The efforts to develop research and training on physical rehabilitation in regards to disasters is considered recent worldwide. In the late evening of the 11th up until the 12th of January of 2011, the most massive natural disaster occurred in Brazil with extremely heavy downpour, abrupt flood, as well as landslides on multiple areas of the Mountain Region of Rio de Janeiro. The objective of this research was to investigate the challenges in terms of physical rehabilitation provided by this eventThe cross-sectional mixed method’s study, which was conducted in the city of Nova Friburgo, used two different data sources: hospital records on traumatic injuries pre and post disaster, and interviews with key informants - victims who suffered injuries related to the disaster, professionals from rehabilitation services in the municipality, and also the city’s health service management. Pearson’s chi-squared test was performed to evaluate statistical significance between the week of a given incident and the type of injury. Interviews were transcribed and analysed through content analysis.p <  0.0001). Different injuries were identified: multiple fractures, crushing, amputation, perforation of soft tissues, inhalation of dust and establishment of chronic conditions through stress. Despite this scenario, out of the 16 health professionals interviewed, twelve did not observe an increase in the demand for outpatient rehabilitation services after the disaster. Interviews with the victims revealed that the pathways for care ran into different barriers. From 11 victims interviewed, only one received complete physiotherapy care through the public health services in the city, while all others hired additional assistance, received volunteer services, had assistance in other cities or remained without rehabilitation.A total of 2326 hospital records and 27 interviews were analysed. The proportion of traumatic injury in the municipal emergency service increased from 16% in the prior week, to 40% in the week post-disaster (The needs for rehabilitation increased after the disaster; however, the demand was repressed due to different barriers such as competing needs and possible lack of medical referral. Recommendations were made, including the action of performing a search of victims with rehabilitation needs. Rehabilitation Disaster Relief Symposium, which occurred in 2011. At that time, it was evidenced that the response plans and protocols for acute care in disasters usually did not include interventions of rehabilitation, resulting in negligence of immediate rehabilitation needs and the establishment of medical complications and permanent disabilities which could certainly be avoided (...) I was under a lot of pressure, the physiotherapist that took care of me said that my prosthesis was the best one and that I had to try hard. What made it difficult was my financial situation, pressure from my physiotherapist and my evaluations, which were bimonthly”.“Concerning the victim that continued the treatment in the rehabilitation centre, despite going to a reference centre and having acquired a state-of-the-art prosthesis, according to the patient, the 104 km distance from her home to the rehabilitation centre, as well as the physiotherapist’s demands for her to adapt to the prosthesis, demotivated her attendance to the assistance and recovery process. This patient was retired on disability, is partially dependent on instrumental daily activities and presented depressive and anxiety symptoms related to the disaster trauma which has damaged her well-being., when I started walking, I couldn’t even bend my leg. After therapy. I was satisfied because I had to get my son’s life back on track, buying everything for him again, giving him more attention, because he had lost his father, right? So I left physiotherapy as fast as I could because I only wanted to walk, right? If I could bend the leg, for me that was great!”“SoOn the other hand, the victim who received long-term volunteer assistance at home had fewer technological resources but did in fact receive regular service, incentive up to a certain point and respect for her needs, not only physical but also social, related to child care. The patient regained her stride, was able to go back to her job and became independent for the instrumental daily activities with few adaptations.Only one victim had a private healthcare plan at the time of the disaster and received complete physiotherapy treatment through the plan. Three victims hired healthcare plans after the disaster. One of them affirms having hired it to obtain adequate treatment for chronic back pain, and was undergoing treatment at the time of the interview. The other relates having given up on physiotherapy through the healthcare private plan because the expected results were not achieved. Only one victim paid directly for physiotherapy, the same victim who recovered completely.Only one victim affirmed not searching for rehabilitation physiotherapy services. She had her respiratory system affected the most and was unaware of the existence of pulmonary rehabilitation, as she was not referred by the health services towards this treatment option.For the outline of rehabilitation services supply after the disaster, descriptions of services rendered were brought from health professionals responsible for such services in the public sphere , a sample of health professionals from the PHS (six physiotherapists that provided long-term volunteer assistance after the disaster), and a manager of the public rehabilitation service in the municipality of Nova Friburgo. The perspective concerning demand was composed of explanations brought by health professionals and the manager, as well as the victims themselves.