Hyperglycemia has adverse effects on neuronal recovery after brain injury, but its effects after spinal cord injury (SCI) are understudied. This retrospective cohort study examined the potential effects on outcomes of hyperglycemia in the hyperacute stage after acute traumatic SCI. This study included all individuals enrolled in the National Acute Spinal Cord Injury Study 3 (NASCIS-3). Glycemic levels at 24 h, at 48 h, and at day 7 after acute SCI were examined as potential determinants of survival, neurological outcomes , and functional outcome  within the first year post-SCI. Hyperglycemia was defined using two thresholds (140 mg/dL and 180 mg/dL). Study subjects were 76 females and 423 males with an overall mean age of 36 years who sustained mostly cervical SCI due to motor vehicle accidents or falls. Hyperglycemia diagnosed at day 7 post-injury was associated with significantly greater mortality rates post-SCI. Among the survivors, hyperglycemia during the hyperacute stage was not significantly correlated with neurological recovery post-SCI. Hyperglycemia persistent until day 7 was significantly correlated with lower functional scores post-SCI. These results suggest that hyperglycemia at day 7 is correlated with greater mortality rates within the first year post-SCI. Although hyperglycemia during the hyperacute stage was not associated with neurological recovery, hyperglycemia at day 7 may adversely affect functional recovery within the first year post-SCI. Future investigations are needed to determine the optimal glycemic target in the management of patients with SCI. The worldwide incidence rate of traumatic SCI varies from 8 to 246 cases per million inhabitants per year, and in the literature its global prevalence rate ranges from 236 to 1298 per million inhabitants per year.9 This has led clinicians to promote vigilant adjustment of hyperglycemia during the management of patients in the hyperacute stage following TBI.9 The same clinical approach has been applied in many acute spine centers in the management of individuals with acute traumatic SCI, even though most of the current evidence for this approach has actually been transferred from knowledge on managing individuals with TBI.The potential effects of hyperglycemia after neurotrauma have been studied primarily in patients with traumatic brain injury (TBI). Pre-clinical and clinical evidence indicate that hyperglycemia and hypoglycemia can adversely affect neurological recovery after TBI.10 First, stress-induced hyperglycemia is considered an adaptive immune-neurohormonal response to stress involving two mechanisms: 1) enhanced glycogenolysis and hypermetabolism induced by secretion of stress hormones such as catecholamine, cortisol, glucagon, and growth hormone after activation of the hypothalamic-pituitary-adrenal axis and the sympathetic autonomic nervous system; and 2) transient increase in insulin resistance as another catecholamine-related effect.10The etiology of hyperglycemia after central nervous system (CNS) injury is multi-factorial.10Second, both TBI and SCI are often accompanied by a systemic inflammatory response syndrome that can cause hyperglycemia due to: 1) release of several cytokines  provoking hyperglycemia and insulin resistance; and 2) release of the corticotrophin-releasing hormone and release of adrenocorticotropic hormone from the anterior pituitary.11 Fourth, pituitary and hypothalamic dysfunction has multiple effects on the stress response, and directly affects glucose homeostasis, hepatic gluconeogenesis, and insulin sensitivity.10Third, pre-injury diabetes mellitus, which had an estimated prevalence of 4.9% in the United States in 1990 and an estimated global prevalence of 9.3% in 2019, is another potential cause of hyperglycemia in individuals with neurotrauma.10Finally, many other factors inherent to the management of individuals with an acute CNS injury can incite hyperglycemia including fluid infusions, surgery, anesthesia, and drugs .With this background, a retrospective cohort study was carried out using a large cohort of individuals with SCI to examine whether hyperglycemia during the hyperacute stage post-injury is associated with the individuals' survival, and their neurological and functional recovery within the first year following traumatic SCI.The Institutional Ethics Board approved the use of the database from the National Acute Spinal Cord Injury Study 3 (NASCIS-3) to address research questions other than the original aims of that trial. A Data Use Agreement between Yale University and the University Health Network was instituted in 2006.12 The NASCIS-3 also included a third study arm in which individuals received 48-h intravenous administration of tirilazad mesylate , a non-glucocorticoid potent lipid peroxidation inhibitor that showed neuroprotective properties in pre-clinical studies.12 All participants in the NASCIS-3 were randomized within 6 h from the time of SCI to receive intravenous MPSS for 24 h or 48 h, or intravenous tirilazad mesylate for 48 h. Treatment was initiated within 8 h from SCI onset.12 Individuals age 14 years or older with acute traumatic SCI were considered eligible to be recruited into the NASCIS-3. Complete inclusion and exclusion criteria are documented elsewhere.12This retrospective cohort study included all 499 individuals who were enrolled in the NASCIS-3. Of note, that trial was designed to compare a 48-h methylprednisolone (MPSS) protocol with 24-h MPSS administration in the management of patients with acute traumatic SCI.12 The NASCIS sensory score was determined by testing pinprick and light touch sensation in each dermatome from vertebral level C2 to S5 bilaterally. Each sensory response was classified as: 1 if absent, 2 if dysfunctional (including hypoesthesia and hyperesthesia), or 3 if normal response. Each sensory score ranged from 29  to 87 .12 The NASCIS pain score was bilaterally established according to the responses to deep pain and pressure at the wrist, thumb, little finger, knee, ankle, and great toe areas. Each response to deep pain and pressure was classified as: 1 for absent, 2 for decreased, and 3 for normal response. The NASCIS pain score ranged from 6  to 18 .Survival data within the first year after SCI were analyzed in this study. In all enrolled individuals, the degree of impairment was assessed using the NASCIS motor, sensory, and pain scores at admission to the emergency department as well as at 6 weeks, at 6 months, and at 1 year following SCI. The NASCIS motor score was determined by assessing motor function in 14 segments bilaterally, using a manual muscle scoring system as follows: 0 for no muscle contraction; 1 for trace of muscle contraction; 2 for active movement without antigravity; 3 for active movement with antigravity; 4 for active movement against resistance; and 5 for normal function. The NASCIS motor scores ranged from 0 (no contraction in any muscle) to 70 .13 The FIM score includes sub-scores for self-care, sphincter control, mobility, locomotion, communication, and social cognition. The FIM score ranged from 18  to 126 (complete independence). All individuals were assessed for degree of disability at 6 weeks, at 6 months, and at 1 year following SCI.The degree of disability was assessed using the Functional Independence Measure (FIM) score.15According to NASCIS-3 protocol, blood samples for glycemic level and serum creatinine concentration were drawn at 24 h, at 48 h, and at day 7 after SCI onset from all participants. Normal reference values in glycemic testing range from 80 to 140 mg/dL. Given the variability in the literature of the definition of hyperglycemia, all data analyses in this study used the lower and upper thresholds of most commonly accepted definitions for “stress-induced hyperglycemia” as follows: 1) a random glycemic test result greater than 140 mg/dL; and 2) a random glycemic test result greater than 180 mg/dL.Analysis of variance (ANOVA) with Bonferroni post hoc test was used to compare the results of glycemic tests within the first week after SCI using continuous variables. The time effect on glycemic levels was evaluated using repeated measures ANOVA. Survival analysis was carried out using Kaplan-Meier curve and log-rank test.p < 0.05. All data analyses were carried out using SAS software, version 9.4.Multiple regression analyses were used to examine within the first week after SCI the potential effects of hyperglycemia on the neurological outcomes . Those multiple regression analyses were adjusted for the respective neurological status at admission, individuals' sex and age at SCI onset, NASCIS-3 trial drug protocol , level of SCI , Glasgow Coma Scale (GCS) score on admission, and serum creatinine concentration (which was collected at the same time as glycemic testing). Similarly, multiple regression analyses were used to examine within the first week after SCI the potential effects of hyperglycemia on the degree of disability as assessed using the FIM at 6 weeks, at 6 months, and 1 year after SCI. Those multiple regression analyses used for FIM scores as the dependent variable were adjusted for the degree of motor impairment on admission (as assessed using the NASCIS motor score), individuals' sex and age at the SCI onset, NASCIS-3 trial drug protocol , level of SCI , GCS score on admission, and serum creatinine concentration (which was collected at the same time as glycemic testing). Significance was assumed at p < 0.0001). Using repeated measures ANOVA, there was a statistically significant effect of time on glycemic levels, F  = 324, p < 0.0001.Of the 499 individuals enrolled in the NASCIS-3, there were 76 females and 423 males with an overall mean age of 36 years  who mostly sustained cervical SCI due to motor vehicle accident followed by falls . The max2 = 0.0374; p = 0.0001; 2 = 0.1056; p < 0.0001; 2 = 0.0040; p = 0.4444; Mean glycemic levels at 24 h post-injury were significantly lower in the group of patients who received tirilazad mesylate for 48 h than in the groups of patients who received either MPSS for 24 h or MPSS for 48 h . Nevertheless, hyperglycemia at day 7 post-injury was significantly associated with a greater mortality rate within the first year after SCI  presented hyperglycemia (defined as a random glycemic level >140 mg/dL) within 24 h after the SCI onset. Hyperglycemia either at 24 h or at 48 h post-injury was not associated with mortality rates within the first year after SCI (p = 0.4162), hyperglycemia at 24 h post-injury was significantly correlated with higher FIM scores at 1 year after SCI (p = 0.0225). There was also a trend toward an association between hyperglycemia at 24 h post-injury and higher FIM scores at 6 months after SCI (p = 0.0547) (p = 0.0059), at 6 months (p = 0.0020), and at 1 year (p = 0.0048) following SCI  . In contwing SCI .p = 0.3662 and p = 0.4113, respectively). However, hyperglycemia at day 7 post-injury was significantly correlated with higher mortality rate within the first year after SCI  presented hyperglycemia (defined as a random glycemic level >180 mg/dL) within 24 h after the SCI onset. Hyperglycemia either at 24 h or at 48 h post-injury was not associated with mortality rates within the first year after SCI , Jeremitsky and colleagues16 reported that overall hyperglycemia score and hyperglycemia score for days 3 to 5 post-injury were significantly correlated with mortality after severe TBI (GCS score ≤8). In a retrospective observational study in a Level 1 trauma center (n = 429), Liu-DeRyke and associates17 documented that patients with TBI who maintained glycemic levels below 160 mg/dL within the first 24 h post-injury showed significantly better survival rates within the initial 30 days from admission to an intensive care unit than their counterparts with glycemic levels of 160 mg/dL or higher. In a prospective cohort study that excluded individuals with prior