However, no significant difference between the two groups was detected at the end of the follow-up. The tele-AI group had higher TAS than FTF group after 1 year. Conclusions: Telerehabilitation after ACLR seems to provide a superior short-term outcome compared to hospital-based rehabilitation during the COVID-19 pandemic.Background: Due to the rapid spread of the coronavirus disease-19 (COVID-19), most of the patients expressed a reluctance to undergo postoperative rehabilitation at a rehabilitation clinic. Therefore, in this scenario it was necessary to reshape the crucial role of postoperative rehabilitation of these patients. We conducted a telerehabilitation program based on an artificial intelligence brace (AI brace) which can monitor the progress of rehabilitation through an app and an internet server. Our hypothesis was that home-based telerehabilitation might provide clinical outcomes comparable to face-to-face, hospital-based rehabilitation programs in terms of effectiveness. Methods: A retrospective cohort study enrolled patients who received anterior cruciate ligament reconstruction (ACLR) between January and September 2020. Patients were divided into two groups: the tele-AI group received telerehabilitation with an AI brace while the FTF group had face-to-face, hospital-based rehabilitation. Clinical knee functional scores and Tegner Activity Scale (TAS) were assessed and analyzed until 12 months after the operation. Results: The tele-AI group had higher IKDC scores at 3 months ( Performing physical rehabilitation after anterior cruciate ligament reconstruction (ACLR) is crucial ,2. Out oResearch has indicated that home-based telerehabilitation is just as effective as standard rehabilitation after a knee or hip replacement ,7,8,9,10The possibility of telerehabilitation is attributed to the progress of telecommunication technology and delivering rehabilitation services remotely. The concept of telerehabilitation was first described in 1998 by Burns et al. . The defTo date, no study has compared the outcomes of AI-brace-assisted telerehabilitation programs and face-to-face, hospital-based rehabilitation programs conducted after ACLR. Our study aimed to compare the clinical outcomes of these two types of programs. Our hypothesis was that home-based telerehabilitation involving the use of an AI brace produces clinical outcomes comparable to that of face-to-face, hospital-based rehabilitation programs in terms of effectiveness.The present retrospective cohort study was conducted at a single medical center in Taichung, Taiwan. We followed consecutive patients aged between growth plate maturation and epiphyseal fusion to 55 years old who underwent ACLR in our medical center by two experienced surgeons between January and September 2020. Patients enrolled in our study were not specified to a particular sports field. The other inclusion criteria were a preinjury Tegner Activity Scale (TAS) level of at least level 5, the use of a protective knee brace after ACLR, and the completion of scheduled outpatient department (OPD) follow-up visits (at least 12 months) postoperatively. The present study excluded patients who underwent revision surgeries, had multiple ligament injuries, or had concomitant comorbidities during the study period  and severe meniscal damage) and patients did not undergo regular follow-up for at least 12 months after their operation. All patients underwent arthroscopic ACLR, involving the use of autologous quadruple hamstring tendons. All surgical procedures were performed by a single experienced orthopedic surgeon, and their clinical outcomes were evaluated postoperatively at 1, 3, 6, and 12 months.In total, 30 patients were enrolled; 15 patients underwent telerehabilitation assisted by an AI brace (Tele-AI group), and the other 15 underwent face-to-face, hospital-based rehabilitation (FTF group). The participants who underwent telerehabilitation used an AI brace with a built-in rehabilitation guidance system. The participants who underwent hospital-based rehabilitation (FTF group) did so at our institution’s rehabilitation center twice a week (at least once a week) for 3 months after their operation and once a week after 3 months until 6 months post-operation. The advantages and disadvantages of telerehabilitation were explained to the participants. Some participants preferred telerehabilitation because of the decreased number of required hospital visits during the COVID-19 pandemic and the fact that daily rehabilitation could be monitored directly by us through a web portal during the postoperative period. Patients in both the FTF group and the tele-AI group were asked to visit the outpatient department every month in the first three months. We followed up with a radiograph and collected postoperative clinical data after 6, 9, and 12 months. The participants were also provided information pertaining to home-based rehabilitation. Informed consent was obtained from all participants. The present study was performed in accordance with the Declaration of Helsinki. Approval was granted by the Institutional Review Board of Taichung Veterans General Hospital . The identification information of all participants was omitted prior to the analysis being conducted.For all thirty patients in our study, we followed the post ACLR rehabilitation protocol set up by our hospital —partial The participants in the Tele-AI group underwent home-based rehabilitation through the rehabilitation system , which comprised a portable motion tracker, a mobile application , and a web portal . The appThe participants used a mobile device that could be connected to a portable motion tracker through Bluetooth (Bluetooth 4.2 with the 10 Hz sampling frequency) to perform and track their daily rehabilitation. The smart core motion tracker  for AI brace was built with accelerometers, angle sensors, and gyroscopes. Thus, the smart core can track motion in three-dimensional space. The product is certified by Industrial Technology Research Institute, Taiwan.A home-based rehabilitation schedule was designed by orthopedic doctors or physical therapists and set up on the web portal. Patients would download the rehabilitation program on the smart phone by the KNEESUP Care App . The app would supply the demonstration video to prevent inaccurate rehabilitation exercise and track the real-time motion by the sensor on the knee brace. Daily rehabilitation achievements were recorded and stored on a cloud-based database, allowing for orthopedic surgeons to follow up the status of each participant by accessing a web portal. Through this rehabilitation system, the conditions of the participants could be monitored, and their rehabilitation progress tracked. When a problem was detected, the affected patient was promptly provided medical support and the rehabilitation schedule can be modified individually.The participants in the FTF group underwent postoperative rehabilitation by visiting our hospital’s rehabilitation center twice a week (at least once a week) for 3 months after operation and once a week after 3 months until 6 months. The rehabilitation protocol is the same as the telerehabilitation group . The freAfter the qualified patients were selected as participants, their knee function was clinically assessed and scored during patient visits. For all participants, data were collected before surgery (baseline data) and during OPD follow-up visits at 1, 3, 6, and 12 months after surgery. The collected data comprised International Knee Documentation Committee (IKDC) Subjective Knee Evaluation Form scores and knee injury and osteoarthritis outcome (KOOS) scores  . Additiop-value of <0.05 was regarded as statistically significant. Categorical variables were tested using the Fisher’s exact test and continuous variables were tested using the Mann–Whitney U test for intergroup comparisons. The Wilcoxon signed-rank test was performed for intragroup comparisons; specifically, the mean gross grading scores of all participants at 1, 3, 6, and 12 months after surgery were compared with their preoperative scores.Statistical analysis was performed to analyze the aforementioned variables of the 30 participants who completed at least 12 months of postoperative OPD follow-up. All analyses were performed using SPSS version 22.0 , and the results are represented as means and standard deviations. A The average age at surgery was 26.27 ± 8.59 years (range: 17–43 years) for the Tele-AI group and 28.60 ± 9.30 years (range: 17–50 years) for the FTF group. Of the 30 patients, the majority had a sports-related anterior cruciate ligament injury (86.7%). The preoperative participant demographics are presented in In the FTF group, the frequency of visiting rehabilitation center after ACLR is 19.6 ± 2.2 (range: 16–23) times in the first twelve weeks and 9.7 ± 1.5 (range 8–12) times between months 4 and 6. Every patient in FTF group visits the rehabilitation center at least once a week and 6 (40%) patients visit the rehabilitation center below 18 times (75%) in the first twelve weeks. In addition, no patient gets infected with COVID-19 during the follow-up periods, and no outbreaks of cluster infection episodes happened in the rehabilitation center.p = 0.983; KOOS, Tele-AI vs. FTF, 80 ± 3.91 vs. 80.71 ± 7.04, p = 0.405).The patient-reported outcomes  are presented in p = 0.0443) and 6 months (p = 0.0052) after surgery and higher KOOS scores at 1 month (p = 0.0365) and 6 months (p = 0.0375) after surgery. No significant difference between the two groups was detected at 12 months after surgery .At 1 month after surgery, both groups had lower IKDC and KOOS scores than they did at baseline. Relative to the FTF group, the Tele-AI group had higher IKDC scores at 3 months  and 11 participants in the FTF group (73.3%) reached TAS level of 5. In addition, five participants (33.3%) in the FTF group did not adhere to the rehabilitation program conducted at the rehabilitation center , and these five patients also had a relatively low TAS level.The purpose of this study was to compare the outcomes of an AI-brace-assisted telerehabilitation program to those of a face-to-face, hospital-based rehabilitation