“I thought, there is going to be a huge increase after the tragedy, and there wasn’t anything like that.” .“Here in the health centre there was no increase, in other places I don’t know, I don’t have access to those.” .“Nothing came up that would show an increase like this, that would draw so much attention from the professional physiotherapist that could make it reach the management as a demand.” (manager of public rehabilitation services).Out of the 16 interviewed professionals, twelve did not observe an increase in the demand for rehabilitation services after the disaster. The increase was also not perceived by municipality interviewee management of rehabilitation services.Of the professionals that did not identify an increase of demand for rehabilitation services, four answered on behalf of private institutions, two for hospitals that do not offer outpatient rehabilitation services, two for non-profit organizations that do not serve the general public, only children with developmental delay and four by municipal services.What was most evident was the reduction of ongoing treatments, because of either the decrease in demand or situations that impaired the supply. Patients of PHS claimed difficulties of transportation to get to the clinic, financial losses, need to support someone in the family, or other priorities arising, such as the recovery of documents, belongings, and reorganizing their personal life. The three non-profit organizations were in recess due to vacation period when the disaster struck, as they work as a school and this being the reason for the vacation period. Two of these had their structures affected and the renovations took months to conclude.Besides, the emotional shock and difficulty to transit through the city worsened the access and treatment continuity after their reopening. Residents of the rural area had even more trouble to commute. This situation took about 6 months to normalize itself. Professionals from two hospitals reported that the structure of these services was halted totally or partially, and because of that, they needed to transfer patients for care to other health clinics. In one of these services, the outpatient physiotherapy service space was permanently closed in 2007 after another flood destroyed the facilities.“Here we usually treat the neurological patients. After the disaster, we started having a lot of the orthopaedics part too. Something that did not happen before the disaster (...) We stopped working at the time because we were affected a bit here. Then we went somewhere else, to a court. It was very hard to work in my sector. We had to adapt gurneys, the location, devices that we did not have, and this disrupted us a lot at the time.” .“The demand increased a lot due to the people that suffered fractures, sprains and were directly traumatized by the tragedy. In quality, the structure continued the same. Actually, it got worse. Because when the tragedy occurred at daybreak, people who wanted to rescue... the places to rescue were nearby... and people forced the doors to steal – no, not to steal – to get the gurneys. So I had no gurneys to work.” .Only three professionals observed an increase of demand for rehabilitation services in direct consequence to the disaster. This happened in a non-profit association that assists the public and children with developmental delay, in a PHS and in only one health clinic, located in a district that was particularly affected by the disaster. In two of these cases, the increase of demand faced the structural damages which occurred due to the disaster:The cleaning and tidying up of everything that was left also caused a lot of problems for many people, physical problems, injuries to the tendons...” .“Then, concerning the rehabilitation process... This disaster created a lot of psychological stress, right? Most have developed some trauma, it starts to rain and you see people panic. So what did the psychological change that occurred in the population generate? Back pain, headache, so... they somatised it. So the demand increased... Some patients showed up with fibromyalgia... all diseases of emotional background.” .In two PHS, the increase in the demand for rehabilitation by indirect consequences of the disaster was observed, as well as injuries caused by the cleaning and reorganisation of physical spaces, or even by mental stress:“He [the doctor] said that when things calmed down, I should look for an orthopaedist. However, I couldn’t leave my grandson because he couldn’t stay with his dad, so I ended up doing everything (...) What they would do to me was going to interfere with this. I had to look after my grandson, I had to take care of my daughter’s life, she was hospitalized in Rio.”When the increase in the demand was observed, it did not happen immediately after the disaster, but about two to 3 months later. The interviews with victims confirmed and explained this delay between the disaster and search for outpatient physiotherapy services. Before searching for these services, patients had to go through hospitalizations, surgeries and immobilization periods. Additionally, the emotional shock, other urgent needs and, in some cases, the need to aid some other family member even more injured. The speech below illustrates well the situation of the victim that suffered an injury on the upper limbs, needed outpatient rehabilitation services, but postponed this search to take care of his family due to the death of his son-in-law, the daughter gravely injured and a young grandson:“We always worked with agreements, with clinics that weren’t rehabilitation clinics, but offered some service to this area, such as physiotherapy. Anyway, something also with the agreement (...) for some situations, but actually the service was never organized, never had a municipal rehabilitation centre.”The interview with management reveals that the city never had an investment in the rehabilitation sector. The city hall has only some agreements with physiotherapy clinics.It is possible to affirm that municipality management recognised that the municipality’s rehabilitation structure was precarious.