Children with cancer are at high risk of developing physical impairments, which can be minimized through participation in rehabilitation programs. Unfortunately, the literature suggests that pediatric oncology rehabilitation services are underutilized and that few children are referred to the service. This study aimed to explore referral practices to pediatric rehabilitation in Canada, identify healthcare providers’ perspectives of barriers to service provision, and propose a framework for action to support the advancement of rehabilitation programs for children with cancer. Canadian healthcare providers working in pediatric oncology participated in a web-based survey. Results demonstrated that medical referrals to rehabilitation services occur mostly when the child presents with, or is at risk of, severe deficits due to surgery. Healthcare providers identified a large number of barriers to rehabilitation. A framework for action is proposed, comprising key strategies to enhance rehabilitation services for children with cancer and to inform future research.Purpose: To explore pediatric oncology referral practices, gather healthcare providers’ perspectives of barriers to access and provision of rehabilitation service across Canada, and inform a framework for action to optimize rehabilitation care and inform future research. Methods: A cross-sectional survey was conducted with Canadian healthcare professionals (HCPs) working in pediatric oncology. Results: A total of 54 responses were received, and 34 corresponded to HCPs who refer children with cancer to rehabilitation services. Results suggest that approximately 25% of children are referred to rehabilitation services, primarily when the child presents with, or is at risk of, significant functional disability due to surgery. A primary barrier to service provision identified across HCPs included a lack of funding and resources. Medical professionals further identified a lack of specialized pediatric oncology rehabilitation services, whereas rehabilitation professionals identified the lack of pediatric oncology specific space and equipment. Identified themes from open-ended survey questions include the need for (1) dedicated funding and resources, (2) improved access, and (3) the need for specialized pediatric oncology rehabilitation services. Conclusion: Several barriers exist in the Canadian healthcare context that impact the delivery of rehabilitation services for children with cancer. We propose a framework for action to advance clinical care and guide future research. Childhood cancer survival has increased, with a current 5-year survival rate of nearly 85% . It is eThere is some evidence of the potential benefits of physical therapy (PT) for minimizing the severity of adverse effects and improving physical function of children and adolescents with cancer ,14,15. IRecent systematic reviews have evaluated the effects of exercise on physical function outcomes. Coombs et al. , publishWhile research evidence supporting physical therapy and general physical exercise interventions for children with cancer has increased over time, limited literature exists supporting the benefit of rehabilitation disciplines such as occupational therapy and speech-language pathology, and current research evidence suggests underutilization of occupational therapy services in this population . More reEngaging children and adolescents with cancer in early rehabilitation programs may help minimize the occurrence and severity of treatment-related effects ,22,23, aThe purposes of this study were to (1) explore the frequency of and reasons for referrals to pediatric oncology rehabilitation services, (2) identify existing barriers to access including barriers to provision of pediatric oncology rehabilitation programs, and (3) propose a framework to support advanced care and future research in pediatric oncology rehabilitation.We conducted a cross-sectional web-based survey in a secure Research Electronic Data Capture (REDCap) service at the University of Alberta in Edmonton, Canada . REDCap The survey was administered between July and October 2017. HCPs who had provided and/or referred children and adolescents with cancer to rehabilitation services across Canada were eligible for the study. The survey was sent out through professional networking and organizations. Twenty-eight additional organizations, institutions, and facilities that provide cancer care were identified online and contacted. Information about the study was delivered via email to healthcare providers and distributed in the newsletters and e-blasts of the organizations and associations. To increase the response rate, we sent electronic reminders every three weeks, and respondents were asked to forward the survey communication to other HCPs who were working with the pediatric oncology population.n = 62). Given the low incidence of pediatric cancers in Canada  ..39].Advocate: Families of children with cancer play a crucial role in advocating for improvements in cancer treatments, availability of resources, and advancements in research [research . The creresearch .Guide: The lack of high-quality rehabilitation-related research evidence is a current barrier to the development of clinical practice guidelines in pediatric oncology. A proposed interim step is for HCPs, researchers, and leaders in the field to collaborate on the creation of expert opinion guidelines and recommendations specific to pediatric oncology rehabilitation services.Educate: Existing rehabilitation professional programs lack adequate content in oncology, and pediatric oncology, in particular [rticular . To addrrticular . EducatiIdentify: Strategies such as prospective surveillance may help to inform the incidence, severity, and natural progression of impairments and functional limitations of the common childhood cancers [ cancers . As sugg cancers , simply Innovate: To address issues related to access to and transitions in care, research is needed evaluating the potential of eHealth. Telehealth and telecommunication technologies may offer opportunities for virtual delivery of rehabilitation interventions and surveillance for families living in rural and remote locations [ocations ,43 or foocations .Engage: To ensure a patient-centred approach, research is needed exploring the perspectives of parents and families towards pediatric oncology rehabilitation programs and family-reported barriers to and facilitators for accessing rehabilitation services.Several limitations were identified in this study. To protect the right of privacy and confidentiality, we did not ask personal information that identified participants. Therefore, we were not able to follow up with respondents when survey responses were unclear or incomplete. As well, although administered across the country, the survey did not encompass respondents from all provinces, which suggests that our results may not be representative of all pediatric oncology rehabilitation programs in Canada. Although a strength of the study involved the higher than anticipated response rate that exceeded our estimated sample size, the relatively small sample size is unlikely to be representative of all pediatric oncology HCPs across Canada. Our results, however, are similar to previous surveys of HCPs conducted in adults with cancer and those conducted in other jurisdictions ,31.Our study provides an overview of the current referral patterns and barriers to pediatric oncology rehabilitation services across Canada and proposes a framework to action to advance pediatric oncology rehabilitation and guide future research in a Canadian context. Despite limited research in the field, educational efforts specific to pediatric oncology rehabilitation are needed to build capacity for services across acute care and community/primary care locations. Prior to evaluating referral strategies to pediatric oncology rehabilitation programs, further research is needed to understand the needs and perspectives of children with cancer and their families toward rehabilitation services. Given the current paucity of research in pediatric oncology rehabilitation, and the barriers to care identified through this survey, action is needed to advance clinical practice and research efforts.


