 48 frames of images with 0.25 s exposure time for each frame. Total of 3378 micrographs were collected.The frozen-hydrated grids were loaded into a FEI Titan Krios electron microscope operated at 300 kV for automated image acquisition with Leginon21. The local motion within a micrograph was corrected using 5 × 5 patches. Two average images, with and without dose-weighting, from all except the first frame were generated with 2× binning  for further data processing. A total number of 6,392 good micrographs were picked for image processing by visual inspection of the average images and power spectra after the drift correction.The frame images of each micrograph were aligned and averaged for correction of beam-induced drift using MotionCor259. The dose-weighted average images were used for particle picking and subsequent image processing. A total of 2,635,578 particles were automatically picked using Gautomatch60 and windowed out in 192 × 192 pixels. Boxed particles were first subjected to 3D classifications by GPU-accelerated RELION-262, using an oval-shaped disk low-pass filtered to 60 Å as the initial model. The particles were separated into 5 classes for 34 iterations with C2 symmetry applied and the best class contained 1,115,672 particles, which were then sent to a second run of 3D classification to be sorted into 5 classes with a soft mask of the TM region applied. A number of 592,176 particles were found in the best classes after 166 iterations of the second run of 3D classification. The particles were selected and sent to 2D classification using RELION and sorted into 100 classes for 30 iterations. A total number of 405,042 particles that show good features were selected and sent to 3D classification without mask and symmetry applied. Three classes showed well resolved transmembrane helices with or without cytoplasmic domain (CD). Class 2, class 3 and class 5 with different states of CD were refined separately at first.The defocus values of the micrographs were measured on the dose-unweighted average images by CTFFIND463 using two cryoEM maps to independently refine from halves of the data.Class 5 showed one monomer in IF state and the other in OF state. 87,780 particles were selected and refined to a resolution of 6.9 Å. Class 3 with CD showed both monomers in IF state. 90,818 particles were selected and refined to 6.3 Å. Class 2 without CD and class 3 with CD featured both monomers in IF states. 251,871 particles were selected and combined from class 2 and 3. The 251,871 particles were refined using a soft mask of TM region and the resolution was estimated to be 4.4 Å by RELION-2 using the “gold-standard” FSC at 0.143 criterion. The final cryoEM map was sharpened with B-factor and low-pass filtered to the stated resolution using RELION-2. The local resolution was calculated by ResMap64. Aromatic residues were clearly visible in transmembrane helices (TMs) 1–9, TMs 11–14 and were used as landmarks for the following model building. The crystal structure of human AE19 (PDB code: 4YZF) was used as initial model. First, the crystal structure was rigid fitted into the 4.4 Å cryoEM map in UCSF Chimera65. The molecular dynamics flexible fitting (MDFF)66 method was used to flexibly fit the atomic structure into the cryoEM density map in VMD. The fitted structure was inspected in Coot and residue assignment was corrected based on landmarks of aromatic residues. The loops between TMs were similarly refined in Coot without the α-helix restraints. The loop between TM8 and TM9 (residues 690 to 701), TM10 together with the loop connecting TM10 and TM11 (residues 730 to 756) were not built because of their flexibility. The atomic model of AE1 TMD was subjected to further global refinement with simulated annealing using the real space refinement feature (RSR) in the PHENIX software package67. Secondary structure and geometry restraints were applied to prevent structure over-fitting. Residues with poor rotamer or considered a Ramachandran outlier were fixed in Coot. Another round of RSR was performed and MolProbity analysis68 was used to validate the final model. The OF structure was built similar to the approach described above. The CD was not built because of insufficient resolution. The same approach was used to build the TMD of the full-length IF-IF structure. The CD of full-length IF-IF was built by flexibly fitting and editing the crystal structure of the cytoplasmic domain of human AE125 (PDB code 4KY9). The model was further refined using RSR and validated by MolProbity analysis.The overall IF 4.4 Å cryoEM map was inspected in Coot31 makes use of a combination of Monte Carlo and MD simulations in order to “flood” a protein with high concentration of small organic fragments for identification of areas within the protein matrix where the fragments tend to bind, assessed by grid free energy (GFE) maps. SILCS has been used previously for identification of ion pathways and binding sites in SLC4 proteins such as human AE1, human NBCe1, and rat NDCBE27. Detailed protocol of the SILCS simulations can be found in Ref. 69. SILCS Software 31 was utilized in order to construct the grid 3D-free energy based maps (GFE maps) that describe plausible high affinity regions of the