“...we ignored this possibility with the issue of rehabilitation in relation to the disaster because we worried a lot more about the emotional trauma and diseases arising from it, such as diabetes, hypertension, than physical injuries. Somehow, we created an idea that the disaster killed, but did not leave sequelae. It was possibly a very erroneous evaluation of the health service.”After the disaster, the rehabilitation needs did not come as a problem to the management, which acknowledges not having performed an active search because problems of immediate response, disease control, and psychosocial demands were prioritized.Knowing the fragility of the municipality’s physiotherapy public services and having noticed the increase in the need for physiotherapy services, six physiotherapists acting in the PHS offered long-term volunteer physiotherapy service. They communicated the offer of free assistance in their private clinics through personal referrals, at institutions (such as churches), or among medical colleagues. They were expecting a higher demand than what they effectively received. In this sense, there were individual, as well as collective initiatives. A group of about five physiotherapists, with clinics in different districts of the city organized themselves in a network to offer these services to victims of the disaster. According to the interview, a proposal was sent to city hall, but they received no response by the time of the field research. Interview with the management reveals that this proposal never reached its destination.This study showed that there was a proportional increase in traumatic injuries at the Nova Friburgo public hospital after the disaster as is expected in extreme weather events , resultiIt is worth observing that there was no increase in the gross number of service cases of traumatic injuries at the only municipal hospital in Nova Friburgo on the week after in relation to the week before the disaster. It is also important to mention that the absolute numbers of traumatic injury and other health problems had declined in the week following the disaster due to the interdiction on part of the hospital. However, by means of victim reports, it was possible to infer that the assistance occurred at other emergencies, field hospitals or even at other municipalities.This is a pioneer study about physical rehabilitation in situations of natural disasters in Brazil. Most international studies about rehabilitation after natural disasters were performed for earthquakes. In these cases, there is often the occurrence of spinal cord injuries, which was not observed in Nova Friburgo. If there was any case, it did not reach the municipality’s public rehabilitation for treatment or monitoring. Nonetheless, injuries such as crushing, long bones fractures, pelvic fractures, multiple fractures, chest injury, strains, and infectious wounds, normally related to earthquakes , 21, alsThe study of Pereira et al. (2013)  shows thOut of the 11 interviewed victims, three lost capacity to work due to the disaster. In none of these cases, the total loss of capacity to perform the instrumental activities of daily life was observed. Besides being all women, what the three cases have in common is that the loss of capacity for work is not related only to the loss of motor function, but mainly with low educational level, which complicated the aid of possible mechanisms of adaption to other activities. These examples reinforce the importance of understanding rehabilitation beyond the biomedical aspects to reach psychosocial, economic and behavioural dimensions. It is clear within this small sample that to these women, rehabilitation was an incomplete and interrupted process. As Ferdiana et al. (2014)  point ouTwo interviewed victims  reported the worsening of chronic pain after the disaster, but not because of direct injury. Instead, it was attributed to the mental suffering directly related to the disaster. Depression and anxiety symptoms commonly occur after the experience of a traumatic event and these are related to chronic pain in the musculoskeletal system , as YabuIn different contexts, chronic pains may entail situations of physical disability, even making the individual look for emergency medical assistance due to great pain . Many tiThe inconvenience of a symptom diluted in everyday health problems is that once it manifests, the extra resources offered to the municipality to respond to the needs that have arisen due to the disaster, many times are spent, The assistance to chronic pain and physical disabilities demanded a specific rehabilitation care system that did not exist previously in the studied municipality and was not established after the disaster.Out of the 11 interviewed victims, all of them needed rehabilitation services after the disaster. However, their paths to obtaining the treatment was varied. The rehabilitation service through SUS in the city assisted only three of these cases, and only one reported the treatment as successful. The two other cases were interrupted. On