The androgen receptor (AR) signaling axis is the major therapeutic target in prostate cancer (PC). Second-generation antiandrogens, such as abiraterone acetate and enzalutamide, have shown impressive results in pre- and post-chemotherapy settings, prolonging the survival of PC patients. However, nearly all patients ultimately develop resistance to antiandrogen therapy. Our findings establish the role of the acetyl-CoA-dependent adaptive mechanisms in resistance to antiandrogen therapy.The commonly used therapeutic management of PC involves androgen deprivation therapy (ADT) followed by treatment with AR signaling inhibitors (ARSI). However, nearly all patients develop drug-resistant disease, with a median progression-free survival of less than 2 years in chemotherapy-naïve men. Acetyl-coenzyme A (acetyl-CoA) is a central metabolic signaling molecule with key roles in biosynthetic processes and cancer signaling. In signaling, acetyl-CoA serves as the acetyl donor for acetylation, a critical post-translational modification. Acetylation affects the androgen receptor (AR) both directly and indirectly increasing expression of AR dependent genes. Our studies reveal that PC cells respond to the treatment with ARSI by increasing expression of ATP-citrate lyase (ACLY), a major enzyme responsible for cytosolic acetyl-CoA synthesis, and up-regulation of acetyl-CoA intracellular levels. Inhibition of ACLY results in a significant suppression of ligand-dependent and -independent routes of AR activation. Accordingly, the addition of exogenous acetyl-CoA, or its precursor acetate, augments AR transcriptional activity and diminishes the anti-AR activity of ARSI. Taken together, our findings suggest that PC cells respond to antiandrogens by increasing activity of the acetyl-coA pathway in order to reinstate AR signaling. Aberrant AR signaling is a key event in PC initiation, progression, and development of castration resistance ,2,3,4. ETherapeutic failure coincides with different molecular alterations including synthesis of intra-tumoral androgens, upregulation of AR, ligand-independent stimulation of AR, as well as the expression of constitutively active AR variants (AR-Vs) ,29,30,31Acetyl-coenzyme A (acetyl-CoA) is a central metabolic and signaling molecule that participates in fatty acid synthesis and protein acetylation . The twoIn the present study, we demonstrate, for the first time, that treatment with antiandrogens abiraterone and enzalutamide elevates intracellular levels of acetyl-CoA in PC cells. Correspondingly, the supplementation with exogenous acetyl-CoA, or its precursor acetate, diminishes the anti-AR activity of the ARSI. Critically, inhibition of ACLY, an enzyme responsible for cytosolic acetyl-CoA generation, results in a significant suppression of ligand-dependent and -independent routes of AR activation. Our data suggest that the aberrant acetyl-CoA homeostasis may play a critical role in the development of resistance to therapeutics targeting the AR axis.C4-2B human prostate cancer cells were obtained from ATCC . LNCaP, 22RV1, and PC-3 human prostate cancer cells were obtained from the Fox Chase Cancer Center Cell Culture facility. Initial stocks were cryopreserved, and at every 6-month interval, a fresh aliquot of frozen cells was used for the experiments. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, gentamicin (50 mg/L), sodium pyruvate (1 mM), and non-essential amino acids (0.1 mM) under conditions indicated in the figure legends.BMS 30314, enzalutamide, abiraterone acetate, acetyl-CoA, sodium acetate, A-485, and C646 were obtained from Cayman Chemical Company . Antibodies against AR was obtained from Cell Signaling Technology . Antibodies against ACLY and ACSS2 were obtained from ProteinTech . Anti-actin antibodies and R1881 were obtained from MilliporeSigma .Western blot analysis was performed as described previously .Intracellular levels of acetyl-CoA were examined using an acetyl-CoA assay kit .The luciferase reporter assay was performed as described previously . To geneLNCaP cells were transfected using SMARTpool siRNAs targeting p300 and CBP  as described in our publication .PSA levels in cell culture supernatants were examined using a PSA ELISA kit . Expression of testosterone in cell lysates and cell culture supernatants was examined using a testosterone ELISA kit .ORF of the AR was amplified with specific primers using EGFP-AR  as a template; ORF of the AR-V7 splice variant was amplified with specific primers using a AR-V7-pcw107 vector  as a template. Resulting ORFs were re-cloned into EcoRI/BamHI and XbaI/XhoI sites of lentiviral vector pLV-CMV-H4-puro. Lentiviral production, infection of cells, and puromycin selection was performed as described previously .Total RNA was isolated using the Mini RNA isolation II Kit  and purified using/via the RNA Clean and Concentrator Kit . Total RNA (1 μg) was reverse transcribed in a final volume of 20 μL with 100 U of Superscript III Reverse Transcriptase  and 50 ng of random hexamer primers according to the manufacturer’s instructions. After reverse transcription, cDNA samples were diluted 40 and 400 times (for genes of interest and 18S rRNA respectively) and 5 μL of diluted cDNA was amplified by real time PCR using PSA (KLK3) PrimeTime primer mix (Hs.PT.58.38546086); and ACLY PrimeTime primer mix (Hs.PT.56a.1709982.g) and ACSS2 PrimeTime primer mix (Hs.PT.58.28278662) . A custom 18S rRNA Mini qPCR assay  was used as an internal amplification control. The amplicon was detected with a forward primer 5′-GCTCTTTCTCGATTCCGT, reverse primer 5′-CCAGAGTCTCGTTCGTTATC and probe 5′-TTCTTAGTTG GTGGAGCGATTTGT labeled with 6-FAM and quenched with Jowa-Black FQ. Each sample was run in triplicate for genes of interest in a 20 µL reaction mix using iTaq Universal SYBR Green Supermix  and 18S rRNA TaqMan Gene Expression Master Mix according to the manufacturer’s instructions . Reactions were carried out/performed in an Applied Biosystems 7500 Real-Time PCR System. Analysis of relative genes expression was carried out/performed using the 2−ΔΔCt method.Tissues were collected and fixed in 10% phosphate-buffered formaldehyde, dehydrated, and embedded in paraffin. Hematoxylin- and eosin- (H&E) stained sections were used for morphological evaluation purposes. Unstained sections were used for immunohistochemical (IHC) studies. IHC staining was performed on a VENTANA Discovery XT automated staining instrument  using VENTANA reagents according to the manufacturer’s instructions. Slides were de-paraffinized using EZ Prep solution  for 16 min at 72 °C. Epitope retrieval was accomplished with CC1 solution  at 95–100 °C for 32 min. Primary antibodies were tittered with a TBS antibody diluent into user fillable dispensers for use on the automated stainer. Immune complex was detected using the Ventana OmniMap anti-Rabbit detection kit (#760-4311) and developed using the VENTANA ChromMap DAB detection kit (#760-159) according to the manufacturer’s instructions. Slides were counterstained with hematoxylin II (#790-2208) for 8 min, followed by Bluing reagent (#760-2037) for 4 min. The slides were then dehydrated with an ethanol series, cleared in xylene, and mounted. All slides were viewed with a Nikon Eclipse 50i microscope and photomicrographs were taken with an attached Nikon DS-Fi1 camera .t-test. A p-value of <0.05 was considered statistically significant.Statistical analysis was performed using a two-sided Student’s p = 2.81 × 10−6), whereas ACSS2 mRNA is expressed at reduced levels in tumors samples (p = 1.78 × 10−10)  .To further explore the role of acetyl-coA elevation in antiandrogen resistance, we examined whether an increase in intracellular acetyl-CoA content could compromise the efficacy of enzalutamide and abiraterone. Until recently, it was believed that acetyl-CoA cannot cross the cell membrane . HoweverRecent studies by Shah et al. show that combined treatment with ACLY and AR inhibitors suppresses AR target gene expression in PC cells expressing AR-FL . HoweverNext, we validated the results of our studies using wild-type AR-positive PC cell lines, specifically androgen-dependent LNCaP and castration-resistant C4-2B and 22Rv1 PC cells. LNCaP and C4-2B cells express functional T877A mutated AR A 65,66],6665,66][3H]acetyl-CoA could be incorporated into cellular lipids. Rhee et al. suggested that there is a possibility that acetyl-CoA transporter 1 (AT-1), which is expressed on the endoplasmic reticulum (ER) and transports acetyl-CoA into the ER lumen [Despite the initial response to androgen blockade, all patients will eventually progress to castration resistance. Castration resistance is progression of disease, either clinical  or biochemical (three consecutive rises in PSA levels above nadir) in the presence of castrate levels of circulating testosterone (<20 ng/dL) . PreviouER lumen , could bER lumen . In addiER lumen .Our analysis of TCGA dataset shows that only ACLY mRNA expression is significantly increased in tumor tissues compared with normal/benign tissues, whereas ACSS2 mRNA is expressed at the reduced levels in tumors samples . For manIntra-tumoral androgen biosynthesis has been described as one of a key mechanisms of ADT resistance . StudiesOur current studies demonstrate that treatment with enzalutamide and abiraterone elevates intracellular levels of acetyl-CoA in PC cells. Accordingly, the addition of exogenous acetyl-CoA compromises the anti-AR activity of these antiandrogens. Our experiments also established the critical role of the acetyl-CoA pathway for both ligand-dependent and -independent routes of AR activation. Therefore, aberrant acetyl-CoA homeostasis may play a critical role in the development of primary and acquired resistance to therapeutics targeting the AR signaling axis .