history of diabetes mellitus (n = 220), Kafaki and co-workers18 showed that the in-hospital mortality rate in the group of individuals with severe TBI who had hyperglycemia on admission was significantly greater than in the group of patients with glycemia levels below 200 mg/dL . In a more recent study analyzing data from a propensity score-matched population (n = 1798), Rau and colleagues19 reported that individuals with either stress-induced hyperglycemia or diabetic hyperglycemia had a 9.1-fold to 2.3-fold greater odds of death, respectively, than patients with non-diabetic normoglycemia after moderate-to-severe TBI. However, the adjusted mortality was significantly higher in the propensity score-matched patients with stress-induced hyperglycemia, but not in those patients with diabetic hyperglycemia.19 Of note, their definition of hyperglycemia was a serum blood concentration ≥200 mg/dL upon arrival in the emergency department.19Although the effects of hyperglycemia on survival after SCI were understudied, there is a growing body of literature on the detrimental effects of hyperglycemia on survival after more severe TBI. In a retrospective case series in a Level 1 trauma center  is not associated with neurological recovery after acute traumatic SCI, after adjusting for major potential confounders. Among survivors, glycemic levels higher than 140 mg/dL at 24 h post-injury were significantly associated with higher FIM scores at 1 year following acute traumatic SCI. However, persistent hyperglycemia (defined as glycemic levels >140 mg/dL) at day 7 post-injury was significantly associated with lower FIM scores at 6 weeks, at 6 months, and at 1 year after acute traumatic SCI.21 studied the role of hyperglycemia on the recovery of rats that sustained a traumatic SCI at vertebral level T9 caused by a weight-drop impactor. In their series of experiments, the authors found that: 1) elevated glycemic levels were detected only in the more severely injured animals; 2) pre-injury, overnight fasting did not directly affect lesion volume but caused mild acidosis that had a minor neuroprotective effect; and 3) moderate hyperglycemia induced within 1 h after SCI did not significantly influence the lesion volume.21Prior experimental studies using animal models provided conflicting results on the potential effects of hyperglycemia on metabolism dysfunction and tissue preservation after SCI. For instance, Sala and colleagues22 documented that hyperglycemia induced in mice by intraperitoneal injection of streptozotocin was associated with enhanced pro-inflammatory reactivity in the microglial cells , greater apoptosis activity within the spinal cord, smaller area of preserved spinal cord tissue, and poorer behavioral test score after contusion injury at the T10 level when compared with sham mice. More recently, Chen and co-workers23 reported that rats with hyperglycemia  and weight-drop-induced SCI at the T10 level developed prominent endoplasmic reticulum stress, neuronal apoptosis, and increased permeability of the blood–spinal cord barrier within the spinal cord when compared with control groups .On the other hand, Kobayakawa and associates22 However, little is known about the effects of hyperglycemia on neurological and functional recovery after acute traumatic SCI. In a recent retrospective cohort study, Kobayakawa and associates22 reported that individuals with acute traumatic SCI who presented hyperglycemia (defined as glycemic level ≥126 mg/dL on admission) showed a greater proportion of poor outcome , lower ASIA motor scores, and lower Spinal Cord Independence Measure (SCIM) scores at discharge than individuals with normoglycemia post-SCI. However, their data analysis did not control for other potential confounders that can influence that apparent correlation between hyperglycemia and outcomes after acute traumatic SCI.Similar to the present study, prior clinical investigations demonstrated that hyperglycemia is a common finding in the hyperacute stage after traumatic SCI.24 documented that age and sex can influence insulin secretion, insulin action, hepatic insulin extraction, and glucose effectiveness, which can result in substantial differences in the regulation of glucose metabolism. Also, Li and colleagues25 suggested that there are sex-related discrepancies in the glucose metabolism that cannot be only explained by the differences of body composition between females and males. Although sex-related differences in neurological and functional recovery after acute traumatic SCI have been reported previously, the potential effects of individuals' sex on outcomes after SCI remains a matter of debate.26–28 Older age at SCI onset has consistently been correlated with higher post-injury mortality rates. Although a few prior studies have reported that older age at the time of injury could be associated with poorer outcomes after traumatic SCI, the most recent investigations using models adjusted for major potential confounders have challenged that chronologic age is an independent factor for less favorable outcomes following acute traumatic SCI.29–33First, individuals' sex and age can conceivably influence hyperglycemia and outcomes after acute traumatic SCI. Among able-bodied individuals, Basu and co-workers34 Moreover, hyperglycemia post-injury has a negative impact on neurological and functional recovery after moderate-to-severe TBI, which justifies consideration of concomitant TBI as a major potential confounder in any data analysis focused on recovery after acute traumatic SCI.20Second, the frequency of concomitant TBI among individuals with acute traumatic SCI was estimated to be 32.5%  in a recent meta-analysis.35 Therefore, data analyses on the potential effects of hyperglycemia on neurological recovery following acute traumatic SCI ought to account renal function as a potential confounder.Third, the kidneys play the foremost role in glucose and insulin metabolism, and current knowledge suggests kidneys are intimately involved in the development, maintenance, and resolution of hyperglycemia in critically ill patients.36 Further, Torbati and associates37 reported that at an acute care spine trauma in Iran individuals with more severe SCI had higher glycemic levels on admission.Fourth, the severity and level of SCI are well-recognized prognostic factors for recovery after traumatic SCI and, hence, both factors should be considered in the data analyses as potential confounders.38Finally, some pharmacological therapies may have an effect on the glycemic levels and outcomes. In this study, glycemic levels higher than 140 mg/dL at 24 h post-injury were significantly associated with higher FIM scores at 1 year following SCI. Although the actual reasons for these findings remain unclear, one may speculate that the infusion of MPSS could be associated with temporary hyperglycemia at 24 h and better functional recovery.12 documented that infusion of MPSS was associated with improved FIM scores at 6 months after injury. On the other hand, in this study those individuals who had persistently elevated glycemic levels within the first week after SCI showed less favorable functional recovery within the first year after SCI. Although a definite explanation cannot be determined, one may wonder whether this group of individuals with persistently elevated glycemic levels within the first week after SCI had a pre-accident glucose metabolism disorder such as diabetes mellitus or pre-diabetes. In a recent combined experimental animal and clinical study, Park and associates39 examined the effects of pre-injury chronic hyperglycemia on inflammatory markers and functional recovery after traumatic SCI. In the clinical part of the study, hemoglobin A1c (HbA1c) at admission was associated with poor functional recovery, and those patients with chronic hyperglycemia (defined as HbA1c ≥6.5%) had high concentrations of inflammatory biomarkers (IL-6 and IL-8) in the cerebrospinal fluid following SCI.39 Similarly, pre-injury chronic hyperglycemia in rats has been shown to be associated with increased inflammatory responses and oxygen-free radicals in the spinal cord and blood, and consequently resulted in greater lesion volume, less spared tissue, and poorer functional recovery.39Bracken and colleagues40 In the present study it was attempted to address some of those challenges and, after adjusting data analyses for major potential confounders, hyperglycemia within the first week post-injury was found not to be associated with neurological recovery in the sub-acute and chronic stages after traumatic SCI. Nonetheless, hyperglycemia at day 7 post-injury was significantly correlated with greater degree of disability as assessed using the FIM score in the sub-acute and chronic stages following traumatic SCI.Overall, few prior pre-clinical and clinical studies have suggested that hyperglycemia in the hyperacute post-injury stage could be associated with poorer outcomes after acute traumatic SCI. However, the heterogeneity of the SCI population and many potential major confounders represent methodological challenges when carrying out clinical studies in this field.33 Data on pre-injury diabetes mellitus as a potential confounder were missing in the NASCIS-3 database, even though the estimated prevalence of 4.9% in the United States in 1990 was significantly lower than the estimated global prevalence of 9.3% for 2019.41 Also, polypharmacy and rehabilitation intensity can have important consequences on the neurological and functional outcomes following acute traumatic SCI.43This retrospective cohort study analyzed a large data set from the NASCIS-3 that included data on several major potential confounders. However, there are limitations that should be taken into account when interpreting and applying the results of this study. First, the retrospective nature of this study precluded collection of other potential confounders. For instance, pre-existing medical co-morbidities could have an impact not only on survival, but also on functional recovery after acute traumatic SCI.44 Finally, all participants in the NASCIS-3 received a drug that could potentially have a neuroprotective effect in individuals with acute traumatic SCI.Second, the participants in the NASCIS-3 were recruited from 1991 to 1995, when some aspects of pre-hospital, acute spine care, and rehabilitation practices could have been discrepant from the current guidelines. For example, maintenance of mean arterial blood pressure greater than 85 mm Hg, pre-operative evaluation preferentially using magnetic resonance imaging, and early spinal cord decompression have more recently become the best practices in the initial management of patients with acute traumatic SCI.45The results of this retrospective cohort study suggest that hyperglycemia commonly occurs among individuals with acute traumatic SCI. Although hyperglycemia within the first 48 h post-injury did not adversely affect survival after SCI, persistent hyperglycemia at day 7 post-injury was significantly associated with reduced survival rates within the first year following acute traumatic SCI. Additionally, hyperglycemia post-injury did not influence neurological recovery within the first year after acute traumatic SCI. Although hyperglycemia within the initial 48 h post-injury did not negatively affect functional recovery after SCI, hyperglycemia at day 7 post-injury was significantly correlated with greater degree of disability as assessed using the FIM score within the first year following acute traumatic SCI. Those results endorse the current practice guidelines that recommend glycemic control during management of patients with acute traumatic SCI. Nevertheless, the optimal glycemic target in the management of patients with acute traumatic SCI remains to be determined, whereas hypoglycemia should be prevented at all times given its risk for irreversible CNS injury.