one of the cases, the interruption happened after 10 sessions without full recovery. On the other, the patient had given up due to the precariousness of the assistance. These findings reveal the situation of fragility that the public rehabilitation services in the municipality undergoes.The prescription of 10 physiotherapy sessions is a custom among physicians when referring the patient for rehabilitation, mostly when the physiotherapy sessions are paid by private healthcare plans or by SUS, which determine rules for the use of services. It is a matter of managing the distribution of physiotherapy services that does not articulate with the real needs of the patients. The number of physiotherapy sessions cannot be defined because it depends on a series of factors that interfere with the functional recovery, including location, nature, extension of the injury, the patient’s age, clinical condition, and daily routine, as well as the level of education , 27.The PHS was responsible for the treatment of seven of the eleven interviewed victims. Among them, only one abandoned the treatment  because improvement was not observed. Another was still undergoing treatment in the interview period. The other five interviewees assisted by other means, had success in relation to their functional recovery. Three of them were serviced by long-term volunteer assistance. In Brazil, the PHS should work to complement SUS (Law 8.080/90). In practice, however, the contrary may occur, as observed in this sample.Two victims with severe injuries were hospitalized in a national reference Traumatology Centre located in the municipality of Rio de Janeiro. After medical release, one of them continued the rehabilitation treatment at the same location by means of transportation offered by the city of Nova Friburgo. The other received physiotherapy assistance at home offered by a volunteer. These are two cases of severe injuries with the need for rehabilitation monitoring for a long period, and they offer an empirical basis to reflect on the rehabilitation centres versus Community-based Rehabilitation (CBR).To Almeida and Campos (2002)  the modeTechnology does not, necessarily, solve the rehabilitation problem. Well-equipped rehabilitation centres may contribute to functional recovery, but should not be seen as the only choice for rehabilitation assistance. If the treatment is not succeeding, it is necessary to identify and consider with the patient the barriers that are being imposed.It is possible to affirm that not all of those that needed rehabilitation services effectively searched for it. The increase in the proportion of people with traumatic injuries and the examples of interviewed victims indicated that an episode of repressed demand occurred since the disaster created different rehabilitation needs directly and indirectly. Gelberg et al. (2000)  uses theTo access the rehabilitation service, users of SUS as well as private plans, need a medical referral. In this sense, by proposing a model to study the access and use of health services, Dutton (1986)  distinguThe first limitation identified was regarding the quality of hospital records. Many of the hospital records, especially those of the week after the disaster, were incomplete. Thus, the only information we could work with was about the occurrence of traumatic injuries, and it was not possible to associate it with the place of residence or other variables. Some records were torn or wet, which makes us believe that the actual number of calls in the week following the disaster was slightly higher than the records showed.The second limitation was the fieldwork and interviews performed almost 5 years after the disaster. It may have a bias on the memory aspect. At the same time, one may consider that such a notable event would persist longer, being remembered by those that experienced it.Although the disaster has generated new cases of people with traumatic injuries and stress at the same time, leading to an increase in rehabilitation needs, not every need was translated into demand. Some barriers have been identified such as: competing needs ; distance from the rehabilitation centre, unawareness of the rehabilitation services, and possible lack of medical referral. At the same time, those who were treating chronic or less serious conditions were also captured by competing needs and the result was not a large increase in the demand for rehabilitation services. Therefore, it is highly recommended that the health service conduct an active search for people at risk of developing disabilities due to injuries caused by the disaster and avoid the underuse of the health services.The present study showed that the participation of the physical rehabilitation sector in the recovery stage of the disaster was minimal. Rehabilitation is a slow process and thus it traverses the daily issues. In disaster situations, it is also traversed by new needs that come about in life, from economic, social and also emotional areas. Health services could not stop in immediate response and disease control.The present study shows that the outpatient rehabilitation needs after a disaster do not generally appear immediately after it. There is an interval of at least 3 months, considering periods of post-surgery recovery and immobilization. Therefore, there is a period for outpatient services to reorganize the assistance and make vacancies available to victims with physical rehabilitation needs after the disaster. In some cases, it will be necessary to bend the schedule, prioritising cases that need more prompt diagnosis and therapeutics, without abandoning patients being monitored.