Detailed knowledge of the BOLD hemodynamic response function (HRF) is crucial for accurate analyses and interpretation of functional MRI data. Considerable efforts have been made to characterize the HRF in gray matter (GM), but much less attention has been paid to BOLD effects in white matter (WM). However, several recent reports have demonstrated reliable detection and analyses of WM BOLD signals both after stimulation and in a resting state. WM and GM differ in composition, energy requirements, and blood flow, so their neurovascular couplings also may well be different. We aimed to derive a comprehensive characterization of the HRF in WM across a population, including accurate measurements of its shape and its variation along and between WM pathways, using resting-state fMRI acquisitions. Our results show that the HRF is significantly different between WM and GM. Features of the HRF, such as a prominent initial dip, show strong relationships with features of the tissue microstructure derived from diffusion imaging, and these relationships differ between WM and GM, consistent with BOLD signal fluctuations reflecting different energy demands and neurovascular couplings in tissues of different composition and function. We also show that the HRF varies in shape significantly along WM pathways and is different between different WM pathways, suggesting the temporal evolution of BOLD signals after an event vary in different parts of the WM. These features of the HRF in WM are especially relevant for interpretation of the biophysical basis of BOLD effects in WM.  Functional Magnetic Resonance Imaging (fMRI) based on blood oxygenation level-dependent (BOLD) contrast is well-established as a technique to map cortical activity in the brain. BOLD signals indirectly report neural activity and are characterized by a hemodynamic response function (HRF) which describes the effects of transient changes in blood flow, volume, and/or oxygenation . The HRFTo date, most efforts to characterize the HRF have focused on measuring the transient, task-evoked BOLD responses to known events or stimuli (i.e. event-related fMRI), where timing information is accurately known. However, recent reports have shown how identification of the peaks of relatively large-amplitude BOLD signal fluctuations in resting-state data may also be used to reliably estimate HRFs without a stimulus . This inMost measurements of the HRF have focused on gray matter (GM), as BOLD effects in white matter (WM) have been reported relatively rarely  and ofteThe biophysical origins of BOLD signals in WM have not been identified, but 2 potential explanations include (i) they reflect venous drainage of deoxygenated blood from adjacent active GM or (ii) they correspond to increases in metabolic demand within WM that result from neural activity within GM and thus are similar to vascular effects in GM evoked by a stimulus. While neural signaling processes demand substantial energy consumption in GM , the metWe aimed to provide a comprehensive characterization of the HRF in WM across a population, including accurate measurements of its shape and its variation along and between WM pathways, using resting-state fMRI acquisitions. We also for the first time relate features of the HRF to microstructural and physiological properties of tissue derived from diffusion MRI. The relationships of HRFs between tissue types, and the measured differences in the HRF along and between WM pathways, suggest that there are different energy requirements and hemodynamic responses along different WM pathways which are consistent with the hypothesis that BOLD signals in WM reflect the metabolic needs of different tissue components than those seen in GM.T1-weighted MRI, and diffusion MRI. The imaging protocols have been described in detail in previous reports . T1-weighted images were acquired using a 3D magnetization-prepared rapid acquisition with gradient echo (MPRAGE) sequence .The data and HRF estimates closely followed the approach of  reports . BrieflyT1-weighted images were nonlinearly registered to MNI space using FNIRT  pipelines  of the Hng FNIRT  and subsng FNIRT . For fMRng FNIRT , and folHRFs were estimated from resting-state time courses in each subject using a blind deconvolution approach  implemenAfter the HRF had been estimated for each voxel in MNI space, features of the HRF were extracted and visualized as separate parametric maps as shown in Diffusion MRI scans were used to derive approximate measures of average tissue microstructure, using Diffusion Tensor Imaging (DTI) and the Neurite Orientation Dispersion and Density Imaging (NODDI) technique .DTI characterizes the magnitude, degree of anisotropy, and orientation of directional diffusion. From this, measures of fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity were derived. The measures are sensitive to a variety of microstructural features. For example, FA may reflect the coherence of membranes and restrictions, whereas RD is sensitive to myelination and axonal number and packing density . The NODOf particular importance, the quantity (1-NDI) represents the relaxation weighted volume fraction of all nonneurite components, which includes anything that does not display stick-like diffusion signal properties such as glial cells and some extracellular spaces.To investigate HRF features of specific WM pathways, we analyzed a set of expertly delineated bundles in MNI space . For thin = 20 points. Along-pathway mean and standard deviations of each HRF feature were derived for each position along the pathway.HRF features were quantified along each major WM pathway using the along-fiber quantification technique on the population averaged WM tracts defined by Linear mixed-effects modeling was used to ask whether HRF features change along pathways: the regression equation used for each feature, y, wasFinally, to assess relationships between different HRF features or between HRF and microstructural metrics, we performed 3 analyses (i) for each pair of features, we plotted one feature against the other for all WM and all GM voxels in MNI space, (ii) calculated the slope of line of best fit using total least squares regression (which minimized fitting error in both dependent and independent variables), and (iii) calculated the correlation coefficient between each pair of variables, again using all voxels in MNI space. Our primary purpose was to establish whether these relationships differ between GM and WM.The results provide information in response to several questions we sought to address.r = +0.13) and with increasing FA (r = +0.11). However, this dip integral shows the opposite trend in GM, becoming more pronounced  with increasing NDI (r = −0.19) and increasing FA (r = −0.17). Likewise, the FWHM covaries with NDI and RD but with opposite relationships between WM  and GM . The slopes and correlation coefficients for relationships between all HRF feature and all microstructure features are given as The HRFs for 15 WM pathways are shown in P-values for the F-tests are given as We aimed to extract a measure of changes in each HRF feature along pathways by taking the ratio of the range of that feature over the maximum value, calling this “% Variation.” The % Variations of each feature, for all features along every pathway, are shown in We have quantified variations of the HRF of resting state fMRI within and across WM pathways and cortical GM by creating population-averaged parametric maps of HRFs and their derived features. We find that, following transient fluctuations in resting-state signal, WM BOLD responses have smaller peaks and are slower to change, than GM responses, and also feature a prominent negative initial dip. Second, we find strong links between different features of the HRF, with similar patterns observed in both WM and GM, suggesting similar factors affect the dynamics and evolution of the response function. However, the tissue microstructural environment differs significantly between these tissue types, and the dynamics of the HRF show different dependencies on the underlying structure. Next, we find that features of the HRF change significantly along WM pathways and are much different in the deep WM than at the superficial WM. The variation observed along pathways also differs between pathways. Altogether, this suggests that, much like in GM, changes in flow and/or oxygenation related to variations in baseline conditions are different for different parts of the WM. These differences in HRF features may be relevant for understanding the biophysical basis of BOLD effects in WM.The origins of BOLD effects in WM are not well understood though they share many features with BOLD effects in GM and thus a common explanation of their biophysical basis is plausible. The HRF features include an initial negative dip, indicating an increase in the concentration of deoxyhemoglobin within tissue, followed by a positive peak that extends for several seconds, indicating an increase in blood flow and volume, a net decrease or washout of deoxyhemoglobin, and then a return to equilibrium. The times for flow to increase and subsequently return to baseline are likely determined by the overall density and dimensions (diameters and spacings) of the vasculature involved . There iAs pointed out by several previous reports , the metTo investigate this potential connection, we produced atlases of microstructure features derived from diffusion MRI. From the diffusion models, it is possible to derive voxel-wise maps of parameters including FA and NDI. FA is highly sensitive to the fractional composition of tissue that is myelinated neurons. Complementing the neuronal fractional volume, the extracellular volume fraction (ECVF = 1 − NDI) is a measure of the volume fraction within a voxel that is not neuronal and thus is a surrogate metric of the glial cell volume fraction. The use of diffusion tractography and along-fiber quantification  has provIf the negative dip in WM reflects the passage of deoxygenated blood draining from activated cortex, and if the vasculature runs parallel to a tract, we would expect the times to dip and to peak to be progressively delayed along a tract, as found here. On the other hand, it is not clear on this model why the area under the dip should increase along a tract from both directions, and peak in the center.In addition to showing feasibility of along-tract profiling of HRF features, our results also suggest that BOLD responses are different across different pathways in a resting state. This parallels the existence of resting-state networks in the cortex. Just as various networks may be characterized for aspects of attention, memory, motor, sensory systems, the WM fibers are structures often associated with unique functional roles, so it is unsurprising that they may require different energy processes on different time-scales. In fact, recent work  shows thThere are several directions to pursue to extend this research. First, the use of the HRF as a potential biomarker would be strengthened if WM HRFs were shown to be changed in altered states/disorders as shown in GM , and if We have characterized the HRF of resting state fMRI within and across WM pathways by creating population-averaged maps of the HRF and features of the HRF, and show that the HRF shows significant differences between WM and GM tissue types, features of the HRF show strong relationships with both other HRF features and with microstructural features, and that these relationships differ between tissue types. In particular, the dependences of some features of the HRF on neural content are reversed between GM and WM, suggesting that the BOLD response in WM may be related to different cellular types than in GM. We further find that the HRF varies significantly along pathways and is different between different WM pathways. Altogether, this suggests that, much like in GM, changes in flow and/or oxygenation related to variations in baseline conditions are different for different parts of the WM. These differences in HRF features may be relevant for understanding the biophysical basis of BOLD effects in WM.R1_Supplementary_Table_1_tgac035Click here for additional data file.