bovine AE1 transporter which attract chemically different ions/molecules.The Site Identification by ligand Competitive Saturation (SILCS) method70. First, systems were energy minimized and equilibrated with a six-step scheme where constraints imposed on protein backbone, sidechain and lipid heavy atoms were gradually relaxed. Then, 5 ns MD simulations were run with 50.208 kJ·mol−1·nm‒2 harmonic force constants on the protein Cα atoms followed by 25 steps of Grand Canonical Monte Carlo (GCMC) simulations where water and probe molecules were inserted, deleted, translated or rotated following Metropolis criteria69. Afterwards, 100 steps of hybrid GCMC/MD simulations were done with an MD step between the GCMC steps to enhance the conformational sampling. Considering the average of the simulation data generated from the 10 discrete simulations, certain solute atoms were binned onto a grid of 1 Å3 voxel followed by a Boltzmann-based transformation to yield the GFE maps representing the affinity sites of the proteins targeted by the probe solutes71. The convergence of the simulations was evaluated by an overlap coefficient value which was found to be 0.70 in all cases, indicative of appropriate sampling.SILCS simulations consist of a series of calculations initiated with embedding the protein in a 120:120 Å POPC membrane bilayer with a 9:1 lipid:cholesterol mixture, followed by solvation of the systems with TIP3 water (molarity of ~55 M) and eight different fragments , randomly. 10 discrete systems were prepared as described and a series of simulations of each of them was started in conjunction with the GROMACS (v. 2018) simulation package72 and CGenFF73 force fields, CGenFF Program74 and LINCS algorithm were used with a 2-fs time step. Verlet cut-off scheme and Particle Mesh Ewald techniques were used to handle the Lennard-Jones and Coloumbic interactions. The MD protocol of GCMC/MD consists of 5,000 steps of energy minimization with steepest descent followed by a 100 ps equilibration and a 1 ns production run in NPT ensemble coupled with Nose-Hoover and semi-isotropic Parrinello-Rahman temperature and pressure controlling schemes, respectively. A total simulation time of 1 μs (10.1 ns·100 steps) was achieved at 300 K and 1 bar.Throughout the simulations, CHARMM3675 was applied for the refinement of the cryoEM structures of bovine AE1. A preliminary bovine AE1 structure with included H1 helix was used for the modeling studies. The position of the H1 helix in bovine AE1 was modeled after its position in human AE19 and further optimized using the cryoEM maps as described below. To couple the structures with their corresponding cryoEM density maps, MDFF Module in CHARMM-GUI75 was used for the generation of three-step protocol input files where coupling strength factor values were gradually increased as 0.3, 0.5, and 0.7. Each step was run for 1 ns. Secondary structures, cis-peptide bonds and chirality restraints were used in the fitting simulations. Simulations were done with NAMD 2.1376 at 303.15 K, controlled with Langevin dynamics, and the CHARMM36m force field77 and 1 fs time step. 12 Å and 10 Å distances were applied for the cut-off and switching for the non-bonded interactions. Refined structures obtained from the MDFF simulations were used for the preparation of the membrane bilayer systems and further MD simulations, herein.Prior to the MD simulations, MDFF method3 mixture with 20 Å water layers on both sides of the membrane. Additional details for the simulation systems are provided in Supplementary Table 78. 20 ns long unconstraint production runs in semi-iso-thermal-isobaric (NPaT) conditions, at 310.15 K and 1 atm, were then performed with NAMD 2.1376 using the CHARMM36 force field  and the available CGenFF parameters for the HCO3‾ ions77. Long-range electrostatic interactions were evaluated with the Particle Mesh Ewald method with cutoffs of 12 and 10 Å for the electrostatic and non-bonded interactions, respectively. Three replicas per dimer, with three different relaxed structures extracted from the 20 ns MD runs were prepared and submitted for 1 µs long all-atom MD simulations with the Anton2 supercomputer and the Anton2 software version 1.31.0 from D.E. Shaw Research in an NPT ensemble at 1 bar and 303.15 K (controlled with an MTK barostat coupled to the Nosé-Hoover thermostat)27. The nonbonded interactions were computed with the RESPA multiple-time-step algorithm. Temperature and semi-isotropic pressure coupling were performed with the multi-integrator (multigrator) algorithm and the long-range electrostatics interactions were evaluated with the available u-series algorithm. Wrapping centering alignment and analysis of the produced trajectories was done with VMD 1.9.379. The first 50 ns of the 1 µs trajectories were removed from the analysis. The VolMap tool of VMD 1.9.3 was used for generation of the density maps. The reported density maps were averaged over the three replicas simulated for the IF-IF and IF-OF dimers. RMSD plots calculated for the backbone protein atoms are shown in Supplementary Fig. For the molecular dynamics simulations, the apo-IF-IF and apo-IF-OF dimers were embedded in a POPC bilayer, in a rectangular box solvated with an equimolar 0.75 M NaCl + 0.75 M NaHCOAll statistical tests used, sample sizes, and the number of replicates are described in the corresponding methods, figure legends and tables.Further information on research design is available in the Peer Review FileSupplementary informationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Reporting Summary-New