program; these programs were designed for patients who had undergone ACLR and were conducted during the COVID-19 pandemic. Relative to baseline, the Tele-AI group exhibited more favorable IKDC results at 3 and 6 months after surgery and more favorable KOOS results at 1 and 6 months after surgery. All participants were assumed to have complied with their assigned postoperative rehabilitation program . The compliance of the Tele-AI group was monitored directly through the web portal, and they were observed to have maintained a high level of compliance throughout the postoperative period. By contrast, more than one-third of the participants  in the FTF group failed to regularly attend scheduled postoperative rehabilitation sessions at the rehabilitation center for fear of being infected with COVID-19.At 6 months post-operation, our results showed significant IKDC and KOOS scores in the tele-AI group, which may contribute to the compliance of rehabilitation. To understand why the outcomes are inferior in the FTF group, three patients in FTF had especially poor short-term outcomes and they were also far behind our rehabilitation protocol due to their fear of the pandemic. According to Rodríguez-Merchán et al., a successful recovery from ACLR surgery is not only dependent on surgical technique and surgeon experience but also on postoperative rehabilitation . In the Whether a knee brace is required after ACLR is a debatable topic ,15,16,17Advancements in telecommunications technology have facilitated the implementation of internet-based telerehabilitation . The defBauwens et al. showed short-term results with 32 patients after ACLR surgery during the COVID-19 pandemic with self-rehabilitation guided by the app . This liA study by N.J. Collins et al. revealed that the KOOS-ADL subscale had better content validity for old patients with osteoarthritis undergoing total knee arthroplasty, while KOOS-Sport/Rec and KOOS-QoL had higher content validity for younger patients with ACL injury . This coTo the best of our knowledge, the present study is the first to evaluate the clinical outcomes of telerehabilitation after ACLR surgery during the COVID-19 pandemic. The Tele-AI group (telerehabilitation) attained more favorable results than the FTF group (standard rehabilitation) did during the COVID-19 pandemic. Despite that, the most perfectly designed telerehabilitation could only have a similar but not superior outcome compared to hospital-based rehabilitation. In our situation, the pandemic and the fear of getting infected with COVID-19 decreased the frequency of people visiting rehabilitation centers. Poor compliance generated unpredictable results.However, the present study has several limitations. First, it adopted a retrospective cohort design, which is inherently susceptible to information bias. Second, the small sample of the present study did not allow for robust subgroup analyses to be performed, and the postoperative follow-up period was limited to 12 months after the index surgery. Third, the generalizability of the present study is low because it was conducted at a single institute. Finally, the high cost of the AI brace used in the present study may have affected the decision-making of patients and led to selection bias.Our findings revealed that during the COVID-19 pandemic, AI-brace-assisted telerehabilitation produced results that were comparable to those of a standard rehabilitation program. Thus, the proposed telerehabilitation program can benefit patients who cannot regularly visit a rehabilitation center. Nevertheless, further studies are necessary to elucidate the benefits of AI-brace-assisted telerehabilitation followed by a regular postoperative rehabilitation program in a pandemic-free environment.


Dogs are one of few species that naturally develop prostate cancer (PCa), which clinically resembles aggressive, advanced PCa in humans. Moreover, PCa-tumor samples from dogs are often androgen receptor (AR)-negative and may enrich our understanding of AR-indifferent PCa in humans, a highly lethal subset of PCa for which few treatment modalities are available This narrative review discusses the molecular similarities between dog PCa and specific human-PCa variants, underscoring the possibilities of using the dog as a novel pre-clinical animal model for human PCa, resulting in new therapies and diagnostics that may benefit both species. The initiation and progression of prostate cancer (PCa) in humans is initially reliant on androgen receptor (AR) signaling ,2,3. DirThe prostate of the dog, unlike that of rodents, is morphologically, histologically, and physiologically similar to that of humans and is under the control of androgens via androgen-receptor signaling ,19,20. DThis review aims to discuss the molecular similarities between dog and specific human PCa variants to further examine the use of dogs as a suitable animal research model.The AR is a ligand-dependent nuclear receptor transcription factor that mediates the action of androgen ligands  in androgen-dependent tissues in health and plays an important role in prostate organogenesis and maintenance in adulthood ,32,33,34The AR in humans is a protein of 919 amino acids consisting of several functional domains, including an N-terminal domain (NTD), DNA binding domain (DBD), and a ligand binding domain (LBD) at the C-terminus ,45. In dThe NTD of the AR is essential for function and the least evolutionarily conserved region of AR; however, there are still similarities between humans and dogs. The polyglutamine  repeat region of the NTD has an average of 21–23 Qs in humans. Longer polyQ repeats are related to decreased AR transcriptional activity, while shorter polyQ repeats are related to increased AR transcriptional activity and are often associated with increased PCa risk ,48,49. TIn the absence of the agonist ligand, the AR is bound to heat-shock proteins  and other co-chaperone proteins in a complex known as the foldosome ,55. ManyThis concept has also been explored in multiple studies in dogs, which have shown that the overexpression of SGTA in vitro abates AR signaling ,60. TherThe CRPC is a hormone-independent but AR-dependent progression of PCa, in which ADT has failed, yet AR signaling axis is still functional and increases growth and progression. Often, this stage is characterized by more aggressive tumor behavior, including the initiation of cancer stem cell (CSC) signaling, epithelial–mesenchymal transition (EMT), and metastasis. A subset of patients develop CRPC secondary to uncontrolled AR signaling due to a mutation in the AR. Splice variants of the AR lacking the ligand-binding domain, such as ARV7, are responsible for this uncontrolled signaling in humans . To dateAdditionally, mutations in AR-independent pathways are more frequent in CRPC and are thought to drive CRPC to become AR-indifferent PCa after ASI therapy. Therefore, AR presence and signaling coupled with new driver mutations in other pathways, EMT, and metastasis is are hallmarks of this progression of PCa (metastatic CRPC (mCRPC)). Because dogs often present with low or null AR expression, are castrated at an early age, develop PCa without the influence of androgens, and often present with metastasis, it has been argued that CRPC in dogs closely resembles CRPC in humans.PTEN loss of function or deletion, and AKT, p-4EBP1, and eIF4E overexpression [The PI3K–AKT–mTOR signaling axis has been shown to play a crucial role in the development and maintenance of CRPC ,63,64. Tpression .PTEN has been implicated in the aberrant activation of this pathway in human PCa, and has also been shown to be lost in dog PCa [This entire pathway has also been observed to be upregulated in samples of dog PCa tissue when compared to the non-malignant prostatic tissue of normal intact dogs . Moreove dog PCa . Because dog PCa ,75,76, dEstrogens have been linked to CRPC progression, and increased levels of estrogens have been correlated with more aggressive PCa ,78,79,80Cancer stem cells (CSC), or tumor progenitor cells, have been linked to the driving of clonal evolution and tumor heterogeneity, growth, and progression via their ability to self-renew, resist apoptosis, and differentiate ,90,91, aStem-cell-marker expression has also been explored in dog PCa ,101. In Epithelial cells can undergo EMT, a phenomenon that is characterized by phenotypic changes in epithelial cells, which begin to appear more spindle-shaped, in the manner of mesenchymal cells. Additionally, this phenotypic change is accompanied by biochemical changes, in which epithelial markers associated with adhesion, such as E-cadherin, are downregulated and mesenchymal lineage markers, such as vimentin, become upregulated . This evEpithelial–-mesenchymal transition has also been shown to occur in dog PCa. One study showed that dog PCa tumors with a more undifferentiated appearance and metastatic lesions had increased TGF-β nuclear positivity on immunohistochemistry (IHC) . E-cadheThe canonical Wnt-signaling pathway is integral to EMT and has been shown to be important in the development of CRPC and the proliferation of CSCs in the PCa microenvironment . BrieflyFor decades, Wnt signaling has been implicated in human PCa progression, and it is critical for CRPC. Furthermore, Wnt signaling has been shown to increase the proliferation and differentiation of prostate cells and promote EMT, leading to more aggressive and invasive disease . This paAndrogen-indifferent prostate cancer (AIPC) is a form of PCa with treatment-resistant phenotypes that do not rely on AR signaling and typically arise from mCRPC following treatment with ASIs ,122, altThe DNPC is characterized by a loss of AR and neuroendocrine (NE) markers, and is sometimes referred to as CRPC subtype DNPC (CR-DNPC) . The DNPSimilarly, dog PCa is often AR- and NE-negative with increased FGF and MAPK signaling , and hasNeuroendocrine prostate cancer may occur de novo or as a consequence of selection pressures on CRPC tumors from ASIs, which is termed treatment-emergent NEPC (t-NEPC) or castration-resistant NEPC (CR-NEPC) ,128. TheTP53, PTEN, and RB1, and is typically responsive to platinum drugs [PTEN, RB1, and TP53, but also in BRCA2 [The AVPC criteria were created to identify patients predicted to have an aggressive disease that is unlikely to respond to ASI therapy and may arise de novo or after pharmaceutical treatment. Any morphological variant of PCa , adenocarcinoma, adenocarcinoma with NE differentiation) may qualify as AVPC if it has a particular molecular signature and/or meets the criteria for an aggressive clinical course , includium drugs . Moreovein BRCA2 .RB1 has not yet been studied in dog PCa. The loss of BRCA1 has been documented in dog PCa, but BRCA2 was not lost or downregulated according to another study utilizing RNA sequencing [Dog PCa is highly aggressive and clinically similar to AVPC, but whether it has the same molecular hallmarks as human AVPC is still unclear. One study showed the decreased expression of p53 in dog PCa tissues when compared to normal and inflammatory prostatic lesions, but not complete loss , while aquencing ,135. BecUndeniably, there are molecular similarities between dog PCa and specific human PCa variants, although there are differences and areas that are understudied. Because the majority of dogs are neutered, present with advanced disease, and have AR null tumors, they are probably not feasible models for androgen-dependent PCa, with the exception of intact dogs in the early stages of the disease. Because of the lack of screening methods for PCa in dogs , establishing which intact dogs have early PCa would be challenging. Dog PCa is clinically and molecularly similar to CRPC and mCRPC, with upregulation in the PI3K-AKT pathway, Wnt signaling, EMT, CSCs, and indifference to AR, although ER expression appears to be different in this disease. Moreover, the importance of splice variants is yet to be determined in dog PCa. Lastly, regarding AIPC, it has been shown that dogs molecularly model DNPC, and may also model AVPC, but are unlikely to recapitulate NEPC. Altogether, dogs are likely to be suitable models for certain variants of advanced PCa, but not all, and additional studies are warranted to further characterize the molecular characteristics of dog PCa.


Androgen receptor (AR) expression is absent in 40–90% of estrogen receptor (ER)-negative breast cancers. The prognostic value of AR in ER-negative patients and therapeutic targets for patients absent in AR remains poorly explored.N = 669) and The Cancer Genome Atlas . We compared AR-defined subgroups by demographics, tumor characteristics, and established molecular signatures .We used an RNA-based multigene classifier to identify AR-low and AR-high ER-negative participants in the Carolina Breast Cancer Study  participants in CBCS and were associated with HER2 negativity , higher grade , and higher risk of recurrence scores , with similar results in TCGA. The AR-low subgroup was strongly associated with HRD in CBCS  and TCGA . In CBCS, AR-low tumors had high adaptive immune marker expression.Multigene, RNA-based low AR expression is associated with aggressive disease characteristics as well as DNA repair defects and immune phenotypes, suggesting plausible precision therapies for AR-low, ER-negative patients.The online version contains supplementary material available at 10.1007/s10549-023-07014-x. Androgen receptor (AR), which is expressed in approximately 30–60% of estrogen receptor (ER)-/progesterone receptor(PR)-/human epidermal growth factor receptor 2(HER2)+ breast cancers and 10–53% of triple-negative breast cancers  , has emeSeveral research groups have reported that non-luminal androgen receptor TNBC molecular subtypes that express AR protein at low levels demonstrate AR dependence for tumor cell growth or viability –21. ThusThe Carolina breast cancer study  is a population-based study of breast cancer that is oversampled for Black and younger women. Using gene expression data for 1202 CBCS participants, we trained a pathway-based classifier to identify AR-low patients and to examine the relationship between AR status and tumor aggressiveness among ER-negative participants. Results in CBCS were validated in TCGA.The Carolina breast cancer study (CBCS) is a population-based study , 23 of whttps://gdc.cancer.gov/). In comparison to cases in CBCS, those in TCGA were older, with larger, higher stage tumors [The Cancer Genome Atlas (TCGA) is a large, publicly available data source containing extensive genomic data on over 30 cancer types. Study details are described elsewhere . For oure tumors .age = 50 vs 52 years), Black women, and ER- tumors . Thus, AR RNA expression  was profiled in 1202 CBCS samples (472 ER-negatives), using a custom NanoString protocol optimized for formalin-fixed paraffin-embedded (FFPE) samples [1649 CBCS tumor samples, chosen based on the availability of cores or slides for analysis, were included on a NanoString RNA panel that included AR, of which 1202 (72%) passed quality control. Samples which failed quality control were more likely to be from CBCS Phase I or II, while included samples were more likely to be from older participants  and 95% confidence intervals by fitting a generalized linear model with binomial distribution and identity link where AR status was the outcome and the variable of interest was the predictor. Because triple-negative status may confound associations between AR and clinical presentation, we also computed models adjusted for triple-negative status and in triple-negative tumors only. As further sensitivity analyses, we assessed demographic and clinical characteristics of ER+ tumors by AR status to further explore whether AR-low phenotype associations also held for ER+ tumors, and used multiple imputation to assess whether missing data biased results.N = 674; 271 ER-negative), we developed a predictor of AR phenotype to identify additional samples with low AR expression. To do this, we split the 472 ER-negative samples with measured AR expression into five groups (“folds”) using stratified random sampling, therefore, ensuring consistent distribution of AR-high and AR-low samples within each fold. In each of five iterations of testing, we retained four of the folds for training  and omitted the last for validation , then repeatedly fit Classification to Nearest Centroid (ClaNC) models that used between two and 150 genes  to distinguish AR-low and AR-high tumors in the training set. For each of these 75 models, we estimated sensitivity, specificity, and the Youden’s index (sensitivity + specificity − 1) in the training and test sets. We selected the final number of genes to use in the classifier by finding the maximum Youden’s index, averaged across the five folds, among the training sets. From this final model, we predicted the AR status of all ER-negative samples, using predictions to calculate a final sensitivity and specificity. We also assessed model performance by conducting a principal component analysis of RNA expression for the selected genes, coloring samples by AR status to visually inspect how gene expression patterns correlated with AR. Finally, we applied the AR classifier to all ER-negative CBCS samples assayed for selected genes (N = 669) and proceeded to compare AR phenotypes to other molecular indicators, described below.To understand overlap between AR-phenotype and deficiencies in immune and DNA repair processes, we analyzed CBCS samples that had RNA expression data on both AR and immune or AR and DNA repair classes. However, because a relatively small number of samples profiled for AR also included information on RNA expression of DNA repair genes  and two DNA repair classes (recombination/Fanconi anemia (HR/FA), and non-HR/FA) according to published methods ). As aboN = 872 with protein and RNA data) to determine how well findings at the RNA level captured post-translation AR protein status.We used TCGA to validate associations between AR status and molecular tumor characteristics. After applying our ClaNC classifier of AR to ER-negative samples from TCGA, we used the composite Homologous Recombination Deficiency (HRD) Scoring method from Kninjenburg et al.  to assesr = 0.68, p < 0.001; Fig. We detected associations between single-gene AR RNA expression classes (high vs. low) and aggressive clinical features. RNA levels of AR were strongly correlated with AR protein as measured by RPPA , women with low AR expression (N = 304) were more likely to be young, Black, HER2 negative, and high grade. Associations were strongest between AR status and high risk of recurrence genomic scores (ROR-P), with AR-low tumors having a 37% higher prevalence of high ROR-P scores than AR-high tumors (95% CI = 28%–45%). These associations, with the exception of Black race, were somewhat attenuated but remained significant after adjusting for triple-negative status and replicated within triple-negative tumors alone, suggesting that low AR is associated with tumor aggressiveness in ER-negative tumors independent of triple negativity. Similar patterns were observed in ER+ tumors, with AR-low/ER+ tumors being more likely to have high grade, stage, or ROR-P scores than AR-high/ ER+ tumors (Supplementary Table 1). Most ER-borderline tumors were also AR low. Results did not substantially differ after multiple imputation.Table Given strong associations between low AR and aggressive tumor features, we developed a classifier to identify low AR status using other independent gene expression data when AR expression was missing Fig. . FivefolApplying the classifier to the 669 ER-negative CBCS tumors with relevant RNA data, we detected 545 82.8%) tumors with AR-low phenotypes. Again, AR-low tumors were more likely to be younger, Black, HER2-negative, grade III, stages II-IV, and have high ROR-PT scores (Supplementary Table 2) as also observed in the TCGA cohort (Supplementary Table 3). ER/AR-low tumors also showed strong evidence of aggressive molecular phenotypes, with 47.0% having enrichment for adaptive immune tumor subtypes, and 85.6% showing enrichment for homologous recombination-related genes  with AR-low phenotypes. AR-low tumors showed higher frequency of homologous recombination deficiency (HRD), with 81 (39.3%) having HRD scores above the clinical cutoff of 42 as compared to 11 (5.3%) of AR-high tumors. We did not find strong evidence of an association between AR and immune expression phenotypes in TCGA , although the TCGA lacks evidence of the immune quiet phenotype due to different selection factors for inclusion in TCGA . HoweverN = 375–389), and red lines represent predictions in the test set (N = 93–97). C. Final performance of Youden’s Index-maximizing classifier using seven genes per group. Correct classifications are shown in green, while incorrect classifications are shown in red. Sensitivity was 86.8%, specificity was 87.5%, and overall accuracy was 87.1%. D. Principal component analysis based on RNA expression of classifier-selected genes in ER-negative CBCS samples. Red samples are those with low AR expression, and blue samples have high AR expression.Fivefold cross-validated Youden’s index and sensitivity of Classification to Nearest Centroids classifier according to number of genes used to predict each AR phenotype. Confidence intervals represent mean plus or minus standard error. Blue lines represent predictions in the training set  [Our findings are consistent with what has been previously reported for demographic factors. Park ctively) . Severalctively) , 34–36. ctively) –39.Our findings also corroborate prior evidence suggesting that AR-negative TNBC is associated with aggressive disease features such as advanced stage and high histological grade , 10, 11.Another distinction observed herein between AR-negative and AR-positive TNBC was with respect to immune profiles. Consistent with our findings, Davis and colleagues previously reported that AR-negative tumors are upregulated in T-cell marker (CD4 and CD8), immune checkpoint , and immune cell-signaling pathway marker  RNA expression compared to AR-positive tumors in TNBC . These fPrevious studies have not evaluated associations between AR and specific DNA repair pathways. We observed a higher prevalence of homologous recombination deficiency in AR-low (versus AR-positive) ER-negative-breast cancer. This finding is consistent with previous studies showing increased genomic instability in AR-negative (versus AR-positive) TNBC. It has been shown that AR-negative TNBCs have 1) increased epidermal growth factor receptor, cyclin-dependent kinase 6, Ki-67, and topoisomerase 2a but 2) downregulated PTEN and HER4 –55. It wDue to study-specific differences in normalization methods and RNA sequencing protocols, the cutoffs used in our analysis may not translate directly to other analyses, though RNA-based classifiers and mixture models should yield similar results. Another limitation was the small sample size of ER-negative breast cancer patient participants, particularly and TNBC patients. QNBC is a subgroup of TNBC that lacks AR expression and has been reported to be more biologically aggressive and distinct from AR-positive TNBCs . A largeOur study’s findings suggest RNA-based methods may be valuable for identifying AR-low, ER-negative breast cancer patients. This gene expression-based approach may capture AR-dependent tumor biology that has been inconsistently observed based on IHC alone. Other research groups have designed multigene signatures that predict response to AR-therapy better than biomarker-based IHC alone , 58. In Supplementary file1 (DOCX 24 KB)Supplementary file2 (PDF 79 KB)Below is the link to the electronic supplementary material.


P < 0.05). Marital status, annual income, medical insurance, health problems, and disability might be factors independently associated with the unmet needs for rehabilitation services. Attention should be paid to the financial burden of the population on rehabilitation services, and in addition to the disabled, the slow patients should also be given priority.To investigate the unmet needs for rehabilitation services among middle-aged and older adults in Chengdu, Sichuan, China, and identify the associated factors. This cross-sectional study was conducted on middle-aged and older adults in Chengdu, Sichuan, China, between 2015 and 2016. The questionnaire included demographic data and questions about rehabilitation needs. Multivariable logistic regression analysis was used to identify the associated factors of unmet needs for rehabilitation services. Among 663 participants, 91.70% needed medical rehabilitation (608/663), 26.55% of who need auxiliary equipment (176/663), 77.07% of who need daily care and social participation (511/663), and 79.34% of who need recreational therapy activities (526/663), while < 30% required auxiliary equipment. Multivariate logistic regression analysis showed that residents who were married, had annual income < CNY 80,000, had no medical insurance, had three or more health problems, were aged ≥ 60, and the disability status were independently associated with unmet needs for rehabilitation services (all  Community rehabilitation and home rehabilitation both have great demand and development potential in terms of physical function and subjective will of older adults1. Community schizophrenia patients have rehabilitation needs in many areas, including social security, emotional management, treatment compliance, and symptom management2. It is due to multiple reasons, such as high costs, lack of training in medical rehabilitation, and doubts related to the therapeutic benefits of rehabilitation3.Many rehabilitation organizations, such as the International Society of Physical and Rehabilitation Medicine, have been formed in response to the recommendations of the United Nations and the World Health Organization for the active promotion of global rehabilitation medicine. This healthcare aspect in the People’s Republic of China has been associated with an increasing focus on improving the overall and long-term rehabilitation service. The status of rehabilitation medicine in China currently reflects the conclusions of existing studies on the need for rehabilitation services. China has established its three-tier service network, which has constantly been improving. However, despite the great demand for patients for rehabilitation services in Mainland China, these services, including community-based rehabilitation, seem inadequate. Nearly 90% of the older adults in Guangzhou are willing to receive rehabilitation treatment4. Eldar et al.5 have demonstrated that China’s rehabilitation services are relatively closely aligned with clinical practice, while several previous studies reported a high need for rehabilitation services in Mainland China9.Previous studies on rehabilitation needs in China and other countries have concluded that it is important to consider the public’s perceptions in developing rehabilitation services10 studied the older adult population in Ontario, Canada, reporting that their needs for rehabilitation services were not effectively met11. These population-based studies are lacking in China, particularly those focusing on middle-aged or older adults.One approach to learning about the need for rehabilitation services in communities is to study large samples of community populations. For example, Armstrong and colleagues12. Previous studies identified financial factors that could predict the use of rehabilitation resources, including physical dependence, major illness, time spent in medical care, having a family physician, having illnesses, age, and intact cognitive functions14. No similar study on rehabilitation needs in Mainland China has been conducted.Previous research has mainly focused on community groups with particular needs for rehabilitation services. For example, a study on the need for rehabilitation services among people with disabilities being discharged from hospitals in Poland revealed that orthopedic equipment and home renovation service did not sufficiently meet their needsTherefore, this study investigated the need for rehabilitation services among all middle-aged and older adults in three Qingyang District communities in western Chengdu, the capital city of Sichuan Province, as well as the factors associated with the unmet needs for rehabilitation.This cross-sectional study investigated the unmet needs for rehabilitation services among middle-aged and older adults in three Chinese communities in Qingyang District, Chengdu, Sichuan, China, between 2015 and 2016. The inclusion criteria were (1) adults > 45 years with good cognitive ability, (2) permanent residents of Qingyang District, Chengdu, or outpatients, and (3) people with some need for rehabilitation. The exclusion criteria were (1) acute illnesses or (2) an incomplete questionnaire. This research was approved by The Chengfei Hospital Ethical Review Board . Written informed consent was obtained from all participants. All methods were carried out in accordance with relevant guidelines and regulations. All procedures were performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.This study was based on a survey administered using a structured interview. The potential respondents were sent invitations in advance, saying that the investigators would contact them and that they were free to participate or not. Then, within 1 month of the invitation, the investigators met the respondents, obtained written informed consent, conducted the face-to-face interview, and filled in the interview table.22. In the MMSE, a score < 24 is considered cognitive impairment23. In the CCSE, a score < 27 was considered cognitive impairment24. As long as a score on one of the scales meets cognitive dysfunction, it is considered cognitive dysfunction. (3) Those with intellectual disabilities were excluded from the sample according to the statistical principle of full consideration of the population sample.The determination procedures of the cognitive abilities of the participants were as follows. (1) Confirm whether participants had clear consciousness. The Glasgow Coma Scale (GCS) was used to determine the degree of consciousness disorder. Clear consciousness of the participants was a prerequisite for cognitive function assessment. (2) Screening for cognitive dysfunction. Under the condition of clear consciousness, the participants were screened for cognitive dysfunction using the Brief Psychiatric State Examination