In rat developmental and reproductive toxicity studies, nipple/areola retention (NR) in male offspring is a biomarker for reduced androgen signaling during development. This is because nipples normally regress in male rats in response to androgen signaling during critical stages of development. NR is thus included as a mandatory endpoint in several OECD test guidelines for assessment of chemicals, particularly as a readout for anti-androgenic effects relevant for reproductive toxicity. With the growing interest in developing Adverse Outcome Pathways (AOPs) to aid in chemical risk assessment, a more pragmatic approach has been proposed, whereby essential units of knowledge could be developed independently of complete AOPs, not least emergent key event relationships (KERs). Herein, we have developed a KER linking “androgen receptor antagonism” and “increased areola/nipple retention”. The KER is based on a literature review conducted in a transparent semi-systematic manner in peer-reviewed databases with pre-defined inclusion criteria. Twenty-seven papers were included for development of the KER. The results support a qualitative relationship between the two key events (KEs) with a high weight of evidence; i.e., a causal relationship between androgen receptor (AR) antagonism and nipple retention in male rats exists. Adverse Outcome Pathways (AOPs) aim to depict causal toxicological pathways relevant for risk assessment, starting from a molecular initiating event (MIE) and culminating in an adverse outcome (AO) in an intact organism, or population. By providing mechanistic knowledge and links to measurable key events (KEs) that are essential for progressing through the causal pathway, the AOP framework can provide risk assessors with valuable information from which they can infer causality by using data from alternative test methods/assays. In essence, the unit that allows for inference is the key event relationship (KER) that links individual KEs along the causal pathway.Since the development and peer-review of complete AOPs is very labor- and time-intensive, a more pragmatic approach to develop, review, and endorse AOPs using a more modular approach has been proposed . This apin vivo, it is desirable to describe in detail the molecular and cellular mechanisms driving the effect, both qualitatively and quantitatively, in order to strengthen the predictive power of non-animal test data for chemical safety assessments.Areola/nipple retention (NR) in male rat, or mouse, offspring is considered a biomarker for incomplete masculinization during fetal development. This is because male rats and mice normally do not display nipples, in contrast to female rats and mice that have 12 and 10 nipples, respectively . This seSeveral mechanisms may lead to NR through reduced androgen signaling. These include disrupted steroidogenesis, inhibition of 5-α-reductase which will prevent conversion of testosterone to the more potent androgen receptor (AR) ligand dihydrotestosterone (DHT), and direct AR antagonism. These events may in themselves be both molecular initiating events (MIEs) or KEs depending on the chemical interactions with biomolecules. Thus, the ultimate goal for risk assessment purposes is to construct an AOP network that includes different MIEs or KEs (and AOPs) that, when affected, can lead to a common AO, in this case NR. Such AOP networks will aid the assessment of single chemical compounds, but also chemical mixtures, thus potentially accounting for cumulative effects in male reproductive toxicity , or any https://aopwiki.org/). This is a non-adjacent KER enabling weight of evidence evaluation of studies that do not report on intermediate steps of the causal pathway; however, the overall aim is to eventually construct a robust AOP network for integrated assessment of anti-androgenic modalities leading to adverse outcomes. The modular descriptions will be openly available in AOP-wiki. The sections corresponding to entries in AOP-wiki are denoted with an asterisk (*) in the heading.In the following, we have described the relationship between decreased androgen signaling by AR antagonisms and NR following AOP development principles (Developer’s Handbook available at https://aopwiki.org/aops/344). The KER described in this report is part of an AOP that links AR antagonism with NR. The AOP identifier is 344 and it is available on AOP-wiki (In vivo studies were included if (i) the study was carried out in mice or rats, (ii) NR in males was investigated as an endpoint, (iii) AR antagonism was the suspected mechanism of action and (iv) anti-androgenic effects of single substance exposures  were investigated. In vitro studies were included if they contained mechanistic information on AR inhibition by chemical stressors.A semi-systematic literature search was conducted during March 2022 in the peer-reviewed databases PubMed and Web of Science, using the search terms “(Nipple) AND (retain* OR retention) AND (androgen)” as well as “(Androgen receptor OR AR) AND (active*) AND (nipple OR areolae) AND (retain* OR retention)”. These searches resulted in 138 papers in total . Upon reKER 2133 connects KE 26: AR antagonism with KE 1786: areola/nipple retention. This is a non-adjacent KER, as depicted in Level of Biological Organization: Molecular.Cell term: Eukaryotic cells.N-terminal domain, the DNA-binding domain, and the ligand-binding domain, with the latter being most evolutionary conserved. Apart from the essential role AR plays for male reproductive development and function  and dihydrotestosterone (DHT). T and the more biologically active DHT act by binding to the AR , with hufunction , the AR function .The main function of the AR is to activate gene transcription in cells. Canonical signaling occurs by ligands (androgens) binding to AR in the cytoplasm which results in translocation to the cell nucleus, receptor dimerization and binding to specific regulatory DNA sequences . The genin vitro by transient or stable transactivation assays to evaluate nuclear receptor activation. There is already a validated assay for AR (ant)agonism adopted by the OECD, Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals , fetal (high), development through to adulthood (high).-Cyproterone acetate: Using the AR-CALUX reporter assay in antagonism mode, cyproterone acetate showed an IC50 of 7.1 nM .-Epoxiconazole: Using transiently AR-transfected CHO cells, epoxiconazole showed a LOEC of 1.6 µM and an IC50 of 10 µM .-Flutamide: Using the AR-CALUX reporter assay in antagonism mode, flutamide showed an IC50 of 1.3 µM .-Flusilazole: Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.8 µM and an IC50 of 2.8 (±0.1) µM .-Prochloraz: Using transiently AR-transfected CHO cells, prochloraz showed a LOEC of 6.3 µM and an IC50 of 13 µM .-Propiconazole: Using transiently AR-transfected CHO cells, propiconazole showed a LOEC of 12.5 µM and an IC50 of 18 µM .-Tebuconazole: Using transiently AR-transfected CHO cells, tebuconazole showed a LOEC of 3.1 µM and an IC50 of 8.1 µM .-Triticonazole: Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.2 µM and an IC50 of 0.3 (±0.01) µM .-Vinclozolin: Using the AR-CALUX reporter assay in antagonism mode, vinclozolin showed an IC50of 1.0 µM .in vivo studies showing anti-androgenic effects  evident by e.g., short anogenital distance (AGD) in males  or 5 (mice) pairs of nipples along the bilateral milk lines. In contrast, male rats and mice do not have nipples. This is unlike e.g., humans where both sexes have 2 nipples .In laboratory rats, high levels of dihydrotestosterone (DHT) induce regression of the nipples in males . FemalesNipple retention (NR) is visually assessed, ideally on postnatal day (PND) 12/13 . HoweverNipples are detected as dark spots (or shadows) called areolae, which resemble precursors to a nipple rather than a fully developed nipple. The dark area may or may not display a nipple bud . AreolaeNR is occasionally observed in controls. Hence, accurate assessment of NR in controls is needed to detect substance-induced effects on masculine development . It is rReproducibility of NR results is challenged by the measure being a visual assessment prone to a degree of subjectivity. Thus, NR should be assessed and scored blinded to exposure groups and ideally be performed by the same person(s) within the same study.The applicability domain of NR is limited to male laboratory strains of rats and mice from birth to juvenile age.A statistically significant change in nipple retention should be evaluated similarly to an effect on AGD as both endpoints indicate an adverse effect of exposure and should be considered in setting a NOAEL”.NR is recognized by the OECD as a relevant measure for anti-androgenic effects and is mandatory in the test guidelines Extended One Generation Reproductive Toxicity Study, TG 443  and the An overview of chemical stressors causing nipple retention in male rats is provided in the main text and supplementary data S1,  of SchwaTaxonomic applicability: Rats and mice.Life stage applicability: Developmental.Sex applicability: Male.in vitro, resulting in decreased AR activation. Decreased AR activation can lead to incomplete reproductive development in males, which can be expressed in several ways. One endpoint affected is areola/nipple retention (NR), which in vivo studies have shown to be linked to suppressed AR activation. NR in rat and mouse toxicity studies is considered an adverse effect .Several chemicals can antagonize the androgen receptor (AR) in vitro induce NR in vivo. However, in the literature review conducted for this KER, no studies in mice were found to fulfill the inclusion criteria. The present KER is hence exclusively a description of the situation in rats, although it is believed that the link also exists in mice.The biological plausibility of a link between decreased AR activation and increased NR in male rats is high. The relationship is supported by numerous studies showing that several potent AR antagonists The AR is activated through binding of either testosterone or dihydrotestosterone (DHT), the latter having the highest affinity for the AR. Upon binding, the AR translocates to the target cell nucleus where it acts as a transcription factor .in vitro studies  which in rats is between gestational day (GD) 15 and 19 .n-butyl phthalate (reduces testosterone levels) also showed that GD 16–17 was the most sensitive period for increased NR in male rats  and rodent reproductivity toxicity studies assessing NR   antagonism” and “areola/nipple retention” in male rat offspring. These are non-adjacent KEs essentially linking a MIE with an AO. Based on a semi-systematic review of available literature, evidence for this qualitative relationship is strong. Methods for reliably quantifying how much the AR must be inhibited before significant NR manifest remains insufficient. Available 421/422) .This work was funded by grants from The Danish Veterinary and Food Administration (DVFA), Project “Feminix”, and The Nordic Working Group on Chemicals, Environment and Health (NKE) sub-group Nord UTTE (project 2020–027 and 2021–020).Emilie Bak Pedersen: Methodology, Investigation, Data curation, Writing – original draft, Writing – review & editing. Sofie Christiansen: Conceptualization, Supervision, Investigation, Data curation, Writing – review & editing, Funding acquisition. Terje Svingen: Methodology, Investigation, Data curation, Supervision, Writing – original draft, Writing – review & editing, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