Musculoskeletal problem, such as traumatic injury, osteoporosis, and osteoarthritis, is one of the leading causes of disability worldwide and is therefore a crucial branch of public health. Biomechanics has been proven to play a critical role in musculoskeletal pathology, treatment, and rehabilitation. Rehabilitation describes specialized healthcare dedicated to improving, maintaining, or restoring physiological strength, cognition, and mobility with maximized results. It helps an individual achieve the highest level of function or greatest independence and quality of life possible after illness, injury, or surgery using physical training, instruments, devices, or other methods. Advances in rehabilitation, including the theory, paradigm, protocol, modality, invention, and engineering, can improve the development of modern medicine and also the quality of life especially for the disabled and elderly people. The main focus of this special issue is on innovative theory and application of biomechanics to understand musculoskeletal pathology and to improve the techniques for the treatment and rehabilitation.As the variety of topics presented in this special issue suggests, there are still many difficulties related to the mechanism of human injury and its rehabilitation and invention or development of the rehabilitation device. M. Zhang et al. explored the effects of scan resolutions and element sizes on QCT/FEA outcomes. They showed scan resolution and element size should be chosen optimally to improve the accuracy of QCT/FEA. To characterize treadmill exercise, N. Guo et al. calculated muscle forces in full gait cycle by inverse dynamic analysis. They found the constraint loading mode had an impact on the muscle forces in treadmill exercise, thus could be manipulated to enhance the effect of the muscle training in spaceflight. J. Gao et al. concluded that effects of treadmill exercise on joints may be associated with different additional weight-bearing levels, and exercise intensities during joint growth and maturation should be selected reasonably. Some new cutting-edge technologies such as robotics, wear devices, and Artificial Intelligence have been applied for the treatment and rehabilitation. S. J. Biggar et al. have developed and evaluated a wearable robotic glove for hand rehabilitation. Regarding the effect of design parameters of personalized orthopedic insole, S. Su et al. showed insole material and support design is positively affecting the correction of orthopedic insole, but negatively resulting unreasonable stress on the stress in the joint and ligaments.Rehabilitation to stroke patients has been constantly receiving close review. Two research teams came up with cutting edge rehabilitation devices for stroke patients to regain, respectively, upper and lower limb motor functions. Q. Miao and colleagues designed a compact and user-friendly robotic exoskeleton for upper limb rehabilitation, which featured a real-time kinematic and dynamic-based system, and allowed both passive and interactive training. Their study demonstrated satisfactory positioning as well as force tracking performance and will head for field study for poststroke patients. Besides, X. Zeng et al. integrated the motion intention of stroke patients via steady-state visual evoked potential with virtual reality technology to develop a rehabilitation robot for the ankle joint. The motion intention-directed passive training was conducted on five subjects with a high success rate. The strategy enabled active engagement for subjects without the ability to conduct active training.The complex interactions between neuromuscular prefatigue and brain control were investigated by L. Wang and colleagues. The research team induced anatagonist muscle prefatigue by isometric elbow flexion. The influence of the fatigue on the cortico-cortical coupling and central modulation was assessed though the technique of EEG-EEG phase synchronization index. Their findings revealed an enhanced intracortical signal which could be an indication for the compensation for the prefatigued-induced joint instability. On the other contrary, Y. Men et al. concerned the microdefect behavior of articular cartilage which implicates the problem of osteoarthritis. Precisely, Y. Men et al. applied a rolling load on porcine articular cartilage and observed the strain of the samples. They identified that the shear strain could be the main factor in cartilage destruction, while rolling velocity also played an important role on the destruction of the superficial and middle layers.Furthermore, recent criteria for orthopedic and cardiovascular rehabilitation affect not only the diagnosis but also the treatment and possibly involves the innovation of supportive or protective devices. This is the reason why, not only will future research focus on mechanism and criteria but also it will increasingly include therapies with rehabilitation and its effects.Lizhen WangChia-Ying LinWei YaoDuo Wai-Chi WongRui Zhu