Treatment of prostate cancer confronts resistance to androgen receptor (AR)-targeted therapies. AR-associated coregulators and chromatin proteins hold a great potential for novel therapy targets. Here, we employed a powerful chromatin-directed proteomics approach termed ChIP-SICAP to uncover the composition of chromatin protein network, the chromatome, around endogenous AR in castration resistant prostate cancer (CRPC) cells. In addition to several expected AR coregulators, the chromatome contained many nuclear proteins not previously associated with the AR. In the context of androgen signaling in CRPC cells, we further investigated the role of a known AR-associated protein, a chromatin remodeler SMARCA4 and that of SIM2, a transcription factor without a previous association with AR. To understand their role in chromatin accessibility and AR target gene expression, we integrated data from ChIP-seq, RNA-seq, ATAC-seq and functional experiments. Despite the wide co-occurrence of SMARCA4 and AR on chromatin, depletion of SMARCA4 influenced chromatin accessibility and expression of a restricted set of AR target genes, especially those involved in cell morphogenetic changes in epithelial-mesenchymal transition. The depletion also inhibited the CRPC cell growth, validating SMARCA4’s functional role in CRPC cells. Although silencing of SIM2 reduced chromatin accessibility similarly, it affected the expression of a much larger group of androgen-regulated genes, including those involved in cellular responses to external stimuli and steroid hormone stimulus. The silencing also reduced proliferation of CRPC cells and tumor size in chick embryo chorioallantoic membrane assay, further emphasizing the importance of SIM2 in CRPC cells and pointing to the functional relevance of this potential prostate cancer biomarker in CRPC cells. Overall, the chromatome of AR identified in this work is an important resource for the field focusing on this important drug target. Androgens and androgen receptor (AR), a hormone-activated transcription factor (TF), are key factors driving the development and progression of prostate cancer (PCa). The AR is therefore the primary molecular target for the hormone therapy of advanced PCa . AndrogeMost of the currently known nuclear receptor (NR)-interacting proteins, including those of AR, have been identified through genetic screens, such as two-hybrid systems, co-immunoprecipitation, and peptide fragment-based in vitro methods –5. Even Coregulators often reside as subunits in protein complexes and participate in the regulation of transcription in multiple ways, e.g. by modulating histone modifications and chromatin structure. Mammalian BRG1- or BRM-associated chromatin remodeling complex  changes the chromatin accessibility landscapes in cancer cells. The complex is a crucial regulator of cell cycle and proliferation , and a dIn this work, we utilized chromatin immunoprecipitation coupled with selective isolation of chromatin-associated proteins (ChIP-SICAP)  to captuTo complement our BioID-derived interactome of AR , we usedThe R1881-induced AR chromatome was categorized to different functional protein groups Fig. . DNA- anTo explore the more detailed role of SMARCA4 in AR-associated chromatin environments, we first performed ChIP-seq with SMARCA4 and AR antibodies in VCaP cells in the presence or absence of 5α-dihydrotestosterone (DHT). We focused our interest on sites co-occupied by SMARCA4 and AR and how androgen affects the co-occupancy. Most chromatin-binding sites of SMARCA4 (61534) were not affected by DHT  compared with the C1 sites, further supporting androgen-induced recruitment of SMARCA4 onto the chromatin. Motifs for FOXA1, ERG and HOXB13 were in turn equally enriched at the C1 and C2 sites Fig.  and e. HTo gain more insight into how SMARCA4 influences chromatin accessibility at ARBs, we depleted SMARCA4 from VCaP cells  showed only small differences upon SMARCA4 depletion, but the effect of DHT remained  were associated with siSIM2-affected sites in ATAC-seq data , but a larger portion of the ChIP-SICAP quantified proteins was significantly enriched with AR in androgen-dependent fashion . VCaP cells, but not LNCaP cells, additionally express an AR variant, AR-V7, devoid of the ligand-binding domain (LBD) [Here, we utilized ChIP-SICAP which purifies exclusively chromatin-bound functional interactors  to cast in (LBD) . MoreoveSMARCA4 is an established chromatin remodeler  and one To the best of our knowledge, the impact of androgen-activated AR on the chromatin occupancy of SMARCA4, the chromatin accessibility and the AR target gene expression has not previously been investigated in an integrative fashion in PCa cells. Our data from VCaP cells show that despite a marked number of SMARCA4 chromatin-binding sites responded to androgen (>40%) and a large overlap (75%) between SMARCA4- and AR-binding sites, the depletion of SMARCA4 changed the chromatin accessibility <10% of those sites. This relatively small effect of SMARCA4 depletion may be explained by two interchangeable ATPase subunits, SMARCA4 and SMARCA2, the latter of which was not however detected in our AR ChIP-SICAP. Moreover, our data did not indicate any compensatory increase in the expression of SMARCA2 upon SMARCA4 depletion. Since enhancers recruit remodelers with different affinity , removalRegardless of the relatively limited effect of SMARCA4 on the chromatin accessibility on ARBs, SMARCA4 depletion affected chromatin accessibility at a larger number of sites (~11000). The depletion did not only decrease the accessibility, but the accessibility at ~20% of the SMARCA4 depletion-affected sites was increased, with FOXA1 being enriched at decreased sites. The FOXA1 has been assumed to create open chromatin environment by displacing linker histones , 43. OurThe SMARCA4 depletion in VCaP cells had interesting effects on AR-mediated gene expression, as e.g. the expression of androgen-regulated genes involved in the extracellular matrix organization and morphogenesis of branching structures were sensitive to SMARCA4. These pathways include possible connections to EMT that has previously been indicated as a cellular process regulated by SMARCA4 –49. SimiBRCA1, BRCA2 and ATM. These roles are in line with SIM2’s growth-promoting role [SYP, CHGA and NSE [Our chromatin-directed proteomics approach revealed SIM2, a basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) TF , and itsing role –58, abiling role , 60 and ing role . Moreoveing role , 22, theing role , 63 and  and NSE , 65 are Our ATAC-seq showed that, although most of the ARBs were unaffected by SIM2 silencing, the accessibility at >2000 ARBs was altered. Chromatin at most of the affected sites was accessible before androgen exposure, suggesting that SIM2 preferentially binds to and acts at open chromatin regions. In addition, binding of AR was affected - attenuated or enhanced - at ~2000 sites by SIM2 silencing. However, there was no simple relationship between the changes in chromatin accessibility and the AR binding upon SIM2 silencing. SIM2 could display some pioneer factor properties with AR in certain chromatin environments, but these properties are much weaker than those of the FOXA1. Moreover, the majority of ARBs affected by SIM2 silencing do not overlap with those altered by FOXA1 depletion. Together, our results indicate that the SIM2 is a TF co-operating with AR in CRPC cells.In conclusion, our results confirm the chromatin opening role of SMARCA4 in AR-mediated gene regulation, which is interestingly reflected in the expression of genes involved in pathways potentially connected with EMT in PCa. In addition, our results indicate that SIM2 plays an important, AR target pathway-selective role in the regulation of CRPC cells. Finally, the chromatome of AR in CRPC cells identified herein forms an important resource for the AR field, focusing on this important drug target.VCaP cells were obtained from ATCC, tested negative for mycoplasma, and verified by Institute for Molecular Medicine Finland. Cells were maintained in DMEM (Gibco #41965-039) supplemented with 10% FBS (HyClone #AYE161472) and 1 U/µl penicillin and 1 µg/ml streptomycin (Gibco #15140-122).6 cells/ 15-cm culture dish, Supplementary materials) were cultured for 72 h, before adding 10 nM R1881 to heavy-labeled  cells and ethanol (vehicle) to light-labeled  cells for 1 h. Cells were immunoprecipitated as in [SILAC-labeled VCaP cells  were reverse-transfected with ON-TARGETplus SMARTpools for SMARCA4, SIM2 and non-targeting control  were cultured in maintenance medium for 48 h. Medium was changed to DMEM, 2.5% charcoal-stripped FBS for 48 h prior to 1 h exposure with DHT 100 nM or vehicle. In siRNA-experiments, SMARCA4, SIM2 or FOXA1 were silenced by reverse transfection with ON-TARGETplus SMARTpools for 96 h prior hormone treatment. ChIPs were performed as in [®Ultra DNA™ II kit  and sequenced with Illumina NextSeq 500 (75SE). (Details and data analysis in VCaP cells (5 × 10ed as in  with αSMed as in . The libSupplementary materialsSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4


The paper presents the design optimization of the ASPIRE spherical parallel robot for shoulder rehabilitation following clinical evaluation and clinicians’ feedback. After the development of the robotic structure and the implementation of the control system, ASPIRE was prepared for clinical evaluation. A set of clinical trials was performed on 24 patients with different neurological disorders to obtain the patient and clinician acceptance of the rehabilitation system. During the clinical trials, the behavior of the robotic system was closely monitored and analyzed in order to improve its reliability and overall efficiency. Along with its reliability and efficiency, special attention was given to the safety characteristics during the rehabilitation task. One of the most common neurological diseases of our century is stroke . GloballThere are a series of consequences that can occur after a stroke. Most common are post-stroke depression (PSD), vascular cognitive impairment (VCI), and post-stroke fatigue (PSF) . Stroke All the above data predicts a future crisis around 2030 when the medical system will be unable to provide the specific care for each stroke survivor because of the continuous increase in the number of stroke patients correlated with the aging of population. It is foreseen that the population aged over 65 will achieve 30% of the world population in 2060 . To prevSince 1990, robotic-assisted medical rehabilitation has become a valuable solution in overcoming the qualified personnel shortage from the medical system . The phyThe advancements in kinematics and control ,17,18,19In 2009, Kai et al.  performeIn 2015, Fong et al.  performeIn 2021, DeBoon et al.  proposedA wide number of upper limb rehabilitation robotic solutions consist of exoskeletons ,41. ThesAmong the robotic solutions for the rehabilitation of the upper limb, some platforms were designed and tested regarding the environment of the rehabilitation task, and using virtual reality, the rehabilitation procedure was placed in an environment more stimulating for the patient.Saposkik et al.  conducteSome other studies recorded proof of the efficiency of a virtual environment used in stroke rehabilitation. Estapa et al.  used a KGiven the above solutions for post-stroke rehabilitation, a valuable solution should include both aspects: a robotic system and virtual environment to provide significant improvements in the quality of life of stroke survivors. Based on the already proven paradigm that patients that are focused on the task during rehabilitation recover faster, the development of additional stimuli like VR and interactive games can have benefits, as long as they succeed in addressing the needs and interests of the patient.The focus of this paper is on the design optimization of a spherical parallel robot for shoulder rehabilitation. The optimization is a two-stage process. The first stage is the preclinical evaluation, where the design of the robotic structure is improved based on the clinicians’ feedback before performing clinical trials. The second stage improvements are based on the clinicians and patients’ feedback based on the clinical trials. In the second section of the paper, the ASPIRE robotic structure is presented in terms of the initial experimental model and control system, and the planning of the clinical trials and the experimental tests using patients are described. The third section of the paper presents the results obtained and design optimization of the robotic system, followed by the discussions and conclusions.ASPIRE is a spherical parallel robotic system that targets the adduction, abduction, flexion, and extension rehabilitation motions of the shoulder joint and the pronation and supination of the forearm . The folThe main design feature of the robotic system consists of two circular guides that lead to a spherical motion of the characteristic point of the mechanism. Starting from the concept stage, the patient is embedded in the design of the robotic structure, and the center of the sphere given by the two circular guides is placed in the center of the patient shoulder, allowing the entire arm motion with respect to the shoulder rehabilitation motion. The targeted motions of ASPIRE are flexion/extension of the shoulder with a motion range of +/− 80 degrees, adduction/abduction of the shoulder with a motion range of +/− 80 degrees, and pronation/supination of the forearm with a range of +/− 80 degrees –(3).The