In the last decade, research focused on rehabilitation robotics has progressed from proposing restricted or rigid solutions in a clinical setting to portable devices compliant with the user and also adapted to the their requirements, based on their disability and the rehabilitation training program. Novel techniques have inspired the evolution of rehabilitation devices from hard and bulky to soft, lightweight, and fully wearable. For example, biologically inspired actuators have relaxed the constraint of having to rely on rigid supports, as the skeletal system can be used to that end. Furthermore, the use of synergies havehas led to a reduction of in the number of actuators and improved their control. Moreover, the latest advances in modeling and simulation have allowed for assessing and compensating for fatigue, as well as simulating the use of assistive devices out of a clinical environment. All these research achievements have enabled a new generation of portable rehabilitation devices. In the present Frontiers Research Topic, novel techniques for the design, simulation, sensing, and control of rehabilitation devices are presented for rehabilitation devices such as powered exoskeletons, neuroprostheses, and equipment for moving the rehabilitation environment out of the clinical setting.in vivo. That is, robots that provide as-least-as-needed assistance to neuromuscular targets while promoting voluntary execution of functional movements. In this context, the robot must be transparent and intuitive to prevent an increase of cognitive loads during the rehabilitation process. This objective is not easy to accomplish, as there are many challenges to overcome and many approaches to tackle. Examples of that, among others, are include the simulation of the human-machine interaction, the design and control of the rehabilitation device, the validation in a clinical environment, or the adaptation of the neuromusculoskeletal system to the rehabilitation process. Based on them, we present the contributions to this Research Topic according to the aforementioned challenges, although they are usually found intertwined in this research field.A key challenge in rehabilitation robotics is to create devices that facilitate positive neuroplasticity in the intact moving human Febrer-Nafría et al. simulate crutch-orthosis-assisted walking to choose the optimal active orthosis controller parameters for a specific subject. Their findings improve the traditional trial-and-error approach to select the best maximum knee flexion angle. The simulation provides optimal values to achieve a more balanced assisted gait pattern, making the process fully personalized to the patients' needs and less time-consuming. Cuerva et al. show a predictive simulation study to improve control in power-assisted wheelchairs. Their work investigates the advantages and disadvantages of an impedance control strategy, which that is more natural and effective than other alternatives. They employed predictive simulations of locomotion with power-assisted wheelchairs in different scenarios by using a realistic physiological model of the user's musculoskeletal system and its interaction with the wheelchair. Their results confirm this control strategy as the most useful, but their simulations also found a waste of energy during the propulsion cycle.Simulation-based studies in rehabilitation robotics provide a time- and cost-effective approach to preview real world scenarios. In this sense, Laffranchi et al. present the TWIN, a modular exoskeleton for SCI subjects. In their work, users' needs drove an iterative process to improve the system's design and construction. Sensing and control approaches are also presented. Supervised tests in a clinical setting demonstrated a stable gait pattern for rehabilitation, improving cost effectiveness. Regarding the upper limbs, Secciani et al. propose an original mechatronic design of a hand exoskeleton for both home assistance and telerehabilitation. It uses a real-time intention detection algorithm, but can also perform exercises preset by therapists in remotely supervised or unsupervised rehabilitation sessions. Surface electromyography (sEMG) signals are used to detect the user's intention, leading to a customizable, compliant, and comfortable design. In the opposite direction, Rätz et al. establish their design of a robotic hand by a set of clinical, anatomical, and mechanical requirements established before the development of the device. This novel clinical-driven robotic hand rehabilitation device is capable of fine haptic rendering, offering an effortless setup that supports physiological full flexion/extension of the fingers while providing high mechanical transparency. Lastly, Shi et al. develop a cable-driven three-degree-of-freedom wrist rehabilitation exoskeleton actuated by the distributed active semi-active (DASA) system. The proposed design has a larger workspace than current wrist rehabilitation training robots, able to cover a broader range of the activities of daily living, with an improved cable-driven design able to increase the effective torque and reduce the parasitic force.The personalisedpersonalized design of rehabilitation devices has been a challenge for researchers worldwide. Design requirements are different depending on the body segment part to be treated. In addition, these design specifications may change depending on the neuromuscular impairment. For example, assisting a patient with spinal cord injury (SCI) is not the same as helping a patient who has suffered a stroke. In addition, the designed device must be focused on the user's needs. It also must consider the evolution of the rehabilitation process, which may imply adaptations in the structure of the device or its control. In this Research Topic, Dalla Gasperina et al. present a cooperative control framework that promotes compliant motion and implements a variety of high-level rehabilitation modalities, including six actuation modes: passive, corrective, weight counterbalance, resistive, transparent, and hypergravity. The purpose is to change the haptic behavior perceived by a human when interacting with the rehabilitation robot by tuning different impedance control parameters. That variety of physical human-robot interactions helps the user to accomplish the task while exploiting physiological muscular activation patterns. Moving on to the work presented by Copaci et al., a new classifier for sEMG signals is presented. The algorithm is based on a Bayesian Neural Network in parallel with an Artificial Neural Network, which the results of which are connected in series with a Layer Recurrent Network. By doing so, the accuracy of the hand gesture recognition based on sEMG signals is improved. The authors' main purpose is to prove that the device control algorithm fits the patient's features and needs. The authors demonstrate that the proposed classifier could achieve high accuracy in hand gesture recognition. Last in this section, Castro et al. present a new approach for a 3D- printed hand prosthesis commanded by a simple sEMG system aided by a fully embedded computer vision system. The results show high percentages of accuracy, sensitivity, and specificity for grasping objects from neutral and pronated palmar grasp, tripod pinch, key grasp, and index finger extension gesture. This study shows that using a vision system is a promising alternative to traditional methods, as the pattern of grasping and manipulating objects is better defined.Control strategies are quite significant to achieveimportant in achieving proper rehabilitation. In some cases, the rehabilitation routine imposes a pre-defined trajectory to be followed and, therefore, the control strategy must account for it. In others, the primary purpose is to regain neuroplasticity, and, thus, the patient's intention imposes control actions, and establishes how the system acts to achieve a proper trajectory. In this sense, Godfrey et al. present the first poly-articulated, electrically-actuated, and body-controlled artificial hand, called SoftHand Pro-Hybrid,; and compared its performance against the Hand Pro-Hybrid benchmark. Their results on a limited number of subjects to assess prototype's functionality, with and without limb loss, confirm the possibility of using hybrid solutions as a valid alternative to myoelectric control, especially in situations requiring high versatility of the device. Finally, the work by Sierra et al. present the assessment of the AGoRA Smart Walker in daily living scenarios with older adults with Parkinson's disease. This kind of device represents a valuable tool for assisting people with gait motor deficits. The actuators of the AGoRA Smart Walker adapt their assistance level to the subject's demands, improving the rehabilitation process. The authors also compared the performance of using their device by between two groups of older adults with different physical and cognitive characteristics (Parkinson's disease vs. other conditions).The assessment or validation of the designed prototype is a valuable step in developing rehabilitation devices before clinical testing. It allows the implementation of possible improvements before evaluating their actual effect on the target population. In this sense, Chen et al. present a prospective, multi-center, and cross-over trial to evaluate the AIi-robot's safety, walking efficiency, donning