AR) gene alterations, including ligand-binding domain mutations and copy number (CN) gain, have yet to be fully established as predictive markers of resistance to enzalutamide and abiraterone in men with metastatic castration-resistant prostate cancer (mCRPC). The goal of this study was to validate AR gene alterations detected in cell-free DNA (cfDNA) as markers of enzalutamide and abiraterone resistance in patients with mCRPC.Androgen receptor , and overall survival (OS).Patients with mCRPC (N = 62) were prospectively enrolled between 2014 and 2018. Blood was collected before therapies—enzalutamide (n = 25), abiraterone (n = 35), or enzalutamide and abiraterone (n = 2) —and at disease progression. We used deep next-generation sequencing to analyze cfDNA for sequence variants and CN status in P = .031), PFS , and OS . AR ligand-binding domain missense mutations  were associated with a shorter PFS in multivariable models. AR CN gain was associated with a shorter PFS; however, significance was lost in multivariable modeling. Genetic alterations in tumor protein p53  and phosphoinositide 3-kinase pathway defects  were associated with a worse OS in multivariable models.Elevated tumor-specific cfDNA (circulating tumor DNA) was associated with a worse prostate-specific antigen response (hazard ratio [HR], 3.17; 95% CI, 1.11 to 9.05; AR status associations with outcomes were not robust, and additional validation is needed.These findings support the conclusion that high circulating tumor DNA burden is associated with worse outcomes to enzalutamide and abiraterone in men with mCRPC. Tumor protein p53 loss and phosphoinositide 3-kinase pathway defects were associated with worse OS in men with mCRPC.  Resistance mechanisms are not fully understood; however, some forms of resistance likely involve alterations to AR, including amplification and ligand-binding domain (LBD) missense mutations. Although rare in primary prostate cancers,7AR gene alterations are highly prevalent in mCRPC.13 Metastatic tissue biopsies as a sole means to detect and observe changes in AR status is impractical, and thus cell-free DNA (cfDNA) is gaining traction as a minimally invasive and easily obtainable tumor biopsy surrogate. Previous studies using cfDNA from the blood to evaluate the association of AR gene aberrations with resistance to abiraterone and enzalutamide are inclusive.17AR copy number (CN) gain19 and/or amplification20 or detection of two or more AR mutations20 have been associated with worse outcomes to such therapies as abiraterone and enzalutamide. In contrast, a recent study demonstrated that neither AR CN gain, nor AR LBD mutations, were significantly associated with time to progression on abiraterone and enzalutamide therapies in multivariable models.17 Thus, the role of AR gene aberrations in mediating resistance to androgen–AR axis therapies has not been fully determined, and additional prospective studies are needed for clinical validation.Next-generation therapies that target the androgen–androgen receptor (AR) axis, such as abiraterone and enzalutamide, have improved survival outcomes for men with metastatic castration-resistant prostate cancer (mCRPC),AR gene alterations are only detected in a subset of patients who have either primary or acquired resistance to androgen–AR therapies, thereby highlighting the need to determine other mechanisms that mediate resistance. The AR splice variant AR-V7 is associated with resistance to enzalutamide and abiraterone23 and is also associated with increased AR CN.24 In addition to AR, alterations in other genes, including tumor protein p53 (TP53), phosphatase and tensin homolog (PTEN), and breast cancer gene 2 (BRCA2), are enriched in lethal prostate cancer.11 Studies support the idea that lineage plasticity from an AR-dependent to an AR-independent state through loss of TP53 and retinoblastoma-associated protein 1 (RB1) mediates resistance to AR-targeted therapies.28TP53 defects have been shown to be associated with worse outcomes with abiraterone and enzalutamide therapies.17 The role of BRCA2 and other homology-directed repair (HDR) genes in mediating resistance to enzalutamide and abiraterone has not been definitively determined. Although it has been reported that truncating mutations in BRCA2 and ataxia-telangiectasia mutated (ATM) gene are associated with a shorter time to progression on enzalutamide and abiraterone,17 other studies have indicated that HDR defects may be associated with a better response to therapy.30Consistent with this, AR CN gain and/or LBD mutations detected in cfDNA were associated with enzalutamide and abiraterone resistance in patients with mCRPC. The secondary goal was to determine if alterations in other genes that are enriched in