kinematic scheme of the ASPIRE robot is presented in The initial experimental model of ASPIRE is presented in For easy acceptance from the patient, the actuating mechanisms were embedded in the Plexiglas case, and only the moving elements that come into direct contact with the patient for performing the rehabilitation motion were visible. For easy maneuverability, the entire robotic structure was equipped with locking wheels.The control architecture of ASPIRE is presented in The entire rehabilitation procedure is controlled through a graphical user interface  that comThe control strategy of the robotic system is presented in The rehabilitation motion parameters are parsed into the PLC of the robotic system, which identifies the rehabilitation motion and sends the motion parameters to the drivers that control each motor of the robotic structure. The hardware architecture  presentsPerforming the clinical trials was one of the final steps of the robotic system validation. Before performing tests with patients, the robotic system underwent a lab testing phase to check the safety of the user during the rehabilitation procedure. Additionally, before any tests were performed, a medical rehabilitation protocol was developed and successfully used during the lab tests and clinical trials.Before the clinical trials, a series of in-lab tests were performed with healthy subjects to validate the functionality of the robotic system. During the functionality tests, the 3D-printed circular guides proved to be inefficient, introducing some unwanted vibrations into the mechanical system, so the guides were redesigned to embed rolling bearings to reduce the friction coefficient a. DuringA specific robotic rehabilitation protocol was required to perform the clinical trials , which wWhen admitted to the hospital, the patient should go through an initial assessment performed by a physical therapist and receive an initial score indicating his impairment levels for the upper limb before rehabilitation treatment. The data is recorded by the therapist for future analysis and for the setup of the starting motion parameters for the robot (evolution/involution record). A rehab program is defined for 7–14 days with one or two daily sessions.Before starting the rehabilitation, the involved patient must sign an informed consent and be conscious and stable hemodynamically, present no fever symptoms, and the brain lesion confirmed via CT (Computer Tomograph) or MRI (Magnetic Resonance Imaging) to exclude other diseases than the neurologic one.The data recorded in a previous stage are used to compute the robot motion amplitudes for the first rehabilitation session. For the next sessions, a daily increase of the amplitudes is applied in the range of 5–10%, aiming to reach the targeted amplitude around day 5.The patient will be caried to the robotic rehabilitation room by a stretcher-bearer using a wheelchair or walking, depending on the health state of the patient.The patient is strapped into the robotic device .The robotic rehabilitation procedure is performed based on the therapist’s recommendations using a predefined exercise of 3 × 10 repetitions for each rehabilitation motion.After the predefined exercises are performed, the robotic device is detached from the patient.The patient undergoes a physical evaluation performed by the physical therapist to check the integrity of muscles and of the ligaments.Repeat steps d–h for every rehabilitation session (4–5 patients × 2 sessions/day × 7 days).At the end of the entire program, the patient is re-evaluated to determine the achieved progress.The rehabilitation motions are performed entirely by the robotic system after the patient’s impaired limb is strapped into the attaching devices. The motion amplitudes are introduced into the user interface by an operator. The amplitudes were previously defined by a kinetotherapist based on the patient spasticity and overall health. Additionally, the interface allows speed and repetition control, parameters also given by the kinetotherapist.After the development of the experimental model, the functional validation test, and the definition of the robotic rehabilitation protocol, the ethical approval for performing clinical trials was obtained in August 2019, in accordance with the Helsinki principles regarding biomedical research, and the robotic system was installed in a room at the Municipal County Hospital Cluj-Napoca within the Neurology Department.A number of 24 patients were admitted to the clinical study, half of them with bilateral upper limb impairment (12) and the other half with right- (6) or left (6)-side hemiparesis. The physical state of the robotic system at the time of clinical study allowed only right limb rehabilitation, and the group of 24 was divided in two groups: 18 patients  that performed the rehabilitation with the robotic system and 18 patients  that performed classical rehabilitation performed by a kinetotherapist. Every patient from the clinical study was admitted into the hospital, evaluated before the therapy, and programmed for two rehabilitation sessions per day for a time span of 7 consecutive days. At the end of the 7 days, the patients were reevaluated to establish the results of the rehabilitation procedure.The tests were performed in hospital between October and November 2019 over a time span of 8 weeks. During the tests, each patient performed shoulder rehabilitation with ASPIRE for 35 min/day. Two therapy sessions were scheduled every day for each patient, one session in the morning and the other one in the afternoon .Snapshots from the videos recorded during the rehabilitation can be seen in Starting from the initial evaluation by the clinician of each admitted patient, the initial motion amplitudes were imposed with respect to the neurological disorder and specific comorbidities of each patient. The amplitudes were increased 5–10% per day, reaching the maximum amplitude on day 5 .At the end of the rehabilitation program, each of the patients that performed rehabilitation with the robotic system were asked to complete a questionnaire regarding their experiences with the robotic system. The questions were divided based on the rehabilitation motions performed. The analyzed characteristics and their recorded values for the height adjustment module are given in The score obtained by each characteristic is highlighted in Using the same scale, a questionnaire was provided for the clinicians in order to evaluate the robotic system. The clinicians had different aspects to consider regarding the User Interface, the Operating Speed, the Safety, and the Amplitudes. The questionnaire was sent to 10 clinicians that also saw the robotic system while operating. The marks given by each clinician are shown in During the experimental tests, a series of characteristics was observed by the clinicians and by the operators and recorded to be improved. Some of the characteristics recorded were similar with the ones identified by the questionnaires. The main issue and the solution to solve every one of them is given below.Observation 1: Times in adjusting the height mechanism were taking too long, extending the total time of the rehabilitation process.Solution: To reduce the setup time, the height adjustment mechanism was not used during the rehabilitation; instead, a liftable chair was used. After the experimental trials, the robotic system underwent the second-stage optimization process, and the screw–nut mechanism was replaced with a ball screw mechanism, and in order to ensure the locking of the mechanism, a worm gear box was designed .Observation 2: Due to the circular guides displacement, sometimes, the flexion–extension mechanism was jammed or slowed down by the patient.Solution: The slides were properly greased at the beginning of the rehabilitation procedure, and a two-DOFs mechanism was used to overcome the angular displacement between the circular guides .Observation 3: The shoulder anchor was problematic during the rehabilitation process.Solution: The shoulder anchor was redesigned to eliminate any edges of the component and allow easy attachment of the shoulder. Additionally, a synthetic foam was used to cover the part where the patient was in direct contact.Observation 4: The forearm anchor mechanism was uncomfortable for the patient.Solution: The entire part was covered with synthetic foam, and to avoid the times needed to sterilize the component, the entire part was covered with single-use medical cotton for every patient.Observation 5: During the use of the system, the motor of the adduction–abduction mechanism heated up.Solution: The heating of the motor was determined to be caused by the momentum created by the weight of the mechanism transmitted through the reduction box; to overcome this situation, the same worm gear box  was adapObservation 6: The edges of the circular guides were exposed and could hurt the patient or the operator.Solution: The slides were covered with a soft material (synthetic foam).The robotic system optimized after the clinicians’ feedback and clinical trials can be viewed in The fully robotic-assisted rehabilitation group, consisting of six patients with right limb impairment.The mixed rehabilitation group, consisting of 12 patients with bilateral impairment, having their right upper limb treated with the robot and the left side with the help of a kinetotherapist.The classical rehabilitation group, consisting of six patients with left limb impairment, treated by a kinetotherapist.The clinical study performed on 24 patients with different neurological disorders with upper limb impairment aimed to evaluate the performance of the robotic system compared to the human therapists. The patients were split into three groups:The patient evaluations were performed at admittance to the hospital and after the rehabilitation therapy. Besides the determination of the therapeutic efficiency of the exercises, the initial data was used as the input motion amplitudes parameters for the robotic system as individual values for each patient.(1)Ranges of motion and muscle strength—goniometry and dynamometry.(2)Multimodal neurophysiological motor system assessment—quantitative electroencephalogram, motor conduction times, and turn/amplitude analysis .The patient evaluation consisted of two types of measurements:U test), with a significance threshold of p < 0.05.Based on the size of the group, for the statistical analysis of the data, the chosen method was nonparametric testing  suffered a shoulder fracture 40 years ago, and he was unable to perform the physical rehabilitation with the robot. Another patient (the one suffering from gouty arthritis) manifested completely different from other patients, succeeding in performing the rehabilitation motion at the maximum amplitude since day 1.Some of the patients (44.44%) used a wheelchair instead of the chair provided by the robotic system due to their general condition. Using a wheelchair during the clinical trials proved to be a challenge due to restrained space provided by the room where the robot was placed. The time spent by each patient performing the rehabilitation procedure was longer than the one spent performing manual rehabilitation, but the longer time spent performing the rehabilitation turned to be a positive aspect of the rehabilitation. Contrary to general opinion that a patient will manifest reluctance when robotic devices substitute medical personnel, all the patients were rather excited to work with the robotic system and eager to test the new technology. During the clinical trials, to create a comfortable environment for the patient, music was played in the background, this aspect proved to be a motor positive factor for the patients and a reinforcement for performing the rehabilitation exercise.Based on the results and the feedback from the patients, the clinical trials were a real success, motivating the research team to continue in improving the rehabilitation system. At the time of the clinical trials, only simple motions were performed, but the obtained results created the premise for improving the robotic system in terms of a control system to embed more safety characteristics for the patients and for the system and developing an improved and reliable user interface.Future works will target the extension of the robotic system functionalities via the control system to enable human–robot interaction modalities and develop a multilevel user interface that can be easily and safely used by every person, with the next set of clinical trials being scheduled for the second half of 2021.The robotic system for shoulder rehabilitation ASPIRE was successfully tested in a hospital environment using patients with real neurologic disorders. The work performed with real patients revealed a significant difference from the initial tests performed in the laboratory with healthy subjects and provided a series of critical characteristics required to be improved in the development of the robotic system. ASPIRE was initially designed for patients with upper limb impairment post-stroke, but the clinical trials proved its usefulness for multiple neurologic disorders . After the successful completion of the clinical trials, the entire robotic structure underwent a detailed analysis, and a series of improvements were made to improve the functionality of the robotic system and the quality of the patients spent during the rehabilitation task. After the design improvements were made, a new series of clinical trials are planned as soon as the COVID-19 pandemic will allow them.