and doffing time cost, and user satisfaction. They conclude that subjects with paraplegia below T6 level could ambulate safely and efficiently with that device, although its use should be learned under the guidance of experienced medical personnel. Marín-Méndez et al. present data from a two-arm, single-blinded, randomized, and controlled clinical trial. The objective was to evaluate the efficiency of a therapeutic massage robot (ADAMO) in reducing non-specific low back pain. Their main finding is that the ADAMO robot is at least as efficient as a regular treatment in reducing low back pain. However, it may be more beneficial for specific patients, such as those with who are overweight. Lastly, Koyama et al. present a prospective study for the Wearable Power-Assist Locomotor (WPAL). This device has been updated seven times, from the first validated prototype in 2005 to the latest in 2020. This study includes updated results from previous reports from July 2007 to December 2020 for 1785 different subjects. These results confirm that the WPAL improves walking independence for a wide range of spinal cord injury levels, and that further refinement of the WPAL will enable its long-term use at home.A final step in assessing rehabilitation devices is their evaluation in a clinical setting. In this way, it is possible to evaluate the effects of the assistive device on a suitable target population. In this Research Topic, Koelewijn et al. expose an overview of research directions regarding interfaces with the peripheral and central nervous systems, and the requirements of interface- computing architectures. Their work guides the research on modular and adaptable interfaces that can assist as needed and process all data recorded in real-time while accounting for signal variations among subjects. Furthermore, biomechanical models and simulation techniques are pointed out to predict motion and interactions between the human and the rehabilitation device. This work summarizes the main challenges in designing and using neuroprosthetic devices.To close this Research Topic, The design of rehabilitation devices results from tight cooperation between engineers, industrial designers, physical therapists, physiatrists, and patients. Therefore, a lot of effort is required to achieve transparent and clinically efficient neuroprostheses and rehabilitation robots that assist neurological patients as needed. From the publications in this Research Topic, we foresee that biomechanical modeling and simulation will be increasingly used to optimize the design of such devices and study human-device interaction, as it allows virtual testing in different scenarios. Moreover, the design of efficient control strategies, and the development of data acquisition and processing techniques will lead to adequate and timed actuation to improve rehabilitation routines. The reduction in the dimensions and energy requirements of the actuation units will also lead to portable devices and increase the rehabilitation process at home. Lastly, the information gathered from biosignals or the possibility to interact with the neuromusculoskeletal system, such as in functional electrical stimulation or spinal cord stimulation, will make possible a new generation of rehabilitation devices able to overcome the current challenges faced in the rehabilitation of subjects with motor disabilities.FR-S wrote the first draft of the manuscript. All authors have made substantial and iterative contribution to the work and approved its final version for publication.The authors, in order of appearance, wish to thank the following support given by: the Ministry of Science and Innovation—Spanish Agency of Research (MCIN/AEI/10.13039/501100011033) through project PID2019-107491RB-I00 (FR-S), the National Council for Scientific and Technological Development (CNPq) though the Productivity Grant No. 311715/2021-4 (LL), the Ministry of Science and Innovation—Spanish Agency of Research (MCIN/AEI/10.13039/501100011033) and ERDF A way of making Europe through project RTI2018-097290-B-C33 (JF-L), and the European Union's Horizon 2020 Research and Innovation Programme as part of the European Research Council (ERC) Starting Grant INTERACT (803035) (MS).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cassiopea , are found in shallow coastal habitats in tropical and subtropical regions circumglobally. These animals have previously been demonstrated to produce flow both in the water column as a feeding current, and in the interstitial porewater, where they liberate porewater at rates averaging 2.46 mL h−1. Since porewater in Cassiopea habitat can be nutrient-rich, this is a potential source of nutrient enrichment in these ecosystems. This study experimentally determines that porewater release by Cassiopea sp. jellyfish is due to suction pumping, and not the Bernoulli effect. This suggests porewater release is directly coupled to bell pulsation rate, and unlike vertical jet flux, should be unaffected by population density. In addition, we show that bell pulsation rate is positively correlated with temperature, and negatively correlated with animal size. As such, we would predict an increase in the release of nutrient-rich porewater during the warm summer months. Furthermore, we show that, at our field site in Lido Key, Florida, at the northernmost limit of Cassiopea range, population densities decline during the winter, increasing seasonal differences in porewater release.Upside-down jellyfish, genus  Cassiopea sp.  uses regular pulsation of their bells, Cassiopea sp. to draw water towards themselves horizontally along the substrate surface, pulling it into the bell below the oral arms1. As the water containing prey flows radially inward, it is redirected upwards through the oral arms, which are covered by nematocyst-laden digitata which capture prey items2. The filtered water is then ejected in the form of a vertical jet at velocities averaging between 1 and 2 cm s−13, away from both the animal and the feeding zone  to 0.04 cm day−1 in mangrove habitat7.In addition to transporting water through their feeding currents, Cassiopea bell pulsing behavior. In the first hypothesis  were performed using linear regression. In addition, for 6 animals, after recording the pressure, a 6 mm circular perforation was made through the bell Fig. C, taking3, we measured the rate of porewater release over four treatments: (1) A control in which an animal was allowed to settle on the sand for the duration of the experiment; (2) A plastic lining covering the entire sand surface except for a hole of the same diameter as the animal, in which the animal was placed  methods from3, such that both the incurrent and excurrent flow of the feeding current could be observed. The imaging setup consisted of a 45 × 45 × 45 cm aquarium, filled with artificial seawater. The water was seeded using 10 μm reflective hollow glass spheres for particle image velocimetry (PIV). An Edgertronic high-speed camera filming at 50 frames per second provided a field of view ca. 30 × 30 cm. Two 2-W continuous wave DPSS lasers (wavelength = 520 nm), spread through cylindrical lenses to produce narrow light sheets, were staggered one above the other to illuminate a single coronal plane across the entire field of view. One jellyfish at a time (n = 9) was placed on the bottom in the center of the aquarium, such that the laser sheet crossed the center of the animal. After allowing it to settle for about 10 min, 30 s of video were recorded at 50 frames per second. Analysis using the LaVision software package produced a PIV time-average over the 30 s. PIV was processed with interrogation windows between 48 and 64 pixels, at 50% overlap.The flow around healthy Bell pulse rates were aggregated from a large number of experiments and field observations dated between 2016 and 2021, for which temperature, salinity, and bell diameter were available. These data represent a total of 330 pulse rate observations. We tested for correlation between bell pulse rate, temperature, salinity, and bell diameter, using multiple linear regression, where diameter was linearized through a semilog transformation.Cassiopea sp. over the winter of 2019–2020. We surveyed a grid of 20 quadrats of 0.25 m2 at Lido Key, Florida (27°18′11.8"N 82°33′56.6"W), and recorded jellyfish population densities and bell diameters. Quadrats were placed in 5 m increments along rows every 10 m perpendicular to a 60 m transect line. In addition, to track temperature fluctuations between field excursions, we incorporated daily minimum air temperatures reported by the Sarasota-Bradenton International Airport, 10 km North of the field site.In order to validate theoretical temperature minima we measured population densities of 2 = 0.002) or surface area .Pressure fluctuations were expectedly minimal in the absence of an animal Fig. A and visBell perforation was followed by a short period of increased bell pulsation rates and mucus production, but after a brief recovery period there appeared to be no effect on the behavior of the animals. A decrease in the mean magnitude of pressure fluctuations from 37.0 Pa (± 15.6 s.d.) to 28.3 Pa (± 7.2 s.d.) was observed within individual jellyfish following bell perforation , while this average was reduced to − 0.11 mL h−1 (± 1.024 s.d.) in the absence of Cassiopea sp. indicating negligible rates of fluorescein absorption, rather than release, over time. Between all treatments, porewater release varied significantly . Post-hoc pairwise comparisons  showed that control and ring treatments were not significantly different from each other, and that disk and perforation treatments did not release fluorescein at rates significantly different from trials without jellyfish.Porewater liberation rate, as measured through fluorescein concentrations Fig. D in the −1 and continued to rise to a peak velocity of 5.5 mm s−1 at a distance of 12 mm from the bell margin. After this point, the flow was diverted upward, reducing horizontal velocity.Particle Image Velocimetry (PIV) demonstrates that the incurrent flow is of low velocity and restricted to a narrow region near the bell less than 5 cm from the bottom Fig. . Horizon2 = 0.57). While all independent variables were statistically significant to the model (p < 0.05), bell diameter (R2 = 0.34) and water temperature (R2 = 0.42) had a much larger effect on BPR than salinity (R2 = 0.07) (Fig. Bell pulse rates fit a multiple linear