Background: Hyperglycemia is commonly seen in critically ill patients. This disorder was also seen in coronavirus disease 2019 (COVID-19) patients and was associated with a worse prognosis. The current study determined the prevalence, risk factors, and prognostic implications of hyperglycemia in COVID-19 patients.Method: This was a retrospective observational study performed in an intensive care unit for COVID-19 patients. Electronic data of COVID-19 patients admitted to the intensive care unit from August 2nd to October 15th, 2021, were collected. Patients were divided into non-hyperglycemia, hyperglycemia in diabetic patients, and hyperglycemia in non-diabetic patients. Primary outcomes were 28-day and in-hospital mortalities. Multinomial logistic regression and multivariable Cox regression models were used to determine the risk factors for hyperglycemia and mortality, respectively.Results: Hyperglycemia was documented in 65.6% of patients: diabetic patients (44.8%) and new-onset hyperglycemia (20.8%). In-hospital and 28-day mortality rates were 30.2% and 26.1%, respectively. Respiratory failure, corticosteroid therapy, and a higher level of procalcitonin were risk factors for hyperglycemia in diabetic patients, whereas cardiovascular diseases, respiratory failure, and higher aspartate aminotransferase/glutamate aminotransferase ratio were risk factors for hyperglycemia in non-diabetic patients. The risk of the 28-day mortality rate was highest in the new-onset hyperglycemia , which was higher than hyperglycemia in type 2 diabetes mellitus patients .Conclusion: Hyperglycemia was common in COVID-19 patients in the intensive care unit. Hyperglycemia reflected the disease severity but was also secondary to therapeutic intervention. New-onset hyperglycemia was associated with poorer outcomes than that in diabetic patients. Since December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has spread rapidly across the globe and claimed more than 511 million infected cases and 6.22 million deaths, making it one of the deadliest plagues in the human history [Chronic comorbidities are common in severe and critical COVID-19 patients . Type 2 Hyperglycemia in critically ill patients is caused by multiple pathological conditions, including inflammatory reactions. Cytokine storm plays a central role in the pathology of COVID-19 . It is nStudy designThis retrospective observational study recruited all COVID-19 patients, confirmed with positive reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2, admitted to our COVID-19 intensive care center from August 2nd to October 15th, 2021. The diagnosis of COVID-19 was based on a positive RT-PCR for SARS-CoV-2 following the World Health Organization (WHO) interim guidance [Data collectionThe relevant data were extracted from the electronic medical records. Data of each patient were collected by two investigators. Any collected information that required further clarification was reviewed by the most senior investigators or by the whole team.We mostly used capillary blood glucose (BG) values in clinical practice. The venous BG from the central laboratory was issued on a daily checkup or on demand, usually in combination with other hematological, biochemical, and immunological tests. Should the venous BG report an abnormal result, we used the capillary BG for closer monitoring. This study used the capillary BG values for analysis. All patients had their capillary BG values measured on admission to the ICU.Hyperglycemia was defined when a patient had at least two random BG values >180 mg/dL in 24 hours. Based on the BG level during the hospital stay and history of T2DM, we divided the patients into three groups as follows:Non-hyperglycemia: non-diabetic or T2DM patients who did not meet the criteria of hyperglycemia during their hospital stay;Hyperglycemia with T2DM: at least two random BG values >180 mg/dL in 24 hours and the patient had been previously diagnosed with T2DM; andNew-onset hyperglycemia: at least two random BG values >180 mg/dL in 24 hours and the T2DM was excluded.The primary outcome was 28-day and in-hospital mortality in the three groups mentioned above.Statistical analysisThe statistical analysis was performed using the SPSS Statistics, version 28.0.1.0 . Data are expressed as frequency (percentage) for categorical variables; mean ± standard deviation, and median (interquartile range) for continuous parameters. The one-way analysis of variance (ANOVA) was used to compare three groups. In univariate analysis, the chi-square test was used for categorical parameters and the Wilcoxon rank-sum test for continuous parameters to compare the survival and death groups. The variables that had significance in the univariate analysis were included in multivariate Cox  regression to identify the independent risk factors of in-hospital mortality. All statistical tests were two-tailed, and