Scale (MMSE) and Cognitive Ability Examination Scale (CCSE), which is a key step in the assessment of cognitive dysfunction. The two scales are currently equally effective, and all participants completed both scales25, and sampled twice as many residents as necessary for stable statistical analysis. Thus, 820 residents were sampled using the simple random sampling (preliminary survey) and systematic sampling  methods with SPSS. The respondents were randomly selected from the official residency lists.At the end of 2014, the three communities had 69,771 residents, 10,933 of whom were > 45 years old. For sampling, this study assumed a response rate based on the usual response rate of questionnaire surveys in China, about 50%26. The “unmet needs for rehabilitation” that individuals might experience refer to any needs for rehabilitation services identified by a healthcare provider that the individual is unable to get due to different barriers26.In this study, the “need for rehabilitation” refers to any need that an individual with a health condition may have that requires rehabilitation management, interventions, and/or the use of specialized rehabilitation services or programs27, including medical rehabilitation, auxiliary equipment, daily care, social participation, and recreational therapy activities. At the end of the CRNQ, the respondents were asked to list all their unmet needs for rehabilitation services in order of importance.The City Rehabilitation Service Needs Questionnaire (CRNQ) includes questions about residents’ characteristics and rehabilitation needs. Appendix 26. In addition, living in a residential home means better adjustment to the living environment and better provision of orthopedic and rehabilitation equipment28. Regarding reliability, the data used in the analysis were tested, and Cronbach’s reliability coefficient α was 0.92 for the sample (n = 663). The response rate was 80.8%.A pilot study was conducted to ensure the questionnaire’s validity and reliability. A total of 30 middle-aged and older adults participated in the study. Based on discussions with the middle-aged and older adults who participated in the pilot study, categories were developed to help respondents understand the definitions of rehabilitation medicine and the need for rehabilitation services. After the preliminary survey, categories were developed to help respondents understand the definition of rehabilitation medicine and the need for rehabilitation services. During the questionnaire development process, the unclearness of the description was found for the questions of “family income” and “whether there is an elevator in the building” through the pre-survey, and then the questionnaire was revised after discussion with the expert group. Five experts in neurology, disability, public health, and rehabilitation medicine reviewed and approved the items. The experts found correlations between “service cost”, “home environment”, and “rehabilitation needs according to the research on rehabilitation needs” according to the following viewpoints. The unaffordability of the services, especially in low-income countries, represents an additional barrier for people in need of rehabilitation29. The disability certificate from the Ministry of Civil Affairs was required to be provided by the respondents.The definition of disability in the teaching materials of Rehabilitation Medicine of the Ministry of Health of China is “Disability refers to obvious physical and mental dysfunction caused by trauma, disease, developmental defects, or mental factors, affecting the ability to live, work, and learn normally to varying degrees. Disability in a broad sense includes sickness and disability, which is a general term for physical and mental dysfunction of the human body”P < 0.05 was considered statistical significance.SPSS version 23.0 software  was used for statistical analysis. Chi-square for contingency or Fisher’s exact test was used for the categorical variables. Multivariable logistic regression analysis was used to estimate the odds ratios (ORs) and 95% confidence intervals (95% CI) of the relationships between independent variables and the perception of unmet needs for rehabilitation services. Only the statistically significant independent variables in the bivariable analysis were included in the multivariate regression model, and variables lost the significance in the multivariate analysis and were excluded from the final model and not shown. All This research was approved by The Chengfei Hospital Ethical Review Board . Written informed consent was obtained from all participants. All methods were carried out in accordance with relevant guidelines and regulations. All procedures were performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.There were 157 residents excluded: 145 did not complete the questionnaire , six had an acute stroke, and six reported no need for rehabilitation  of the respondents had unmet needs for rehabilitation service, 26.55% (176/663) needed auxiliary equipment, 77.07% (511/663) needed daily care and social participation, and 79.34% (526/663) needed recreational therapy activities (Table P = 0.046), had annual income < CNY 80,000 , were without medical insurance , and had three or more health problems  were more likely to have unmet medical rehabilitation needs. The factors that had a significant impact on unmet auxiliary equipment needs were age ≥ 60  and disabilities . Unmet recreational needs were significantly higher among respondents who were married , had annual incomes < CNY 80,000 , or had no medical insurance  . Policymakers and healthcare professionals should adjust their efforts to ensure to meet the rehabilitation service needs of the older adults in the community. These findings are consistent with the results of Kołłątaj et al.12; nonetheless, they are still limited in Mainland China and have a limited application to individuals with disabilities5.The respondents who reported more than three health problems were also perceived as having more unmet medical rehabilitation needs than healthier individuals, contrary to the findings of a previous study39. However, our results did not show any impact of education on unmet rehabilitation service needs; thus, further studies are needed to clarify the relationship.Some previous studies found correlations between educational attainment and rehabilitation service needsThe findings of the present study can assist in raising public awareness and education initiatives in the Rehabilitation Medicine Department of Chengfei Hospital and community health centers. This study may further the integration and guidance of management departments responsible for community rehabilitation resources. Moreover, it may further the efforts to increase medical insurance coverage and rehabilitation service projects, as well as improve facilities for people with disabilities.Based on the obtained results, we would like to propose several public health recommendations: firstly, the government should establish rehabilitation quality control centers in the three-tier health system, secondary hospitals, and rehabilitation institutions, emphasizing the necessity to meet the needs of married people, those with low income, and the uninsured population in the community. Secondly, this study might serve as a valuable reference for the Community-Based Rehabilitation Committee of the Chengdu Society of Rehabilitation Medicine (a folk medicine organization) for its work in community rehabilitation. Thirdly, the reported data highlight the vulnerabilities of uninsured people, encouraging Chengdu Human Resource and Social Security Bureau to address these concerns.32. Future studies should address these two demographic design flaws to provide a complete idea of the rehabilitation needs of older adults in Chengdu. Second, we examined fewer rehabilitation service types compared to previous studies in developed countries41. For example, we did not test unmet needs for physical therapy, occupational therapy, physiotherapy, traditional or basic rehabilitation, speech, swallowing, or psychological rehabilitation. Third, some social and financial factors were not tested. For example, a previous study in Nepal found that mountain geography, poor road infrastructure, and inconveniences influenced access to community rehabilitation services42. All our respondents were residents of urban Chengdu, located on the Chengdu Plain, where the altitude varies by no more than five meters. However, additional social and financial factors should be analyzed. Fourth, the rehabilitation service project implementers might have biased the research results43, which we did not control in this study. Finally, using established questions to assess rehabilitation service needs provides only general information. Future studies employing qualitative methods to understand better the needs for rehabilitation services and individual expectations for rehabilitation are required.Despite the aforementioned contributions, the current study has several limitations. First, the sample was not representative, considering the sampling was randomized in the three Qingyang District communities. The demographics of this city’s middle-aged and older population require further investigation. Nonetheless, this kind of community differentiation was used in previous studies44, we administered questionnaires in person, considering that such an approach has been identified as the optimal approach in epidemiological research45.However, although similar studies have used different methods in other populationsThis study found that > 75% of residents had unmet needs for rehabilitation, including medical rehabilitation, daily care, social participation, and recreational therapeutic activities, while < 30% were in need of auxiliary equipment. Marital status, financial factors, and medical insurance affected the respondents’ rehabilitation service needs. Rehabilitation programs should target these vulnerable groups and acknowledge that the lack of auxiliary equipment for older adults and individuals with disabilities is a major problem in current rehabilitation efforts.