regression with the equation:Cassiopea sp. produces several hydrodynamic effects capable of altering the ecosystem which it inhabits. Not only do Cassiopea produce feeding currents capable of turning over the water column above them several times per hour3, they are also capable of releasing interstitial porewater from the benthos5. The rate of porewater release, on the order of mL h−13, is capable of increasing water column NH4 levels by almost 30% under certain conditions3. In this study, we investigated two hypothetical mechanisms for this porewater release, and found that a combination of the morphology of the bell and the pulsing behavior of the jellyfish was responsible for releasing porewater from directly below the bell via a suction-pumping mechanism.The upside-down jellyfish, 4, a low-pressure zone surrounding the animal due to a velocity gradient between the substrate boundary and the incurrent flow of the Cassiopea sp. feeding current, predicted porewater release from the substrate surface surrounding the perimeter of the animal. While porewater is entrained from the perimeter of the bell into the feeding current4 lateral expulsion of porewater due to the suction pump mechanism would produce a visually similar flow of porewater. A horizontal flow of water does occur near the bottom1, but this flow is restricted to a narrow region near the bell and velocities were low compared to the vertical excurrent jet (Fig. .3, which were adapted from those of Jantzen et al.5 (Fig. −1 ± 1.27 s.d., Fig. The Bernoulli hypothesisjet Fig. A using ll.5 Fig. . If the 5, a mechanism using the exumbrellar cavity as a suction pump that draws porewater vertically upward beneath the bell and then expels it laterally, would expect to see the majority of porewater released from directly under the bell of Cassiopea sp. This mechanism is supported by bell morphology5 and the appearance of deep porewater at the benthic surface of the exumbrellar cavity5. In our, an impermeable disk was placed underneath the animal to obstruct the flow predicted by the suction pump hypothesis (Fig. The suction pumping hypothesissis Fig. B. Additisis Fig. C. Both tsis Fig. D, suppor3. Note that the muscles responsible for bell contraction in Cassiopea sp. are roughly 2-dimensional sheets13 with a thickness of one cell14 and therefore the cross-sectional area also does not scale with diameter. Our experiments were performed on smooth acrylic rather than sand, so that the conditions here were optimal for the formation of a tight seal with the bottom. However, the magnitude of this difference is likely to be small, as Cassiopea sp. produce copious amounts of mucus, which can compensate for small-scale surface roughness. In addition, the duration of each individual bell pulse is short1, so given the fine pore size of a sand or mud substrate, it is unlikely that subambient pressure would have the opportunity to dissipate enough to affect the high suction impulse produced.Since the suction pumping mechanism requires pressure fluctuations in the exumbrellar space, we also directly measured the water pressure below the jellyfish. The initiation of the power stroke of bell pulsation coincides with a sudden decrease in water pressure in the exumbrellar space Fig. A,B of a Cassiopea to produce copious amounts of mucus, which could have acted to minimize the impact of bell perforation. These parallel lines of reasoning firmly suggest that suction-pumping is, in fact, the dominant mechanism by which Cassiopea sp. release porewater.While not statistically significant, bell perforation did lead to data suggesting a decrease in exumbrellar pressure fluctuations Fig. C, which Cassiopea can occur at high densities of up to 100 animals m−23. Furthermore, while the Bernoulli mechanism predicted that interstitial water movement would be limited to the upper layers of the benthos, the suction pump mechanism has the potential to release porewater from deeper sediment strata. This deep flushing should expand the oxygen penetration depth downward, affecting factors such as respiration and sediment stability15. Given the fact that Cassiopea are capable of moving along the substrate16 this also means that the oxygen penetration depth is likely to fluctuate over time, favoring organisms that are able to adapt their metabolism or are able to relocate themselves17.The suction-pumping mechanism for the release of porewater has broad-ranging ecological implications. Release rates should increase additively with population density, and the rate of bell pulsation should correlate with the rate of porewater liberation. The additive relationship to population density is important, since 11, any benthic-pelagic coupling mechanisms in this habitat could alter nitrogen dynamics, especially given the fact that many marine primary producers preferentially take up ammonium, the most reduced state of nitrogen available, as a nitrogen source18. Cassiopea sp. animal size and population densities are known to correlate with anthropogenic disturbances, and it is suggested that this is due to an increase in nutrient availability in these areas6. In addition to prey capture, Cassiopea sp. could be supplementing their nitrogen demand through the release of nutrient-rich interstitial porewater, from which Cassiopea sp. can directly absorb ammonium and other nutrients such as phosphate and trace metals5. In fact, jellyfish presence significantly reduced porewater ammonium levels near the animal5, suggesting that nutrient-rich porewater was replaced by down-welling low-nutrient surface water. The observed benthic locomotion of Cassiopea16 may be a mechanism to avoid remaining in locations where they have depleted this nutrient resource3. It has been reported that Cassiopea sp. affect benthic nutrient transport on a more general level, increasing ammonium uptake and decreasing nitrate uptake of the bottom sediments19. Water column nutrient levels also varied significantly between presence and absence of Cassiopea sp., and also between light and dark treatments in the presence of Cassiopea sp.20. The addition of jellyfish increased the efflux of ammonium from the benthos during the dark treatments, but reduced ammonium concentrations in the water column during light treatments20. It is entirely possible that absorption of nutrients by Cassiopea sp.5 in order to meet daytime metabolic demand resulted in the animals reducing water column ammonium concentrations in these experiments20.Given that porewater at the field site in Long Key, Florida, from which the animals in this study were collected, has mean ammonium concentrations of 72 μM, 160 times higher than the surrounding water columnCassiopea sp. have been shown to increase spatial heterogeneity of interstitial oxygen and nutrient fluxes20, making it comparable to other biogenic processes like bioturbation. Bioturbation typically oxygenates the upper layers of substrate, increasing the nitrification zone21, and also increases 3-dimensional heterogeneity of oxygen and nutrient concentrations, allowing for more complex nutrient dynamics21. The transport of interstitial porewater from specific regions under individual jellyfish could well produce a similar effect. The porewater release rates can also be compared to that of abiotic processes, such as wind-wave driven flow over sediment wave ripples, which have been shown to liberate porewater at rates of up to 140 L m−2 day−1, or three orders of magnitude greater than diffusion alone, on shallow, exposed coastlines such as beaches22. Environmental mixing would be lower in the sheltered mangrove habitats where Cassiopea sp. are found, since at our study site wind wave height was reduced from 5.4 cm in the bay to 0.07 cm in the mangroves3. In these coastal habitats, the sediment often acts as a nutrient sink, causing certain nutrients to become limiting to primary producers. Some fringe mangrove forests along coastlines in both Florida and Belize have been shown to be N-limited, for example24. If these nutrients are then released back into the water column, they potentially increase primary productivity in the system occupied by Cassiopea sp. Depending on the system, this could either increase production or cause eutrophication, potentially altering productivity on a local or regional scale, as has been observed when nutrients are released from the benthos by winds25 or bioturbation26.In addition, Cassiopea sp. can release greater quantities of nutrient-rich porewater during the summer months. This was confirmed by a recent study on the related species, Cassiopea medusa from Lake Macquarie, Australia8. By extension, our model suggests that pulsing, and therefore porewater release, should cease entirely below 18ºC. In fact, at our site in Lido Key, population densities of Cassiopea sp. declined rapidly once water temperatures dropped this low (Fig. Cassiopea sp. polyp feeding was inhibited10. As such, it is likely that winter minimum temperatures of 18ºC represent a limiting condition on Cassiopea sp. range expansion. Studies on Cassiopea medusa, suggested thermal stress and bell degradation at 16 °C8. As global climates warm, we can expect both a poleward shift of Cassiopea sp. Range27 and an increase in transport rates of porewater and its associated benthic nutrients throughout this range, leading to increased productivity, and potentially exacerbating eutrophication in some regions.The mechanics of suction-pumping also imply that interstitial porewater release rate will correlate with bell pulse rate. Pulse rate correlates with water temperature Fig. B, which low Fig. . This saCassiopea, suggesting that release rates are independent of population density, but affected by pulse rate. The potential role of bell pulse rate was investigated further, and we found correlations between bell pulse rate and both animal size and water temperature. As a result, we expect that porewater liberation would demonstrate seasonal variations, with lower rates during the winter and reaching a maximum during the summer months. Cassiopea are able to release nutrient-rich porewater in the shallow quiescent habitats they inhabit, and through their feeding current mix these nutrients throughout the water column. Since this effect varies seasonally, it is likely that further study will show that these jellyfish are responsible for a complex system of nutrient dynamics in their ecosystem.We determined that a suction-pumping mechanism is responsible for the interstitial porewater release by