a p-value of less than 0.05 was considered statistically significant.Research ethicsThe present study was approved by the Institutional Review Board of University Medical Center, Ho Chi Minh City, Vietnam . The informed consent for participation was obtained from the patients or their family members. All methods were performed in accordance with the Declaration of Helsinki.Demographic, laboratory, and therapeutic characteristicsAfter screening, 517 patients who met the inclusion criteria were recruited for the study. The demographic and clinical characteristics of the three groups on admission are presented in Table As well recognized as a factor inducing hyperglycemia, CST was also investigated in the current study. Briefly, the new-onset hyperglycemia group seemed to receive slightly higher doses of corticosteroids than the two other groups.The laboratory results on the admission of the three groups of patients are presented in Table Risk factors of T2DM hyperglycemia and new-onset hyperglycemiaWe further investigated the independent risk factors of T2DM hyperglycemia and new-onset hyperglycemia. The analysis results are presented in Table Patients’ outcomes based on the glycemic statusOut of 517 patients enrolled in the study, 156 (30.2%) patients died in the hospital. The 28-day mortality was 3.9% (7/178), 34.5% (80/232), and 44.9% (48/107) in the non-hyperglycemia, T2DM hyperglycemia, and new-onset hyperglycemia group, respectively.The hazard ratio (HR) for death with adjustment for other risk factors was significantly increased among patients with hyperglycemia with or without T2DM compared to those of the non-hyperglycemia group Figure .We also noticed that the percentage of patients requiring invasive mechanical ventilation was significantly higher (70%) in the new-onset hyperglycemia group than in the T2DM hyperglycemia group (51%) and in patients without hyperglycemia (9%).Independent predictors of COVID-19 mortality2/FiO2 ratio on admission, PCT ≥ 0.5 ng/mL, and average daily corticosteroid dose were independently associated with increased risk of mortality. Interestingly, the new-onset hyperglycemia was associated with an important increase in in-hospital mortality .We also performed a multivariable Cox regression model to identify the risk factors of 28-day in-hospital mortality. The results are presented in Table It is well established that diabetes significantly increases the risk of developing and dying from infectious diseases ,17. HypeThe incidence of hyperglycemia was noticeably high (65.6%) in the studied population. Hyperglycemia-associated mortality in critically ill patients and the beneficial effects of glycemic control have been intensively studied since the breakthrough work by Van den Berghe et al. . These sThe new-onset hyperglycemia was documented in 20.8% of all patients and 41.0% of non-diabetic COVID-19 patients. This incidence was lower than that (28.4%) in the retrospective study by Li et al. . More imThe 28-day and the in-hospital mortality rates were 26.1% and 30.2%, respectively. Our center was the tertiary referral hospital receiving the most severe COVID-19 patients during the zenith of the pandemic. This might explain the higher mortality rate compared to previous studies from China and United States. Besides a higher 28-day mortality, the new-onset hyperglycemia was also related to an increased need for mechanical ventilation during the hospitalization (70%) compared to the T2DM hyperglycemia group (51%). The current study confirmed once again the findings in previous works , increasHyperglycemia was documented in approximately two-thirds of severe and critical COVID-19 patients admitted to ICU. Hyperglycemia in patients with T2DM was more frequently seen and two-fold higher than the new-onset hyperglycemia. The new-onset hyperglycemia group had the highest 28-day and in-hospital mortality rates, followed by the T2DM hyperglycemia group. The lowest mortality rates were documented in the non-hyperglycemia group. Similarly, the percentage of patients requiring invasive mechanical ventilation was significantly higher in the new-onset hyperglycemia group than in the T2DM hyperglycemia group and in patients without hyperglycemia. CST was the strongest independent risk factor for hyperglycemia in T2DM patients, whereas respiratory failure, reflected by the proportion of patients requiring invasive mechanical ventilation upon admission, was the strongest independent risk factor of hyperglycemia in non-diabetic patients. New-onset hyperglycemia was the most important risk factor for death in COVID-19 patients. The present study suggests that BG levels should be actively monitored in severe and critical COVID-19 patients. The occurrence of hyperglycemia in these patients, especially those without a previous diagnosis of T2DM, should be considered a marker of severity and worse outcomes. More studies are required to elucidate the likely multifactorial mechanisms of new-onset hyperglycemia in COVID-19 and the bidirectional interaction between new-onset hyperglycemia and severity in COVID-19 disease. We also need a prospective study investigating the impact of glycemic control in this group of patients and other similar diseases.