Low T3-(/T4-) syndrome, also known as non-thyroidal Illness Syndrome (NTIS) describes a decrease in free serum thyroid hormones without a concomitant increase in TSH, frequently observed in critically ill patients. However, whether NTIS is only a metabolic adaption to stress in critically ill or plays a crucial role as an independent risk factor for ICU mortality, remains unknown. Our study aimed to evaluate NTIS as an independent risk factor for increased ICU mortality.All patients admitted to the interdisciplinary intensive care unit (ICU) at the University Hospital of Leipzig between 2008 and 2014 were retrospectively analyzed for thyroidal function. Baseline data, information on additional thyroid function tests, disease progression, hospital and ICU length of stay (LOS) and patient outcome were retrospectively analyzed from the hospitals digital information system. For statistical evaluation, univariate analysis, matched pairs analysis and multivariate logistic regression were conducted.One thousand, seven hundred ninety patients were enrolled in the study, of which 665 showed NTIS. Univariate analysis revealed a positive association of NTIS with ICU- and hospital-LOS, need for mechanical ventilation, incidence of sepsis, acute respiratory distress syndrome, acute liver failure and increased ICU mortality. Results of matched pair analysis confirmed these findings. In multivariate logistic regression, NTIS was associated with an increased ICU-LOS, increased duration of mechanical ventilation, acute kidney injury and liver failure, but showed no independent association with increased ICU-mortality.Duration of mechanical ventilation as well as incidence of acute kidney injury, sepsis and acute liver failure were detected as independent predictors of mortality in patients with NTIS. NTIS itself was no independent predictor of increased ICU-mortality.The online version contains supplementary material available at 10.1186/s12871-023-02015-1. Non-thyroidal Illness Syndrome (NTIS) or Low T3-(/T4-) Syndrome describes a decrease in free serum thyroid hormones without a corresponding increase in thyroid stimulating hormone (TSH) in the absence of manifest thyroidal illness , 2. It iWe conducted a retrospective observational cohort study in patients admitted to the interdisciplinary operative ICU at the University Hospital of Leipzig between December 2008 and December 2014. All patients were screened for existing data on serum levels of TSH, fT3 and fT4, which are tested routinely once per week according to our ICU protocol. Patients younger than 18 years, an ICU length of stay (LOS) of less than 24 h, any history of thyroidal or pituitary disease as well as medication affecting thyrotrophic axis  were excluded from the study. The study protocol was approved by the ethics committee of the Medical Faculty of the University of Leipzig (reference number: 362/18-ek).ECLIA) from ROCHE .Baseline parameters included age, gender, severity of disease and body mass index (BMI). For severity of disease, we registered Simplified Acute Physiology Score II (SAPS II) at the timepoint of admission to ICU. Patients were screened for the occurrence of sepsis, pneumonia, acute respiratory distress syndrome, acute liver failure and acute kidney injury (AKI) as well as the need for renal replacement therapy and mechanical ventilation. All data were retrospectively collected from the hospitals´ information system . NTIS was assumed, when fT3 and/or fT4 levels were decreased, with normal or decreased levels of TSH. Patients with known diagnosis of any thyroid illness were excluded. NTIS was classified into two subgroups as follows: the “NTIS Low fT3” group with low serum level of fT3, normal serum level of fT4 and low or normal serum level of TSH, and the “NTIS Low fT3 fT4” group with low serum level of fT3 and fT4 and low or normal serum level of TSH. Patients with any other patterns of discordant thyroid function, were grouped into the Non-NTIS cohort. The normal range of serum hormone concentrations at our laboratory were as follows: fT3: 3.1 – 6.9 pmol/l; fT4: 12.8 – 20.4 pmol/l; TSH: 0.4 – 3.77 mU/l. Quantitative serum thyroid hormone levels were analyzed by immune-assay method by electrochemiluminiscence -immunoassay . For multiple comparison of pair-wise differences, we used Mann–Whitney U-test with Bonferroni-Correction. For the identification of parameters independently associated with NTIS, univariate logistic regression analysis was performed. To increase power of the statistical testing, we used matched pair analysis. Two groups of pairs were formed using a propensity score adjustment: NTIS Low fT3 and Non-NTIS, as well as NTIS Low fT3 fT4 and Non-NTIS. Matching was done, using age, gender, height, BMI and SAPS II-score at the timepoint of admission. Differences between groups were tested using the Mann–Whitney-U-Test or χ2-Test. Risk factors of univariate analysis with p values of < 0.1 were included in the multivariate analysis to identify significant independent risk factors in association with NTIS. A logistic regression analysis was performed to test for independent predictors of ICU-mortality and hospital-mortality. To characterize logistic regression, p value of chi-square (expected to be < 0.05), Nagelkerke’s R2, results of the Hosmer–Lemeshow goodness of fit statistics (expected to be > 0.05), sensitivity, and specificity of the statistical model are given. A p-value < 0.05 was considered statistically significant. Statistical analyses were executed with SPSS 24.0 .With regard to baseline data, continuous variables are reported as median with interquartile range  whereas categorical variables are presented as whole numbers and percentages (%). Univariate analyses and matched pair analyses were performed according to the above-named groups. In particular, NTIS patients were matched with Non-NTIS patients according to age, gender, BMI and SAPS II. Differences between these groups were compared in a multivariate analysis. All numeric parameters were tested by Kolmogorov–Smirnov-Tests for normal distribution and are expressed as median (MD) and interquartile range . Statistical univariate comparisons between patients with and without NTIS were performed using the χp < 0.001 vs. Non-NTIS) and an increased incidence of co-morbidities in the NTIS groups . Incidence of intracranial hemorrhage/lesions and diabetes were comparable between the study groups.Three hundred eighty one Non-NTIS patients were matched to 381 NTIS Low fT3 patients, and 284 Non-NTIS patients were matched to the same number of patients with NTIS Low fT3 fT4 (Table p = 0.002) as an independent predictor for NTIS Low fT3 , ICU-LOS  and acute liver failure  were identified as independent predictors for NTIS Low fT3 fT4  or low fT3-(/fT4-) syndrome, strongly correlates with the severity of critical disease . HoweverIn our current retrospective study, we aimed to enlighten the possible associations between patients’ characteristics, severity of critical disease and NTIS in a large cohort of critically ill patients. Univariate analysis showed significant differences between patients with and without NTIS in numerous analyzed parameters, including age, severity of critical disease and ICU-LOS. No differences were found for gender and BMI. Our current findings are in accordance with data from a variety of former studies –6, howevOur matched pair analysis showed a significant increase in a multitude of co-morbidities as well as an inferior outcome in NTIS Low fT3 fT4 patients compared to Non-NTIS patients. These findings were less evident, when patients with isolated Low fT3 were compared to Non-NTIS patients Table . RegardiAlthough our current study shows interesting results, several limitations should be discussed. Firstly, by implication of the retrospective setup, we had no influence on the timing of laboratory workup and thyroid function testing. Secondly, critically ill patients are a generally inhomogeneous group of patients ranging from severe trauma to infections or intoxications. This data incorporates all groups of patients despite the fact, that the cause of NTIS is likely multifactorial and may also vary in different groups of patients. However, due to the very large number of patients, this might also be a strength as it may still reveal common factors. Thirdly, and probably most importantly, our study can only show associations, but no causalities. The question of causalities, hence the question, whether NTIS is either the sequel or rather one of the reasons for critical illness, may only be answered from this point on by large observational, multicenter clinical studies, correcting for known risk factors. A direct extrapolation of our results to clinical recommendations without prior evaluation in prospective studies cannot be recommended. However, such studies do not yet exist and, to our knowledge, so far, our study is the largest retrospective database analysis addressing this relevant topic.This study is the largest database study on critically ill patients with NTIS. It further enlightens the association between NTIS and a multitude of critical co-morbidities, frequently observed in ICU patients. Our central finding is that NTIS Low fT3 and NTIS Low fT3 fT4 were no independent predictors for increased mortality. This confirms the current assumption that NTIS is merely one part of the adaptation to severe illness and has—according to our current knowledge—no pathological entity of its own.Additional file 1:Suppl. Table 1. Multivariate regression, independent risk factor for NTIS Low fT3. Suppl. Table 2. Multivariate regression, risk factors for NTIS Low fT3 fT4.