Background: Allergic rhinitis (AR) is a chronic respiratory disease. Hereditary factors played a key role in the pathogenesis of the AR. This study investigated the association between CLEC16A variants and AR risk in the Chinese population.Methods: We applied Agena MassARRAY technology platform to genotype five single nucleotide polymorphisms (SNPs) located in CLEC16A in 1004 controls and 995 cases. The association between CLEC16A SNPs  and AR risk were calculated by logistic regression analysis, with odds ratio (OR) and 95% confidence interval (CI). False-positive report probability (FPRP) was also used to assess the significant results to reduce false positives. Multifactor dimensionality reduction (MDR) was completed to assess the interaction between CLEC16A variants to predict AR risk.Results: Totally, CLEC16A  were significantly associated with AR risk. Therein, rs2286973, rs11645657 and rs36045143 were related to a decreased risk of AR in the people ≤ 43 years old, females and the people with BMI≤24, respectively. And rs887864 and rs12935657 were also associated with a decreased susceptibility of AR in the people >43 years old. Meanwhile, in the results of region stratification, rs887864 conferred a reduced risk to AR in the people from loess hilly area.Conclusion:CLEC16A variants conferred a decreased risk to AR in the Chinese population. Allergic rhinitis (AR) is a chronic respiratory disease characterized by allergen sensitization mainly mediated by immunoglobulin E (IgE). It is characterized by runny nose, sneezing, stuffy nose, pruritus and other triggers. Several factors have been implicated in the etiology of AR, including pollen, dust mites, mold, animal dander and other factors . The disCLEC16A (C-Type Lectin Domain Family 16 Member A) was formerly known as KIAA0350. It is located in the chromosome 16p13.13. This gene encodes a family member containing a C-type lectin domain. The protein CLEC16A was shown to be a cytosolic protein that binds to Vps16A and regulates the receptor expression through autophagy. In a CLEC16A knockout mice through Cre/loxP system, they found up-regulation of cytokine and chemokine secretion, imbalance of dendritic cell subsets and change of receptor expression in mice  were reported to be an important genetic factor in abnormal autoimmune response. In one previous study, tes gene . Also, H based on the 1000 genome project. Based on the NCBI website, we found that rs887864, rs12935657, rs11645657 and rs36045143 were located in the intron region of CLEC16A, while rs2286973 was located in the last exon region of CLEC16A. When rs2286973 G mutates into rs2286973 A, the encoded amino acid has not changed. Subsequently, we assessed the relationship between CLEC16A variants  with AR risk in the Han population from Shaanxi, China.In the present study, we selected five variants of The study was approved by the Ethics Committee of Shenmu Hospital and the authorization number was sm004. In this study, all the involved subjects were the Chinese Han population. Totally, 1004 controls  and 995 patients with AR  were consecutively recruited from 5 towns  in Shenmu city. The selected criteria for patients with AR were as follows. ①Clinical symptoms: the patient has two or more symptoms such as sneezing, runny nose, itchy nose, blocked nose and so on. ②Nasal symptoms: pale and swollen bilateral nasal mucosa, inferior turbinate edema, and watery nasal discharge. ③Allergen testing: Blood was drawn to examine specific IgE, and the diagnosis of AR was confirmed if the patient had high specific IgE in serum. The exclusion criteria for AR patients were: no history of asthma, no comprehensive disease such as lung cancer, and no infectious disease such as tuberculosis. Also, based on the routine physical examination, the controls had no history of respiratory diseases, including sinusitis, rhinitis and other inflammatory disease. By reviewing the participant’s hospital records, we collected the information of participants, such as age, sex, height, weight, place of origin, residence, occupation, past medical history, family medical history, etc. In addition, we also collected the blood routine indicators obtained by the blood analyzer, such as red blood cell distribution width_SD (RDW_SD), basophil count (BASO), eosinophil count (EO), hemoglobin (HGB), mean hemoglobin concentration (MCHC), neutrophil ratio (NEUT_per), lymphocyte ratio (LYMPH_per), eosinophil ratio (EO_per), basophil ratio (BASO_per) and red blood cell distribution width_CV (RDW_CV). Biochemical indicators  and renal function indicators  were measured by drawing venous blood. Written informed consent was collected from all subjects. All procedures comply with the regulations of the Department of Health and Human Services (DHHS) on the protection of human research objects.http://www.internationalgenome.org/), CLEC16A variants  with minor allele frequencies> 5% and Hardy-Weinberg equilibrium (HWE) > 0.01 were chosen in the global population. According to the specificity principle of primers, these sites meet the requirements. The SNP-related primers (amplification and extension primers) were finished and listed in CLEC16A variants were proceeded in a 384-well plate. And data management was performed by Agena Bioscience TYPER software, Version 4.0 . Generally, OR and 95% CI were calculated by logistic regression to analyze the association under four genetic models . To further eliminate false positive in the data, we used FPRP to analyze the significant results. Multifactor dimensionality reduction (MDR) was completed to assess the interaction between CLEC16A variants to predict AR risk. P less than 0.05 was considered as significative.IBM SPSS statistical software version 24 and Microsoft Excel were used to analyze the socio demographic descriptive data of the case group and the control group. The continuous variables were expressed as mean ± standard error (SE) and compared by Student’s t-test. Age and sex distribution differences of AR patients and controls were assessed by p = 0.002), but there was no difference in sex and BMI between the two groups . In addition, there was no significant difference between the two groups in the distribution of wind beach area and loess hilly area (p = 0.738). Noteworthy, 1004 controls and 995 patients with AR were collected in this study. The mean age of the 1004 controls  and 995 patients  were 43.77 ± 0.26 years old and 42.81 ± 0.33 years old, respectively . There wCLEC16A variants  were listed, including SNP-ID, gene, chromosome, position, localization, allele, minor allele frequency, major allele frequency and HWE p-value. The genotype distribution of all loci in the control group met Hardy-Weinberg equilibrium. We listed the function of the selected SNPs predicated by Regulome DB scores and HaploReg. And the primers of CLEC16A variants were shown in In p = 0.023). As for rs11645657, controls with the three genotypes  had different clinical parameters-HGB and UA , whereas, cases with A/A, G/A, G/G genotype had significantly different LYMPH_per (p = 0.035).Also, we conducted clinical parameter analysis of different genotype carriers . The resCLEC16A variants  with AR risk (p = 0.040), while rs12935657-G had no significant association with AR risk . In the results of genetic models  analysis, rs887864 was significantly associated with AR risk . The above results illustrated that allele-A and genotype-AA may play a protective role in the pathogenesis of AR. And, a significant correlation between rs12935657 and AR susceptivity was observed in the codominant  and recessive  models, suggesting that genotype-GG may play a protective role in the pathogenesis of AR. We also did the FPRP analysis for the significant associations of CLEC16A variants with AR risk . Rs887864 and rs12935657 were also associated with a decreased susceptibility of AR in the people >43 years old  and the people ≤43 years old  adjusted by age, sex and BMI.We further analyzed the relationship between  and BMI . In Tablp = 0.027; recessive-G/G genotype: p = 0.040; log-additive: p = 0.041; rs887864: allele-A: p = 0.023; codominant-A/A genotype: p = 0.044; dominant-G/A-A/A genotype: p = 0.040; log-additive: p = 0.018; rs11645657: allele-C: p = 0.038; codominant-C/C genotype: p = 0.044; log-additive: p = 0.025), but these loci had no significant association with AR risk in males. The BMI stratification demonstrated that rs2286973, rs11645657 and rs36045143 were correlated with AR risk in the people with BMI≤24 . Whereas, these SNPs were not found to be associated with AR risk in the people with BMI>24.In CLEC16A variants and AR risk stratified by region (wind beach area and loess hilly area) in p = 0.015) and recessive (A/A genotype: p = 0.016) models. Yet, the site was not found to be associated with the risk of AR in the people from wind beach area.Meanwhile, we evaluated the association between CLEC16A variants by MDR method (p < 0.000). In the To predict the AR risk, we did the SNP-SNP interaction among R method . In the CLEC16A is found in the 16p13 area next to CIITA gene. It was reported that CLEC16A lacks a functional Ag recognition domain, and Schuster et al. observed that it can induce autoimmune responses in mice, possibly by stimulating Ag expression in thymic epithelial cells  and AR risk. It showed the importance of CLEC16A polymorphisms in AR. The analysis of the interaction between rs2286973, rs887864, rs12935657, rs11645657 and rs36045143 is helpful to discover the risk factors of AR. Our results showed that the best model was composed of rs2286973 G/A, rs887864 G/A, rs12935657 G/A, rs11645657 C/G and rs36045143 A/G for AR.al cells . More imal cells , primaryal cells , alopecial cells . Also, C T cells . The stu CLEC16A . Among tpulation . In our The incidence of AR increased with age . It has AR was a sex-specific disease . In one 2 . In the people from loess hilly area, rs887864 conferred a reduced risk to AR. Yet, the site was not found to be associated with the risk of AR in the people from wind beach area. A large number of samples were required for subsequent verification.Regionality was also a factor affecting AR. In our previous research , the prehttp://www.regulomedb.org/) and HaploReg predict the function of SNPs  were not included and should be considered for future assessment. Thirdly, we used Regulome DB  conferred a decreased risk to AR in the Chinese population.In a short,