The Biopsychosocial (BPS) model is recognized and widely accepted in the field of health care, particularly in rehabilitation. However, in clinical practice the applicability of the BPS model is a challenge for many professionals.This study aimed to explore the factors that impact the perception of rehabilitation professionals about the BPS model in Ukraine. In addition, the job satisfaction assessment was done to measure whether rehabilitation specialists in Ukraine fulfil their professional roles according to their expectations and values. Participants were 346 rehabilitation specialists from Ukraine who completed the Bio-Psycho-Social Scale (BPS) for Use in Healthcare and the Global Job Satisfaction Scale (GJSS). The ANOVA was used to investigate the outcome differences between the two scales.The highest proportion of responders in this study represented state health services. The BPS subscale outcomes on “assessment and reporting” and “professional knowledge and skills” were significantly higher for specialists from a private sector. The global job satisfaction scale outcomes did not differ between responders from the private and state health sector. The perception of professionals networks was associated with greater job satisfaction.The findings suggest that conception of the BPS approach in rehabilitation system of Ukraine varies across the private and state settings. The professional network plays important role in job satisfaction of rehabilitation professionals in Ukraine. Future research focusing on education strategies to effectively train professionals to apply biopsychosocial approach to practice is of critical importance to prepare rehabilitation specialists in Ukraine. The International Classification of Functioning, Disability and Health (ICF) was launched by the World Health Organization , as a biLinked with the use of the ICF, over the last decades there has been a shift towards a biopsychosocial (BPS) paradigm in health care and has resulted in the introduced concepts such as client-centered-practice, inclusion, shared decision making, coaching and self-management . This haCurrently, Ukraine is one of the countries with high needs for rehabilitation services. The military conflict in the Donbass region since 2014 and the Russian large-scale invasion in Ukraine since February 24, 2022, has increased demands for acute and long-term rehabilitation services for many people injured in the war. According to the President of Ukraine, about 100 military personnel are killed every day and about 500 are wounded . In addiThe noticeable factor of quality in health care systems is employee’s job satisfaction which further heavily influences the performance and clinical outcomes of patients. The studies exploring factors associated with job satisfaction in different groups of health care professionals have reported multiple components including job conditions, the work responsibilities, organisational policies, promotion opportunities, security, professional communication and relationship within the health care team . Researchttp://rehabeukr.eu/) started in 2018 to address the three major goals related to development of innovative master programs in physiotherapy in Ukraine by: (1) building professional capacity of the academic and research staff, (2) development of the teaching/learning/assessment resources, and (3) providing specific educational infrastructure needed to implement the nationally new professional study program in physiotherapy. This project was committed to overcoming the traditional biomedical model, to promote biopsychosocial (BPS) approaches and to promote implementation of the ICF in Ukrainian rehabilitation services linked with the REHAB project. The academic staff and students from the four project partner universities in Ukraine  received more than 20 training sessions  to learn a common language for describing physiotherapy as part of multidisciplinary rehabilitation system that involve different health professionals. The European project partners representing the Josef Pilsudski University of Physical Education in Warsaw (Poland) and European Federation of Adapted Physical Activity have more than 20 years educational and research experience in the rehabilitation field. More than one thousand bachelor and master students from physiotherapy programs and over one hundred academic and research staff from across Ukraine have been involved in the REHAB project training between 2018 – 2022.Several projects funded by the European Commission have been initiated to promote collaboration with international rehabilitation partners aiming implementation of European standards of training for rehabilitation professionals in Ukraine. For example, the project entitled “Innovative Rehabilitation Education—Introduction of new master degree programs in Ukraine” (REHAB) , financed by Erasmus + Programme of the European Commission involves the three Ukraine higher education institutions  and European partners from Belgium  and Portugal (University of Porto). This particular project targets implementation of a new curriculum for occupational therapists according to the standards of the World Federation of Occupational Therapy (WFOT). In the project, the focus is not only on training the teachers but also the staff from hospitals and rehabilitation centers (Caritas and Modrychi), because they have a crucial role as guides and coaches of the internship students. Also, the collaboration with governmental institutions of Ukraine is promoted since their support is needed in