Ziziphus jujuba Mill.) is a commercially important tree native to China, known for its high nutritional value and widespread distribution, as well as its diverse germplasm resources. Being resilient to harsh climatic conditions, the cultivation of jujube could provide a solution to food insecurity and income for people of arid and semi-arid regions in and outside of China. The evaluation of germplasm resources and genetic diversity in jujube necessitates the use of Simple Sequence Repeat (SSR) markers. SSR markers are highly polymorphic and can be used to evaluate the genetic diversity within and between cultivars of Chinese jujube, and are important for conservation biology, breeding programs, and the discovery of important traits for Chinese jujube improvement in China and abroad. However, traditional methods of SSR development are time-consuming and inadequate to meet the growing research demands. To address this issue, we developed a novel approach called Multiple-Genome-Based SSR identification (MGB-SSR), which utilizes the genomes of three jujube cultivars to rapidly screen for polymorphic SSRs in the jujube genome. Through the screening process, we identified 12 pairs of SSR primers, which were then used to successfully classify 249 jujube genotypes. Based on the genotyping results, a digital ID card was established, enabling the complete identification of all 249 jujube plants. The MGB-SSR approach proved efficient in rapidly detecting polymorphic SSRs within the jujube genome. Notably, this study represents the first successful differentiation of jujube germplasm resources using 12 SSR markers, with 4 markers successfully identifying triploid jujube genotypes. These findings offer valuable information for the classification of Chinese jujube germplasm, thereby providing significant assistance to jujube researchers and breeders in identifying unknown jujube germplasm.Jujube ( Ziziphus jujuba Mill.) belongs to the genus Ziziphus of family Rhamnaceae [Ziziphus species, it has the largest cultivation size and the most commercial and ecological importance [Jujube  [Gossypium sp.) [Similarly, molecular markers used for species classification, such as RAPD, RFLP, AFLP, SRAP, and Simple Sequence Repeat (SSR), offer advantages over morphological classification approaches ,10,11,12mmularia , Apricotmmularia , Taxus [mmularia , olive [mmularia , Jinsha mmularia , banana sa spp.) , cotton ium sp.) , Grapes ium sp.) ,23,24, aSSRs, also known as microsatellites, are short, repeated DNA sequences consisting of 1–6 nucleotide motifs . IdentifThe development of SSR markers has become cost-effective now due to reduced sequencing costs and genome assemblies. Based on the genome sequences of three closely related jujube genotypes , this study developed an effective SSR screening approach (MGB-SSR) that decreased experimental costs and streamlined the workflow. The 12 SSRs identified by this method effectively discriminate the three jujube genotypes. In addition, the representativeness of these SSRs among different jujube germplasm resources was determined, and based on the results, barcoding IDs for 249 distinct jujubes were generated using the 12 identified SSRs.The analysis of SSRs in the three jujube genomes yielded comparable proportions of SSRs with the same core unit length. Trinucleotide repeats constituted the majority, at 71.12% of the total, followed by tetranucleotide repeats at 20.09%, pentanucleotide repeats at 5.53%, and hexanucleotide repeats at approximately 3.25%. Notably, the number of detected SSRs exhibited a positive correlation with genome size .By conducting a comparative analysis of the SSR sequences among the three jujube genomes, we identified a total of 286 polymorphic SSR sequences. Initially, sequences that exhibited variations only in two genomes were excluded, followed by filtering based on base mutations, resulting in a final set of 35 SSR sequences . SubsequAmong the 12 pairs of SSR primers that met the criteria, seven contained three-nucleotide repeats, while five contained four-nucleotide repeats. The primers were divided into four groups based on the color of the fluorescent adapter: FAM (green): LSSR-10, LSSR-25, and LSSR-27; HEX (blue): LSSR-17, LSSR-29, and LSSR-26; ROX (red): LSSR-4, LSSR-23, and LSSR-6; TAME (black): LSSR-8, LSSR-28, and LSSR-22 .Using these 12 pairs of primers, successful amplification of the expected fragments was achieved for 249 jujubes. Each jujube individual displayed a diverse range of genotyping results. For instance, the LSSR-22 primer revealed three different genotyping results in the samples ‘PingshunJunzao’, ‘Xiangfenyuanzao’, and ‘Linfentuan zao’ .A total of 106 alleles were identified across 12 polymorphic SSR markers, resulting in an average of 8 alleles per SSR marker. The markers LSSR-22 and LSSR-26 exhibited the highest allelic diversity with 15 alleles, while LSSR-4 and LSSR-6 displayed the lowest diversity with 5 alleles each.The uniformity of allele frequency was assessed using effective alleles, which ranged from 2.0396 to 7.8924 across all markers, with an average of 3.985 . GeneticTo evaluate the polymorphism within the SSR population, the polymorphism information content (PIC) was calculated, which represents the informativeness of a marker, with higher values indicating greater polymorphism and diversity within the population. The highest PIC value was observed for LSSR-26, reaching 0.861, indicating significant polymorphism within this marker.The phylogenetic tree, constructed using SSR data, classified the 249 jujube genotypes into four main groups, with the wild sour jujube dispersed among all groups. The first group comprised the largest number, accounting for approximately 43% (107 jujubes), while the fourth group contained at least 22 genotypes refer to . JujubesPrincipal coordinates analysis (PCoA) is a useful method for assessing genetic relationships among species. In this study, PCoA was performed on 249 jujube genotypes. By utilizing the first and second principal components as horizontal and vertical coordinates, the jujubes were categorized into two main groups, representing cultivated and wild jujube, respectively. Although there were a few scattered genotypes, the majority of jujube and wild jujube exhibited a close genetic relationship. These findings align with the results obtained from the phylogenetic tree analysis, indicating a high level of genetic similarity between jujube and wild jujube .The amplification results for all jujubes were organized in a sequential order corresponding to the SSR markers: LSSR-4, LSSR-6, LSSR-8, LSSR-10, LSSR-15, LSSR-17, LSSR-22, LSSR-23, LSSR-26, LSSR-27, LSSR-28, and LSSR-29. This sequential arrangement generated a 24-digit ID for each cultivar . NotablyThe SSR molecular marker technique is widely utilized due to its co-dominant inheritance and high polymorphism. It finds extensive application in genetic diversity analysis, determination of genetic relationships, variety identification, core germplasm selection, and molecular identification ,35. GeneAdditionally, based on the detected SSR count and proportion in the three jujube genomes, we observed that the proportion of SSRs with the same core repeat unit length varies only slightly across different genomes . Despite being jujube cultivars, ‘Dongzao’, ‘Junzao’, and ‘Suanzao’ exhibit significant genomic differences; for instance, their final assembled genome sizes are 437.7 Mb, 351 Mb, and 406 Mb, respectively. ‘Dongzao’ and ‘Suanzao’ have genome sizes that are more similar to each other compared to ‘Junzao’. According to the statistical analysis of SSR quantity, the genome size, and SSR count show a positive correlation . In conclusion, SSRs are uniformly distributed in the jujube genome, and the genome size significantly influences the quantity of SSRs.Traditional methods for developing SSR markers can be classified into three categories: (1) database and literature searches to identify relevant SSR information, (2) cross-amplification using common primers among closely related species, and (3) utilizing software to identify microsatellite loci in genomic DNA (gDNA), complementary DNA (cDNA), and expressed sequence tag (EST) sequences. With advancements in sequencing technology, new methods for identifying SSRs have emerged ,45,46. F(1)Efficient SSR screening: The utilization of multiple closely related genomes in the MGB-SSR approach allows for the identification and elimination of invalid sites that are identical across genomes, and this avoids the massive selection from thousands of candidate SRRs. This significantly reduces the time and financial resources required for screening.