P ≤ 0.03). For patients with CF, the repeatability was significantly lower at Ao2 compared to Ao1 , as well as the assessment of regurgitation . This was not the case for patients with NCF. The frequency of patients that changed in AR grade from severe to non-severe when the position of the measurement changed from Ao1 to Ao2 was higher for patients with CF compared to NCF (RVol: 5/26 (19%) versus 1/17 (6%), P = 0.2; RF: 4/26 (15%) versus 0/17 (0%), P = 0.09). Our study shows that complex flow influences the quantification of chronic AR, which can lead to underestimation of AR severity when using PC-MRI.This study aimed to investigate if and how complex flow influences the assessment of aortic regurgitation (AR) using phase contrast MRI in patients with chronic AR. Patients with moderate (n = 15) and severe (n = 28) chronic AR were categorized into non-complex flow (NCF) or complex flow (CF) based on the presence of systolic backward flow volume. Phase contrast MRI was performed repeatedly at the level of the sinotubular junction (Ao1) and 1 cm distal to the sinotubular junction (Ao2). All AR patients were assessed to have non-severe AR or severe AR  ≥ 60 ml and regurgitation fraction (RF) ≥ 50%) in both measurement positions. The repeatability was significantly lower, i.e. variation was larger, for patients with CF than for NCF (≥ 12 ± 12% versus ≥ 6 ± 4%,  Aortic regurgitation (AR) is characterized by the diastolic backward flow of blood from the aorta into the left ventricle. Accurate assessment of chronic AR severity is essential for appropriate clinical decision-making, risk prediction, and timing of surgery , 2. Two-Even though PC-MRI is considered to be accurate and the standard MRI method for the assessment of AR severity, it has been questioned under certain conditions . For exaConsequently, the aim of this study was to investigate if and how complex flow influences the assessment of AR severity using PC-MRI in patients with chronic AR.The study cohort has previously been described in another publication, characterizing complex flow patterns in patients with chronic AR . Here, tThe study comprised 43 patients  with moderate n = 15) and severe (n = 28) chronic AR, determined by 2D echocardiography according to the current ASE guidelines [ and seveThe MRI examination was performed on a 1.5 T whole-body Philips scanner  equipped with a multi-channel phase array cardiac coil.2 with a slice thickness of 8 mm [After standardized patient-specific planning, a series of cine-images was performed, first in the short-axis view covering the whole heart without gap from the atrioventricular ring to the apex, followed by cine-images in the common long-axis projections. All cine-images were acquired in accordance with current guidelines using balanced steady-state free precession sequences (TR/TE = 3.4/1.7 ms and flip angle = 60°) with retrospective electrocardiography (ECG) gating (30 phases per cardiac cycle) and parallel imaging (acc factor = 2) during expiratory breath-hold. Typical in-plane spatial resolution was 1.7 × 1.7 mm of 8 mm .2, TR/TE = 4.8/2.9 ms, BW = 477.8 Hz/pixel, flip angel = 12°, phases per cardiac cycle = 40, acc factor = 2, TFE factor = 4, TFE shots = 13, NSA = 1) were performed in accordance with current guidelines at the level of the sinotubular (ST)-junction (Ao1) as well as 1 cm above the ST-junction . Left ventricular volumes were obtained by manual tracing of the endocardial contour in the successive slices of the continuous SA stack. Endocardial contours were subsequently propagated through all phases using a semi-automated tracing algorithm, followed by manual adjustment, if necessary  during systole was calculated from the forward flow rate curve. Also, the eccentricity of the outflow jet was estimated as the flow displacement (FD) parameter described by Sigovan et al. . For thaSubanalyses were performed to study the effect of the measurement position. For that purpose, the percentage difference of RVol between positions was calculated, and then correlated to systolic BFV, FD and aortic dimension.i.e. Ao1 and Ao2, as well as for the repeated measurements, i.e. M1 and M2.All AR patients were assessed to have non-severe AR or severe AR according to current guidelines . Cut-offP < 0.05, was used to test significance between methods and different measurement positions. Mann–Whitney U-test, at a significant level of P < 0.05, was used to test difference between different types of flow characteristic (NCF versus CF)). Correction for multiple testing was performed using the Holm step-down method, where the P value was adjusted accordingly [Values are expressed as the mean ± standard deviation, unless otherwise stated. The repeatability was calculated as the absolute value of the difference of the two consecutive PC-MRI measurements (|M1 − M2|) as percent of their mean ((M1 + M2)/2). The agreement between the methods was evaluated using the Bland–Altman method, calculating the mean difference (MD) and limits of agreement (LoA = MD ± 1.96 SD) . The degordingly . StatistAll subjects were successfully examined with MRI. However, two PC-MRI measurements were not repeated at the distal measurement position Ao2 and were therefore excluded from the repeatability analysis at that position, as well as from the repeatability analysis regarding impact of measurement position.Patient characteristics are shown in Table P < 0.001, Fig. P < 0.001).The grade of flow complexity, as indicated by the systolic BFV, increased with increasing aorta dimension . Overall, the direct method gave significantly smaller RVols than the internal reference method (Table P < 0.001).The direct RVol measure correlated with the internal reference for both CF and NCF patients. This was the case for both measurement positions, od Table . At Ao2,The repeatability was significantly lower, i.e. the variability was larger, for patients with CF than for patients with NCF, at both measurement positions Table . HoweverP = 0.3; RF: P = 0.7; Repeatability of RVol: P = 0.9; Repeatability of RF: P = 0.5; the P values are corrected for multiple testing [P < 0.001; RF: P = 0.01; Repeatability of RVol: P = 0.02; Repeatability of RF: P = 0.02; the P values are corrected for multiple testing [No significant influence of measurement position, neither regarding regurgitation values nor regarding repeatability of regurgitation values, was found for patients with NCF  versus 0/17 (0%), P = 0.09); Fig. The frequency of patients that changed in AR grade from non-severe to severe, and vice versa, when the position of the measurement changed from Ao1 to Ao2 was higher for patients with CF compared to NCF (RVol: 5/26 (19%) versus 1/17 (6%), P = 0.02). The comparison of the percentage difference of RVol between positions and FD showed a moderate association . Similar results were found for systolic BFV .The percentage difference of the RVol between positions and aortic dimension showed only a weak association  this variation corresponds to 6 ml respectively 12 ml and, thus, should be taken into consideration in the evaluation of AR severity.The assessment of AR was shown to depend on the position of the measurement. For patients with complex flow, displacement of the measurement plane to a more distal position in the ascending aorta significantly affected the assessment, which may be due to a higher grade of in plane flow at this position, and consequently a higher grade of complex flow. For patients with non-complex flow, no differences were found in the quantification of the regurgitation values between different measurement positions. Others have shown that the accuracy of regurgitation quantification decreases with increased distance to the aortic valve , 27. HowIn the present study, an internal reference (LVSV–PuSV) was used for comparison with RVol of AR as it is not affected by complex flow in the ascending aorta. The method has a higher variability in comparison to PC-MRI , but hasPhase contrast MRI is an established method that allows non-invasive measurement of blood flow in vessels deep in the body –37. The Present findings show that complex flow is strongly associated with dilatation of the aorta. In our study, most patients with aortaic dilatation (≥ 40 mm ) presentOur study has some limitations. Firstly, the study comprised only a relatively small number of patients, which has nonetheless been sufficient to show significantly differences concerning the impact of complex flow on the quantification of RVol and RF using PC-MRI. Secondly, the selected patient cohort did not include patients with mild AR. Future studies are warranted to include a larger range of AR severity. Thirdly, there is no “golden standard” to determine the true RVol and RF. In the present study, we chose the indirect method (LVSV–PuSV) as internal reference, accepting an offset bias. Finally, 4D flow measurements with retrospective gating may overcome some of the challenges in assessing AR severity in regions of complex flow, but this needs to be evaluated in future studies.In conclusion, this study shows that quantification of AR using PC-MRI is influenced by complex flow, associated with dilatation of the aorta, which can lead to underestimation of the severity grade. This in turn can compromise the clinical decision making and the timing of surgery, and as such, alternative MRI methods e.g. LVSV–PuSV should be included in the examination protocol to ensure diagnostic certainty for patients with enlarged aortas.