state funded rehabilitation centers.Although the use of ICF as a unified language and framework for a BPS description of health in various domains is supported by Ukrainian health policy, there is no data on the perception and understanding of the rehabilitation professionals in Ukraine on how they understand and if they use a BPS model in their clinical practice. Also, to the authors knowledge, no evaluation of job satisfaction of rehabilitation professionals has been carried out in Ukraine.This study aimed to explore the factors that impact the perception of rehabilitation professionals about the BPS model in Ukraine. In addition, the job satisfaction assessment was done to measure whether rehabilitation specialists in Ukraine fulfill their professional roles according to their expectations and values.This is a cross-sectional study of rehabilitation students and professionals in Ukraine. The data were collected through an online survey from a convenience sample of people who volunteered to take part in the study and met the inclusion criteria. The inclusion criteria for participation in the study included master level students in occupational therapy or physical therapy and people employed in rehabilitation. As there is currently no formal professional standard linked with the qualification needed for working in physical or occupational therapy in Ukraine, students were also recruited with working professionals. All respondents were from different regions of Ukraine  and gave their consent to take part in the study. A total of 345 respondents were recruited, however 64 respondents had missed providing data on ‘years of experience’ and were removed from the analyses. After cleaning the data for missing values, there were 281 respondents . To preserve anonymity of the respondents (some were students from partner institutions), we did not track location data and do not know the spread of the distribution. The BPS scores of missing 64 responses  were tested against the remaining 281 responses  and after testing differences by independent t-tests, the differences were statistically significant , hence we conclude there is a potential sample bias with the participants who completed the survey.. This study complies with the Declaration of Helsinki and was performed according to ethics committee approval at the Latvian Academy of Sport Education, Ethics committee in Health Care . Informed consent was obtained from all participants in this study. Data were collected between December 2021 – February 2022.The Bio-Psycho-Social Scale for Use in Healthcare  includesThe Global Job Satisfaction Scale has nine-subscales that measure employee job satisfaction  in 36 itBackground variables included questions about the participant’s gender , qualifications , work place setting , as well as profession . All items underwent a back-translation protocol whereby translators from English to Ukrainian did not share the original work to the translator who carried out the back translation. Where there were minor  and major  discrepancies between original and translated text in the first round (n = 13), the researchers met to discuss the terms that appeared different in the translated text. After confirming with translators of the revised text, the questionnaire was uploaded to an online survey platform in both English and Ukrainian languages for completion. Data converted into English language and downloaded for analyses 28].Data analysed by IBM SPSS version 27.0). Respondents were grouped by the SPSS two-step cluster command, where Bayesian Information Criterion (BIC) were used to find the best ratio between the BIC and the minimum number of clusters in the first step. The second step measures the distances between the clusters to produce the final set of clusters .0. Respo. The cluTo investigate the differences in BPA and global job satisfaction by clusters, mean scores of overall BPS and its components were tested by one way analysis of variance (ANOVA). Homogeneity of variances were tested with Levine’s test and because the null hypothesis was rejected (p = 0.915), the Tukey post-hoc test was carried out to control for Type 1 errors between the clusters . In addition, parametric correlations of the BPA and global job satisfaction were performed to detect possible multicollinearity for linear regression analysis. To examine how BPA components were associated with global job satisfaction, individual linear regression analyses were performed for each cluster, where global job satisfaction was the dependent variable and each BPS component were the independent variables, after adjusting for background variables.The four clusters were labeled as ‘None or Private Practice’, ‘Private health’, ‘OT or PT in Public Health’, and ‘Specialists in Public Health’. The characteristics of the clusters can be seen in detail from Table Across the four clusters, the mean scores for dimensions of BPS_A (Assessment and report)  varied across the clusters Table . There wBetween the factors within BPS, the correlations ranged from 0.278 between BPS_P and BPS_N and 0.55 between BPS_U and BPS_N Table .Table 3BRegression onto global job satisfaction by BPS components can be seen in Table In this study, respondents to the survey were grouped into four clusters, mainly dominated by their workplace setting with about half of responders working in state funded health institutions. The National Health Service of Ukraine (NHSU) monitors and analyses the needs of citizens in health services and finances required for these services across