(2)Enhanced accuracy with capillary electrophoresis: By combining SSR screening with capillary electrophoresis, the MGB-SSR method overcomes the limitations associated with traditional gel electrophoresis. Issues such as uneven distribution of PCR products between lanes due to variations in gel concentration and voltage are eliminated, resulting in more accurate and reliable test results.(3)Universal applicability: With advancements in sequencing technology, it has become feasible to obtain 2–3 closely related genomes for most fruit trees. This means that the SSR markers developed using MGB-SSR can be applied across various species within the same family or even different genera. Furthermore, genetic diversity analysis has demonstrated that the SSR markers developed through MGB-SSR exhibit significant polymorphism, as indicated by the Na, Ne, and PIC parameters meeting the standard criteria.(4)Detectable polyploidy: The MGB-SSR method has shown effectiveness in detecting polyploid jujube germplasm resources. With the increasing number of jujube hybrid varieties, the evaluation and identification of polyploid jujube are becoming more important. In this study, four of the SSR markers utilized effectively characterized polyploidy in jujube, demonstrating their potential for this application.(5)Compared to utilizing markers from the SNP array and the commonly used WGS strategy, which require a substantial amount of genome re-sequencing for genotyping and classifying different jujube germplasm resources, our method using only 12 SSRs significantly reduces both time and financial costs.The MGB-SSR method proposed in this study offers several advantages over traditional methods:In summary, the MGB-SSR method provides a more efficient and accurate approach to SSR screening, offering cost and time savings, improved accuracy through capillary electrophoresis, and broad applicability for genetic diversity analysis in fruit trees and other related species. Additionally, it shows promise in detecting polyploidy in jujube germplasm resources.The utilization of a set of highly polymorphic SSR markers significantly reduces the possibility of concordance between individuals, ensuring accurate molecular identification. This technology has been successfully applied in various crops such as nuts and melons ,48,49,50www.ncbi.nlm.nih.gov, accessed on 11 January 2022.): ‘Dongzao’ genome [The genome data of three jujube genotypes were downloaded from NCBI’s Genome database  was used to identify the SSR loci in the three jujube genomes, and the parameters were set to 3–6 nucleotides for the core repeat unit and the number of repeat times <3. Subsequently, the core unit length, number of repetitions, and repetition frequency were counted, and the distribution properties of SSR in the jujube genome were studied by comparing the identified results of the three genomes.The MISA program  (version(1)Create Blast databases for the three jujube genomes.(2)The SSR sequences were subjected to Blast alignment against the corresponding jujube genome database, and subsequently, non-unique results were removed, retaining only the specific SSRs.(3)The remaining specific SSRs were subjected to a mutual comparison with the other two jujube genome databases. During this comparison, any results that were found to be identical within any of the two jujube genomes were discarded. Additionally, SSRs showing less than 90% consistency across the three genomes were also excluded.(4)All the remaining results were statistically merged, and those with consistent conservative sequences at both ends but differing core repeat units were selected.(5)Eliminate the results that show differences only between two genomes, thus highlighting the variations among the three genomes at the SSR level.Based on the SSR position information obtained from the MISA program, we extracted the core repeat unit and 300 bp conserved sequences at both ends of each SSR from the genome. Using these extracted SSR sequences, we then performed a screening process to identify the polymorphic SSRs in the three genomes. The steps for this screening process were as follows:The pipeline is displayed in (1)They were designed in conserved regions near both ends of the core repeat sequences;(2)The length of the amplicons ranged between 50 and 300 bp;(3)The primers had similar annealing temperatures;(4)To prevent primer dimers, there was no complementary sequence between the primers;(5)Primer specificity was assessed using NCBI-BLAST;(6)The upstream primer had an 18 bp M13 linker sequence added to the 5′ end, which matched with fluorescent linker primers of different colors .The selection of primers for SSRs followed specific criteria using the Primer Premier 6 software (version 6.24) . The choTo evaluate the efficacy of each primer pair, 6-FAM fluorescent adapter primers were employed, and test samples were selected from a diverse range of jujube varieties collected from different locations in China. The sample set consisted of 26 diploid jujubes (numbered 1–26) and 4 triploid jujube cultivars, including ‘Zanhuangdazao’ and 3 triploid progenies resulting from the cross between ‘Dongzao’ and ‘Chenguang’. This selection ensured the representation of a wide spectrum of SSR markers. The PCR amplification results were observed through on-machine detection to assess the characteristics of the samples.The PCR (polymerase chain reaction) system was carried out in a volume of 20 μL: 2× Taq PCR buffer 10 μL; 10× primer 0.6 μL ; genomic DNA 20 ng with ddH20 to 20 μL. The temperature profile was set as 95 °C (5 min); cycles, 20 times ; 72 °C (10 min) for final extension. Then, the product was stored at 4 °C.Following the detection of the PCR product through agarose electrophoresis, 0.3 μL of the PCR product, 0.5 μL of the molecular weight internal standard, and 9.5 μL of deionized formamide were combined and added to the PCR plate. The mixture was then denatured at 95 °C for 5 min, cooled at 4 °C, and centrifuged. For machine detection, 1× buffer was added. The detection findings were entered into Genemarker (version 2.2.0), and subsequently, the electropherogram and site information table were exported.Using the aforementioned system, PCR amplification was performed on all DNA samples, and the results were analyzed. The software Popgen 32 (version 1.32) was utilized to calculate various parameters such as the number of alleles, effective number of alleles, and expected heterozygosity for each sample. The SSR data were then converted into a 0/1 matrix (representing the presence or absence of stripes) and analyzed using the ‘ape’ package (version 5.7-1) in R language . The evowww.chiplot.online, accessed on 6 February 2023.) tool was used to visualize the generated ID cards.The genotyping results for each SSR marker were sorted and assigned numbers. The value ‘00’ indicated undetectable or missing typing, while the remaining values ranged from ‘01’ to ‘99’, ordered in ascending order . SubsequThis study presents a novel method, MGB-SSR, for identifying SSRs in the jujube genome. MGB-SSR offers a rapid and cost-effective approach to screening polymorphic SSR markers with superior efficiency and affordability in comparison with the existing methods. Initially, we individually identified SSR loci in the three jujube genomes. Notably, the number of SSRs in these genomes displayed a positive correlation with their respective genome sizes, while the proportion of SSRs with the same core unit length remained consistent across different jujube genomes. Subsequently, we eliminated duplicate SSRs within the three jujube genomes, yielding a total of 286 SSR loci, with 35 of them exhibiting differences among the three jujube varieties. Through successful primer design, we targeted 12 of these polymorphic SSRs, enabling the generation of digital ID cards for jujube plants. This groundbreaking effort resulted in the successful differentiation of 249 distinct jujube genotypes/germplasms, offering valuable insights for the classification of jujube germplasm resources. Additionally, four of the SSR markers proved effective in detecting triploid jujube genotyping, providing reference data and methodological support for the development of polyploid jujube SSR markers. Furthermore, since MGB-SSR relies on comparative analysis among closely related genomes, it holds the potential for extension to other species. Overall, this study introduces an advantageous and innovative approach to screening polymorphic SSR markers, highlighting the merits of the MGB-SSR method.