Despite increased information showing obesity is an important breast cancer (BC) risk factor, the mechanisms implicated in this association are not well understood. In this review we describe multiple lines of evidence indicating that altered secretion of androgens and adipokines, from dysfunctional adipose tissue, are independently linked with BC development. However, adipokines (adiponectin and leptin) participate in important biological processes in BC cells by modulating signaling pathways similar to those used by the androgen receptor. These similarities suggest that crosstalk between these factors can occur, with a high probability that its interactions may be responsible for modifying the behavior of normal and tumor cells, especially in obesity. The knowledge of how adiponectin and leptin can interact with the androgen receptor signaling may prospectively guide the development of therapeutic approaches aimed at potentiating the inhibitory actions of adiponectin and androgen receptor or interfering with the pro-stimulatory role of leptin in BC.Obesity is an increasing health challenge and is recognized as a breast cancer risk factor. Although obesity-related breast cancer mechanisms are not fully understood, this association has been linked to impaired hormone secretion by the dysfunctional obese adipose tissue (hyperplasic and hypertrophic adipocytes). Among these hormones, altered production of androgens and adipokines is observed, and both, are independently associated with breast cancer development. In this review, we describe and comment on the relationships reported between these factors and breast cancer, focusing on the biological associations that have helped to unveil the mechanisms by which signaling from androgens and adipokines modifies the behavior of mammary epithelial cells. Furthermore, we discuss the potential crosstalk between the two most abundant adipokines produced by the adipose tissue (adiponectin and leptin) and the androgen receptor, an emerging marker in breast cancer. The identification and understanding of interactions among adipokines and the androgen receptor in cancer cells are necessary to guide the development of new therapeutic approaches in order to prevent and cure obesity and breast cancer. Breast cancer (BC) is the leading cause of death from cancer in women worldwide . FurtherIn contrast, hormonal dependency of BC has been extensively studied (estrogen receptor—ER and progesterone receptor—PgR) and found to have significant prognostic and predictive value . However2 is commonly defined as obesity, which is considered an epidemic disease as ~10% of the global population is obese [A body mass index (BMI) ≥ 30 Kg/mis obese . Severalis obese ,14,15,16is obese . Additiois obese ,19,20. Iis obese ,22,23. His obese ,25.The relationships among overweight/obesity and intrinsic BC molecular subtypes, have also been investigated. An analysis of 1676 postmenopausal BC women, revealed significant associations between being overweight (BMI ≥ 25) and luminal A, B and basal-like intrinsic subtypes, and patients with basal-like BCs were more likely to be highly obese (BMI ≥ 35), compared with underweight/normal patients . MoreoveThe pathogenesis of BC involves interactions between malignant cells and the mammary microenvironment, and therefore the tumor phenotype may be regulated by not only endocrine but also paracrine signals in the surrounding stromal tissue, which is mainly formed by adipocytes in BC. In this regard, the dysfunctional obese adipose tissue (hyperplasic and hypertrophic adipocytes) secretes high amounts of molecules and extracellular vesicles, which can target cells and promote cell-to-cell communication ,29. ElucVEGF expression in BC cells. All of this may be explained by the PGRN ability of regulate PI3K/AKT and JAK-STAT signaling [The obesity-related hypoxic and inflammatory state is also associated with a decreased response to insulin and insulin-like growth factors (IGFs) signaling, leading to insulin resistance and hyperinsulinemia . In BC cignaling ,42. In aignaling . NonetheADPN is a monomeric glycoprotein able to trimerize forming a low molecular weight (LMW) isoform, then trimers can combine to form middle (MMW—hexamers) or high molecular weight (HMW until 18-mers) isoforms. Furthermore, ADPN may exist in a globular form, consisting only of the C-terminus domain . ADPN isIn vitro studies have demonstrated the anti-proliferative effects of ADPN on ER+ and ER− BC cells ,49,50, bAlthough ADPN is considered to reduce cellular viability, several studies have shown that ADPN may also increase the proliferation of ER+ cells and even negatively interfere with AMPK activation ,60,61,62Ob) gene, is expressed in several tissues but mainly in breast adipose tissue, possibly explaining why serum LEP levels are higher in women compared with men [LEP protein, encoded by the obese  [In contrast to ADPN, almost all studies have demonstrated that LEP exhibits pro-carcinogenic effects, which are mediated by activation of the obese receptor, a transmembrane protein with at least six alternatively spliced forms (Ob-Ra to Ob-Rf) ,73,74,75tivation . LEP als pathway ,78. In E pathway . Moreoved ACAT2) ,81,82,83Finally, LEP is considered one of the most important obesity-associated proinflammatory adipokines. It has been reported that higher LEP levels may stimulate NF-κB to increase the expression of pivotal inflammatory mediators such as TNF, IL-1b, and COX-2 . This isApproximately 75% of all BCs are ER+. Therefore, most of BC research has focus on identifying the targets and roles of this steroid receptor and its main ligand E2. However, an extensive amount of evidence suggests that estrogens are not the only steroid molecules related to BC ,87,88, aAndrogenic hormones are the major circulating sex hormones in both sexes . In femaIn contrast, 80–90% of ER+ tumors are also AR-positive (AR+) and associated with favorable prognoses  ,98, whicAR signaling it is recognized as a master regulator of gene programs associated with a wide variety of biological processes including reproduction, differentiation, cell proliferation, apoptosis, inflammation, metabolism and homeostasis . AndrogeDifferent reports have shown that DHEA, DHEA-S, 4-dione and their derivates, can influence the proliferative capacity of breast epithelial cells ,111. FurCCND1 and PTEN and activation of CDKN1A, TP53 and BRCA1, which are well-recognized oncogenes and tumor-suppressor genes, respectively [Many in vitro studies have shown that AR signaling consistently inhibits the basal and estrogen-induced proliferation and survival of MCF-7, T47D and ZR-75-1 cell lines  ,116,119.ectively ,122,123.ectively . In suppectively . Furtherectively   mRNA, may benefit from enzalutamide in combination with the AI exemestane [As AR signaling have shown to inhibits estrogen-induced proliferation and survival of ER+ BC cells, natural and synthetic steroidal androgens were used as therapeutic approach, however they were discontinued for induce serious side effects . Furtheremestane . This coemestane ,136.The above results support the idea that the AR and androgens , may play a proliferative role in ER+ tumors. However, combining the data from in vivo and in vitro studies, it appears that a fine-tuning regulation of androgens and estrogens levels, as well as of their receptors, is essential to keep balanced its functions in mammary tissue. Accordingly, two insights could explain the contradictory effects of AR positivity on ER+ BCs: (i) the AR appears to have context-dependent roles as it shifts from an anti-proliferative stimulus to a pro-proliferative one, depending on the high and low expression levels of ER, respectively, and (ii) although AR positivity likely reflects well-differentiated BCs and is thus associated with a good prognosis, the biological effects of AR signaling, when over-stimulated by genomic or non-genomic mechanisms, may confers worse outcomes in BC, which has been recently suggested .In line with the effects of AR in tumors with low ER levels, in vitro studies have shown that the stimulation of the AR signaling can promotes the proliferation of ER− BC cell lines with high (MDA-MB453) or low AR levels  ,142,143.Androgen-induced growth of MDA-MB453 cells has been shown to be associated with activation of the WNT/β-catenin and HER2 pathways . These sp = 0.06) [Regarding biological behaviors, extensive molecular profiling and clustering studies have identified great heterogeneity within ER− tumors  . Among t = 0.06) . In geneAs mentioned above, the ovaries and the adrenal cortex are the main sources of androgens, but in postmenopausal women these sex steroids are mainly synthesized by adipose tissue ,162. ThiBiologically, the increased androgen synthesis induced by obesity may favors the growth of ER+ cells due to T aromatization and augmented E2 production. However, it must also be assumed that the proliferative stimulus may be enhanced by the itself elevation of androgens and their interaction with AR. In line with this insight, a recent study demonstrated that in a preclinical model of obesity, AR transcriptional activity was increased and promoted ER+ BC progression in environments with low E2 availability . Thus, in models treated with enzalutamide (anti-androgenic drug), AR inhibition prevented obesity-associated tumor progression by suppressing the proliferation and survival of BC cells . Hence, AR and its target gene FKBP5, were significantly increased. Bioinformatic analyses further demonstrated that AR and FKBP5 expression were higher in ER+ BC cases with increased IL-6 levels [Although it has been reported that postmenopausal obese women have elevated circulating or mammary adipose tissue androgens levels compared with lean women, some studies have found inconsistent results . Therefo6 levels . To the Most studies indicate that AR signaling plays a positive role in ER+ BC prognoses by blocking ER function. In this context, ADPN has been reported to prevent androgens aromatization , which iADPN can activate multiple intracellular signaling pathways. Through AMPK-independent signaling, ADPN promotes interactions with TKRs, such as the IR, which may increase PI3K and MAPK signaling in both ER+ and ER− BCs ,60. ThesAR gene expression. In fact, several pioneer and general transcription factors  target the AR promoter and can be regulated by pathways stimulated by ADPN [AR downregulation [AR expression might induce BC cell growth, consistent with the reported tumorigenic actions of ADPN [AR expression by decreased levels of ADPN would promote tumor growth because the AR would not be enabled to inhibit ER signaling  via phosphorylation of JAK/STATs or (ii) by activation of several adaptors or membrane-associated proteins  ; Both meAR gene promoter region [AR gene expression, similar to IL-6 [AR expression was downregulated in LNCaP cells cultured in the presence of high LEP concentration [AR gene expression, has led to the hypothesis that high LEP levels might potentiates the oncogenic effects of the ER by reducing AR levels and its inhibitory action on the ER. In contrast, opposite effects on AR expression could be observed in ER− or low ER expression cells, which would be consistent with the confirmed pro-tumorigenic role of both, LEP and the AR, in basal-like BC subtypes [Of note, STAT-family members are transcription factors itself that bind to a gamma-activated sequence or an interferon-stimulated response element, and according to the Signaling Pathways Project web knowledge base, these elements have been identified in the r region ,182. Thi to IL-6 . Indeed,ntration . In lumisubtypes ,103  and in vivo  experimental obesity models are required to study their interactions. These approaches will allow the evaluation of not only the degree to which the adipokines and the AR promote activation or inactivation of each other, but also whether their coordinated actions can modulate the biological processes that they are involved in. Because the epidemiological data are inconsistent, regarding the association of obesity with hormone receptor expression, menopausal status and BC subtypes, it will be interesting to clarify the relationship between lifestyles and clinical associations. To address this topic, further epidemiological studies with a larger number of included patients are needed. Additionally, the assessment of potential correlations among adipokines with androgen levels or AR status will be useful to establish their influence on BC prognosis.Clearly, obesity is a biological variable that merits consideration regarding the therapeutic strategies used to manage BC. Accordingly, and because of the modifiable nature of obesity, the most recommendable approach must be to promote lifestyle changes  to achieve a healthy weight to prevent cancer development and even improve BC treatment efficacy. In line with our literature review, it is logical to assume that drugs targeting ADPN, LEP and even adipokines less studied as PGRN, require a deeper evaluation on their use in BC management. However, regarding AR, clinical trial results indicate that antiandrogen therapies may be effective and needed for AR+ TNBC patients, especially for LAR subtypes or even non-LAR with AR-weak positivity. Furthermore, although AR inhibition seems not to be widely effective in ER+ patients, focus need to be put in the identification of luminal tumors where AR levels are higher respect to ER levels. Preclinical and clinical research suggest that these tumors, as well as AR+ HER2-Enriched BC subtypes, are more dependent on androgen signaling. Therefore, additional studies using antiandrogen therapies, alone or in combination with other strategies, should be considered as an important medicinal target, that in the future, will allow to obtain significant clinical benefits for patients with different BC subtypes.The knowledge of how ADPN and LEP can interact with the AR signaling pathway in breast epithelial cells may prospectively guide the development of new therapeutic approaches aimed at potentiating the inhibitory actions of the antiandrogen therapies in BC. Nevertheless, the use of drugs targeting ADPN and the AR as potential therapeutic tools, will have to be carefully assessed and separately studied according to the ER status, because of the contradictory effects reported of they in each BC tumor subtype.