the country. The NHSU implements the Program of Medical Guarantees since 2020 that covers 31 health benefit packages, including the three specifically related to rehabilitation: (1) medical rehabilitation for children aged 0–3 years, (2) persons with neurological conditions, and (3) persons with musculoskeletal conditions. For each rehabilitation package, the NHSU requires a rehabilitation staff to be available and issues the requirement for a multidisciplinary rehabilitation team, basic equipment, goal-setting principles and use of the ICF . This exRegarding BPS scale results, the scores tend to be higher than in previous studies . It mighMore detailed analyses of the BPS survey outcomes across the four clusters indicated that significant differences were observed between the private and public health institutions for subscale “assessment and reporting” and “professional knowledge and skills” presenting significantly higher values for the rehabilitation specialists from a private sector. These results show that public health institutions do not have sufficient access to assessment instruments or do not use them accordingly. According to WHO reports about the situation of assessment of the rehabilitation system in Ukraine, it was stated that there are financial weaknesses related to limited rehabilitation packages in the NHSU monitored services that also includes availability of assessment instruments . Also, tThe global job satisfaction scale outcomes did not differ across the four clusters.The current study showed that the rehabilitation specialists in Ukraine, whether working in the state or private sector, were uncertain with respect to satisfaction with their jobs.The variables that contributed highly to the total scores of job satisfaction varied by cluster. The most notable predictor for job satisfaction was the BPS_N among the “None”, “OT or PT”, and “PRMS” clusters. In other words, increased perceptions of networks were associated with greater job satisfaction. Networks in physiotherapy can be good for improving cross-sectoral work, learning opportunities  and are The rehabilitation institutions in Ukraine have limited possibilities to meet the standards set by the NHSU for facilities and equipment. Also, there is limited access to continuous professional development (CPD) of in-service rehabilitation professionals. While CPD is required for medical doctors, it is not yet required for other professions within the rehabilitation workforce . All aboThis study presented small to moderate correlations between BPS subscales. It might be linked with the perception of physiotherapists of learning and implementing the BPS approach that requires skills to comprehend the difference of this approach in order to applying their new skills. For example,  listed tThe rehabilitation systems are different in every country and its conditions may depend on the cultural and socioeconomic environment in which specialists are trained, work and live. The implementation of the BPS approach for different group of patients requires team based and integrated service provisions with the holistic focus on clinical reasoning, problem solving and goal-setting . In UkraRegression analyses confirmed outcomes described above. For example, rehabilitation professionals reported higher job association with better access to assessment instruments, using professional network and possibility to use a variety of environment to contribute therapy plan . These outcomes are in line with previous studies reporting that working environment (including access to therapy assessment instruments) and relationship with patients are among important factors of job satisfaction of rehabilitation professionals , 22.The study was cross-section in nature, therefore causality between variables cannot be established. The sampling was from a convenience sample from selected cities in Ukraine, therefore, interpretation of the results require caution prior to generalizing to all physiotherapists in Ukraine. While the study power can be considered sufficient, the number of participants was low and it is possible that a larger sample would have resulted in different proportion across the clusters, not least the differences in BPS scores between those who completed all questions and some  may need to be considered when considering these results. Given the limitations and challenges described above, it seems that a deeper understanding is needed of rehabilitation professionals’ experiences of integrating BPS into clinical practice in Ukraine.The conception of the BPS approach in rehabilitation varies across the different settings and professionals affiliated with the Ukrainian health care system. In general, rehabilitation professionals, whether working in government or private hospitals, were positive about their job, while there were not fully satisfied scores. Overall, job satisfaction was associated with perception of professional networks. To gain further knowledge of the BPS implementation process across different rehabilitation settings in Ukraine, a qualitative study could be conducted in parallel with a feasibility study. Moreover, after the military aggression of Russia in Ukraine the health care challenges are affected by significant day to day increase of inpatient care related to war injuries in military personnel and civilians. The complex nature of military injuries requires unique expertise to develop new, innovative methods for trauma evaluation and treatment followed by decision of appropriate tactics of rehabilitation.