Interactions between individuals are thought to shape evolution and speciation through natural selection, but little is known about how an individual (or player) strategically interacts with others to maximize its payoff. We develop a simple decision-theoretic model that generates four hypotheses about the choice of an optimal behavioral strategy by a player in response to the strategies of other players. The golden threshold hypothesis suggests that 62% is the critical threshold determining the transition of a larger player's strategy in reaction to its smaller dove-like partner. Below this critical point, the larger one exploits the smaller one, whereas above it, the larger one chooses to cooperate with the smaller one. The competition-to-cooperation shift hypothesis states that a larger player never cooperates with a smaller hawk-like player unless the former is reversely surpassed in size by the latter by 75%. The Fibonacci retracement mark hypothesis proposes that, faced with a larger dove-like player, a smaller player chooses to either cooperate or cheat, depending on whether its size relative to the larger player is less or more than 38%. The surrender-resistance hypothesis suggests that, in reaction to a larger hawk-like player, a smaller player can either gain some benefit from resistance or is sacrificed by choosing to surrender. We test these hypotheses by re-analyzing body mass data of full-sib fishes that were co-cultured in a common water pool. Pairwise analysis of these co-existing fishes broadly suggests the prediction of our hypotheses. Taken together, our model unveils detectable yet previously unknown quantitative mechanisms that mediate the strategic choice of animal behavior in populations or communities. Given the ubiquitous nature of biological interactions occurring at different levels of organizations and the paucity of quantitative approaches to understand them, results by our decision-theoretic model represent an initial step towards the deeper understanding of how biological entities interact with each other to drive their evolution. Cooperation, Competition, Golden section theory, Interaction, Fish. These iConsiderable evidence shows that phenotypic variation drives the behavior and functioning of animal interactions . Animal 2Interaction descriptors: Specifically, we consider an animal population in which each individual may choose to cooperate or compete with any other members, driven by resource availability or quorum sensing. Thus, any two individuals  X and Y in a population will produce four types of interactions, i.e., mutualism, altruism, aggression, and antagonism  of roughly similar size tend to act together as a single organism in which cooperation dominates and schooling behaviors produce an egalitarian form of organization . This coFaced with the needs of foraging, reproduction, and defense from predators, dominant animals  display aggressive and courtship behaviors upon subordinates . This agEach type of animal-animal interaction described above can be quantified mathematically. Let altruism B.Biological justification: In our previous study  over cooperation and competition regions in all three experiments EP  and of P < 0.001), indicating the effectiveness of If one strain is aggressive on the other, i.e., the former grows at a cost of the latter, then the relative abundance of the former over the latter should increase through some regulatory systems when the two strains are relocated from their respective isolated environments to the socialized environment . By contOur previous cultural experiments using different species have well supported the biological relevance of How a larger player reacts to a smaller player? Under the assumption  rewards B can be In general, we assume X makes decisions in rows, while Y makes decisions in columns. For varying values of We consider lower-order animals without a fully developed theory of cognition. We hypothesize that such players are not making decisions based on absolute size, but relative size. We can therefore analyze the break-even point if a larger player is confronted with a smaller player that act in a non-aggressive manner (dove). Assuming some decision making ability, the larger player is non-aggressive ifIdentifying the break-even leads to the simple quadratic equation:ϕ is the golden ratio. This is the first testable hypothesis: when a larger player encounters a smaller dove-like player, it acts aggressively when the smaller player is below 62% of its size and cooperatively when it is larger than that. In other words, the golden ratio is a cut-off point that helps a player chooses cooperation or conflict. If a larger player still chooses to compete with a smaller dove-like player when the latter's size is beyond 62% of the former's size, both, especially the larger one, need to invest extra energy.Considering the smaller player's behavior and relativizing to the larger player , the break-even point at which a larger player reacts to a smaller aggressive player (hawk) can be obtained by solving the equationHow a smaller player reacts to a larger player? Based on the reward matrix (1), this question can be answered by detecting the “break-even” points of the following equations:y2 – x2y – 2xy + x2 = 0. Let x = 1 by normalization and, then, we have y2 – 3y + 1 = 0, whose non-extraneous solution isFibonacci retracement mark, the smaller player Y prefers to cooperate with the larger dove-like player X. Yet, if this relative value is larger than 38%, player Y becomes interested in acquiring benefit from player X by cheating the latter, which is a third testable hypothesis. From y = 0 by letting x = 1, which generates a fourth testable hypothesis. Faced with the larger hawk-like player, the small player would be sacrificed if it chooses to cooperate, but it may benefit from its choice of conflict with the larger player.If the relative trait value of player Y to X is smaller than the 38%, the 3Cyprinus carpio) conducted with a full-sib family composed of 71 full-sibs co-cultured in a common water pool. The fish have schooling and aggregation behaviors, making them an ideal model system to investigate how social interactions impact phenotypic differentiation (P < 0.001), which is in agreement with the first hypotheses that a larger fish tends to establish a mutualistic relationship with a smaller cooperative member if the small-large ratio is beyond the golden ratio.We reanalyze a published data from a mapping experiment of fish (ntiation . Using intiation . In the ntiation . SimilarP < 0.05)  and the submissive group (doves). Some fish may play both hawks and doves if they are aggressive to one member but submissive to another member. By comparing the body mass of hawks and doves in the network, we found that the ratio of the hawks-doves to the hawks is 0.502 (the upper tier) whereas the ratio of the doves to the hawks-doves is 0.581 (the lower tier), with both ratios being significantly lower than 0.618 (P < 0.05). This is consistent with the third hypothesis that egoists start to cheat altruists when their relative strength over the latter is beyond the Fibonacci retracement mark. In the antagonism network, each pair of nodes is linked by two antagonists . Correlation analysis of networks suggests that the pattern of social interactions does not depend on how much genetic similarity there is between the fish. Both mutualism and antagonism as bidirectional interactions are negatively associated with directional altruism and aggression, respectively (P < 0.001). Taken together, whether a fish chooses a competitive or cooperative strategy in response to its conspecifics is not associated with their genetic relatedness, rather than with their relative body size.Despite being from a full-sib family, a pair of fish as siblings may still differ in genetic relatedness since they inherit the same alleles from the parents at different likelihoods. We calculated the coefficients of genetic similarity based on genome-wide segregating SNPs and used them to construct a relatedness network center, . We did 4Our decision-theoretic model produces four testable hypotheses regarding how one animal interacts with others to maximize its payoff. The first hypothesis, called the golden section threshold hypothesis, states that a larger animal is aggressive upon a smaller nonaggressive animal if their relative smaller-to-larger size is less than 62%, and otherwise, it would decide to cooperate when the relative size is beyond this number. This hypothesis can be explained by the optimal ratio of benefit and cost . For exaEncountered with a smaller but aggressive animal, a larger animal will never be non-aggressive, but if the smaller animal grows to reversely surpass the larger animal by 75%, the larger one will have no choice but cooperate. This is a second hypothesis, called the competition-to-cooperation shift hypothesis. We name the third hypothesis the Fibonacci retracement mark hypothesis. According to this hypothesis, while a smaller animal benefits from a larger nonaggressive animal through cooperation, it may become cheating to be aggressive upon its benefactor when its relative size outmatches the Fibonacci retracement mark. It is worth noting that Fibonacci retracement also emerges naturally in financial analysis as heuristic strategy for stock management . The fouThese four testable hypotheses were proposed through a heuristic mathematical analysis of ecological phenomena to explain how different organisms choose their optimal behavioral strategies in response to the strategies of their conspecifics in a population or community. We cultured full-sib fish from a mapping population to validate these hypotheses. The first and third hypotheses can be well tested, while the fourth hypothesis partially tested. As an open question, testing the second hypothesis needs a longitudinal experiment of culture, from which dynamic interactions can be monitored.The premise of our hypothesis formulation is the mathematical descriptors of interaction types derived from animal behavioral ecology. Collective behavior and social grouping are omnipresent phenomena in the animal kingdom, ranging from pairs of individuals to communities . The mecOur hypotheses will not only stimulate new thinking about ecological interactions and their resulting evolution and speciation, but also may hold potential implications for cancer cell biology, social network science, and even international political science. For example, a tumor may be composed of many size-varying, interacting cell populations, which can be distinguished by modern single-cell analysis techniques . For two55.1Escherichia coli (E), Staphylococcus aureus (S), and Pseudomonas aeruginosa (P), and grew each strain from each species in monoculture and two interspecific pairs (EP and SP) with two randomly selected strains each from a different species in co-culture. We used quantitative PCR (qPCR) technique to measure the abundance of each strain in both monoculture and co-culture experiments once every two hours during the first 24 h, followed by once every four times till 36 h, after the cultural experiment started.In Mu), antagonism (An), aggression (Ag), and altruism (Al) for each time point at each phase in co-culture.Microbial growth is thought to obey some biological rule described by growth equations . We used5.21 family of Cyprinus carpio including 71 progeny produced by Hebao Red carp and Koi carp. The fish were cultured in the common water pool at the Research Institute for Heilongjiang River Fisheries, Harbin, China, and measured for body mass after anesthesia with MS222 when they reached an adult stage of fish growth. As a fitness-related trait, we used body mass as a measure of the payoff of decision making by each fish. For a given pair of fish, whose body mass is x and y (x > y), we calculated the mathematical descriptors of four interaction types, mutualism, altruism, aggression and antagonism, based on the reward matrix (n(n – 1)/2 descriptor values for each type of interaction. By culling off a proportion of small values, we reconstruct four n-node sparse networks, each for a different type of interaction. 1 family. These SNPs were used to calculate the degree of pairwise relatedness among 71 full-sibs.We used a published genetic mapping data of fish  to test d matrix B. ConsidShuang Wu & Libo Jiang: Analyzed and interpreted the data.Xiaoqing He & Yi Jin: Performed the experiments.Christopher H. Griffin: Analyzed and interpreted the data; Wrote the paper.Rongling Wu: Conceived and designed the experiments; Wrote the paper.10.13039/100000001National Science Foundation (DMS-1814876) (CHG).This work was supported by https://github.com/BeijingCCB/GoldenTheory for public use.Data associated with this study has been deposited at The authors declare no conflict of interest.No additional information is available for this paper.


AR) gene identified a hemizygous de novo mutation (NM_000044.6:c.1621G > T) in exon 2 resulting in a termination codon p.(Glu541*) at the DNA binding domain (DBD). This novel nonsense mutation adds to the compendium of AR mutations which result in complete androgen insensitivity syndrome (AIS).An infant with 46,XY karyotype, and unambiguous female phenotype was found to have testes in the inguinal regions. Capillary sequencing of the androgen receptor ( AR gene is located on chromosome Xq11-12 and codes for the AR, which consists of 919 amino acids in length. The N-terminal domain NTD (amino acids 1–537) is encoded by exon 1 and is the largest domain in the AR, comprising more than half of the receptor, while the DBD is encoded by exons 2–3 and the ligand binding domain (LBD) by exons 4–81. Pathogenic variants in the AR gene cause AIS (OMIM 300068) characterized by complete or partial resistance to the biological actions of androgens in an individual with XY karyotype with normal testes and production of age-appropriate androgen concentrations. The prevalence of AIS is estimated to range from 1/20,400 to 1/99,100 males2. The majority of pathogenic alterations are missense variants causing dysfunctional LBD and DBD3. Recent case reports described nucleotide changes found in deep intronic regions which activate cryptic splice sites and expression of aberrant AR transcripts in AIS patients6.Androgens play a role in sex differentiation and the development of male sexual characteristics. Their action is mediated by the AR, a nuclear steroid transcription factor. The 2. Patients with CAIS do not have a cervix or uterus and have a blind-ending vagina. Serum testosterone concentrations are within or above the normal range for boys, while luteinizing hormone and anti-mullerian hormone concentrations are increased2. The diagnosis of CAIS is usually based on clinical presentation and laboratory findings. Determination of testosterone (T), androstenedione (A), and dihydrotestosterone (DHT) concentrations are required to exclude testosterone biosynthesis defects or 5-alpha reductase deficiency7. Management of AIS, in particular CAIS, requires a multidisciplinary team including an endocrinologist, urologist, gynecologist, and psychologist to address functional, sexual, and psychological issues2. For CAIS presenting in infancy, gonadectomy may be performed with puberty induction, as there is a potential risk of gonadal tumor2.The phenotype of AIS includes (1) normal female external genitalia (complete AIS (CAIS)), (2) ambiguous genitalia or male with hypospadias and micropenis ), and (3) normal male phenotype with infertility (mild AIS (MAIS)) in 46,XY individuals. CAIS often presents in infancy as an inguinal hernia or labial swelling containing a testis in an apparently normal female or may present as a mismatch between prenatal sex prediction (XY) and phenotype at birth We report a 1-month-old female infant who was born full term of normal vaginal delivery, with a birth weight of 2.54 kg. She was discharged well on day 3 of life but was noted to have a bilateral inguinal hernia at 1 week of life. On examination, there were reducible inguinal hernias with normal unambiguous female external genitalia, for which she was admitted for a bilateral herniotomy. At surgery, a biopsy was taken from the mass resembling a testis seen in the left inguinal hernia. Further exploration demonstrated the presence of testes in both inguinal regions in the absence of a uterus, which was subsequently demonstrated with pelvic and inguinal ultrasound. Histopathology of the biopsy showed seminiferous tubules.This is the first child of non-consanguineous parents, with no family history of any disorder of sexual development (DSD) or AIS. Chromosomal karyotyping by G-banding revealed a male karyotype, 46,XY. The human chorionic gonadotropin stimulation test demonstrated a significant rise in testosterone concentration from 0.93 to 19.75 mmol/L. The child also had a normal testosterone (T): dihydrotestosterone (DHT) ratio of 8.9 (reference <20) and a normal testosterone (T): androstenedione (A) ratio of 2.8 (reference >0.8). A preliminary diagnosis of CAIS was made.AR gene was performed with informed consent. A hemizygous variant NM_000044.6:c.1621G > T, NP_000035.2:p.(Glu541*) was identified , ClinVar (http://www.clinvar.com), Human Gene Mutation Database Professional 2020.4, and GenomAD (https://gnomad.broadinstitute.org/). The nonsense variant in exon 2 of the AR gene introduces a termination codon, which is expected to abolish the DBD and beyond. This variant is classified as pathogenic in the context of X-linked recessive AIS  according to the ACMG/AMP guidelines for the interpretation of sequence variants8. Thus the molecular investigation confirmed the diagnosis of CAIS.To our knowledge, this variant has not been previously described in the literature and is not present in the AR database (AR gene is recommended for patients with 46, XY DSD, regardless of family history6. Somatic mutations in the AR gene have also been rarely reported10. Our case illustrates that the presence of an inguinal hernia in a normal unambiguously female infant is a clear indication to screen for CAIS. The limitation in our molecular investigation was that only one tissue type, the blood, was tested. Pyrosequencing is a sensitive platform which has a 5% limit of detection of the minor allele fraction. The absence of the proband’s mutation in the mother’s blood DNA does not therefore completely reassure the family with the absence of recurrent risk of AIS in their future offspring, and future pregnancies should be carefully monitored although the risk is nevertheless low based on the current findings.Since 30% of AR mutations in AIS occur de novo, sequencing of the AR gene in a patient with CAIS presenting with bilateral inguinal hernia.Since the diagnosis was CAIS, the parents decided to raise the child as a girl, with testes removal before puberty. Bilateral orchidectomy was performed when the child was one and a half years of age. During the laparoscopic examination, there was no uterus and there was a blind-ending vagina. Histopathology showed immature testicular tubules with no spermatogonia and no malignancy. In conclusion, we identified a novel de novo nonsense mutation in the The relevant data from this Data Report are hosted at the Human Genome Variation Database at 10.6084/m9.figshare.hgv.3084.Supplementary Table 1


I met a traveler from an antique landWho said: Two vast and trunkless legs of stoneStand in the desert….Near them, on the sand,Half sunk, a visage lies, whose frown,And wrinkled lip, and sneer of cold command,Tell that its sculptor well those passions readWhich yet survive, stamped on these lifeless things,The hand that mocked them, and heart that fed;And on the pedestal these words appear:“My name is Ozymandias, King of Kings;Look on my works, ye Mighty, and despair!”Nothing beside remains. Round the decayOf that colossal wreck, boundless and bareThe lone and level sands stretch far away.Percy Bysshe ShelleyRosalind and Helen, A Modern Eclogue; With Other PoemsLondon: Hollinger. p. 72 (1876)This poem is in the public domain. Frankenstein, 1818), are known to many as classic English Romantic period authors. Percy was a major player in that literary scene with shorter works such as Ozymandias (1818), Ode to the West Wind (1819), and To a Skylark (1820), all well-known and studied still in many introductory literature courses. He also was known for his longer works that included allegorical tales such as Adonais (1821) and Prometheus Unbound (1820), but arguably, he is best remembered for the sonnet Ozymandias. Shelley tragically died in a boating accident at age 29. Although not renowned while alive, he gained fame posthumously and influenced other great poets such as Browning and Yeats.Percy Bysshe Shelley (1792–1822) and his second wife, Mary Shelley , many archeologic wonders were found, prompting consideration of the very colorful and remote pharaonic past. Those days seemed indestructible, but in Shelley’s time, the grand and storied empire was long gone and left in sandy ruin. In an essay dubbing Ozymandias “a poem to outlast empires,”1 David Mikics noted that Shelley was aware of Diodorus Siculus, a Roman-era historian whose writings describe the Ozymandias statue. Diodorus noted that perhaps Ozymandias was the Biblical Exodus pharaoh, Rameses II, and suggested that the inscription on the statue (the largest in Egypt at the time) was “King of Kings Ozymandias am I. If any want to know how great I am and where I lie, let him outdo me in my work.” In Shelley’s poem, Diodorus becomes the “traveler from an antique land” who might have seen the statue. Shelley did not. He was reacting to an announcement by the British Museum that the massive torso and head portion of a colossal Ramesses II statue from 13th century BCE, discovered in 1816 by an Italian explorer in Thebes, was soon to arrive in England. Shelley’s poem ended up being published before the statue arrived, and he died shortly thereafter.Ozymandias, written in sonnet style iambic pentameter with an atypical rhyming scheme. Evolution is history, and history is constantly evolving. Only the pace varies, such as with scientific discovery, and only those things we know about and understand can we know. Philosophers have said that one can dip their toe into a moving stream only once, because the stream is constantly changing. Heraclitus, the weeping Greek philosopher seen crying at the foot of the stairs in the iconic Vatican Library School of Athens painting (Raphael 1509–1511), has been said to weep in angst because of life’s constant change and the folly of mankind. And so it is that the story of Ozymandias and the colossus statue is but a metaphor for change, evolution, and the fate and folly of man. It makes me think of the great natural feats blessing our earth, particularly the evolution of our geologically older mountain ranges  and our newer ones (the Rocky Mountains and Sierra Nevadas); the massive glaciers carving out extraordinary visages such as California’s Yosemite Valley, Kings Canyon, Devils Postpile, and the Minarets as a few examples.It is important to recall the theme of Thinking of what glaciers can do brings up the issue of global warming as another example of change and evolution. Climate change can’t be argued. The ice age left Kansas long ago. Ohio’s “Kettle” lakes and the scarred valleys that I see with my wife during our weekend hikes are another testament of the past. Perhaps the speed of change can be debated, as can the influence of the way we live (Heraclitus’ “folly of man”). And of course, there are many more examples of change and evolution in our profession of medicine. Just think, as we are coming out of pandemic lockdown, about the evolution of coronavirus. Inevitable and fascinating.Ozymandias is a great poem to read, study, and ponder. It is a true classic published over two centuries ago and withstanding the test of time. It is interesting to vision implications of the sands of Egypt slowly blowing over an empire  and changing things forever, particularly the hubris of pharaohs and the very life lived in those times. “I met a traveler from an antique land…” and it made me think of all that is good with change and evolution, but that human sensibility must not be sundered if we expect to beat back blowing sands.Ozymandias—it is best when read aloud.Enjoy