lethal prostate cancer, including TP53, PTEN, and BRCA2, were associated with response to enzalutamide and abiraterone. In this study, high circulating tumor DNA (ctDNA) burden was significantly associated with prostate-specific antigen (PSA) response, progression-free survival (PFS), and overall survival (OS). AR LBD mutations were associated with a shorter PFS, whereas AR CN gain was associated with both a shorter PFS and worse OS, but lost significance in multivariable analyses. TP53 loss and defects in the phosphoinositide 3-kinase (PI3K) pathway were both associated with worse OS. Study limitations, including sample size and patient heterogeneity, necessitate larger and prospective validation of the association of plasma AR status with outcomes.The primary goal of this study was to determine whether Patient information, study end points, sample collection, deep next-generation sequencing (NGS), sequence alignment and analysis of variants, CN variation, estimation of ctDNA fraction, and statistical analyses are found in the Data Supplement.P = .146), PFS , and OS ; however, these associations did not reach statistical significance . We used deep NGS to analyze cfDNA for CN variation and mutations in 46 cancer-associated genes (Data Supplement). Nearly all patients (61 of 62) had detectable CN variation(s) and/or mutation(s) with an allelic frequency above the 1% cutoff before therapy  by logistic regression analyses  and, using proportional hazards regression modeling, a shorter PFS  and worse OS  and a shorter median survival  and OS  were shorter in patients with AR CN gain; however, significance was lost upon inclusion of ctDNA burden in multivariable modeling  of patients before therapy initiation and in an additional 15% (four of 26) of evaluable patients at disease progression  that remained significant in multivariable logistic regression analyses (Data Supplement).Pathogenic raterone . Using lAR LBD mutation than in patients without a detectable AR LBD mutation , even when controlled for ctDNA burden (P = .020) and other variables . Studies support the idea that the AR L702H mutation mediates an acquired response to glucocorticoids, thereby providing rationale to switch from prednisone to dexamethasone.32 In support of this notion, replacement of prednisone with dexamethasone resulted in a greater than 80% PSA decline for this patient (Data Supplement).in cfDNA . Two AR TP53 are highly enriched in lethal prostate cancer11 and have recently been shown to be associated with worse PFS and OS in patients treated with abiraterone and enzalutamide.17TP53 was highly altered in patients’ cfDNA (TP53 defects—pathogenic mutations and/or CN loss—were not associated with PSA response (P = .602) or PFS  that remained significant after adjusting for clinical variables  that remained significant after adjusting for other variables  and worse OS , even after controlling for ctDNA burden  using proportional hazards regression modeling; however, significance was lost after controlling for ctDNA burden , PFS (P = .855), or OS  polymerase or platinum-based chemotherapy.48 In addition, immunotherapy trials have been largely unsuccessful in men with mCRPC49; however, rare responders have been reported.50 A seminal clinical trial demonstrated that microsatellite instable cancers caused by mismatch repair (MMR) gene deficiency were sensitive to programmed death-1 blockade, perhaps because of the formation of neoantigens resulting from increased mutational burden.51 Inactivation of MMR genes and elevated mutational burden have been detected in some men with aggressive prostate cancers.53 One patient in our study had a detectable noncanonical MMR gene mutation in his cfDNA and a correspondingly high mutational burden that suggested that he may be an ideal candidate for checkpoint immunotherapy. This study supports the idea that cfDNA may be a useful analyte for directing clinical decisions in prostate cancer precision medicine.In the current study, many patients had detectable alterations that could serve as potential therapeutic targets. Previous studies have shown that patients with mCRPC with either germline or somatic mutations in HDR genes achieved significant responses to olaparib59 Statistical significance was not reached, likely because of the small overall sample size and the few patients with prior therapy. In addition, the small size precluded any definitive conclusions pertaining to the association of AR LBD mutations with outcomes. Larger prospective studies will be needed to validate our findings. Samples were obtained from two hospitals, and future prospective studies would benefit from the inclusion of a larger number of institutions. Future prospective studies would also be strengthened by radiologic confirmation of progression for every patient. An additional limitation was the variability of cfDNA input for NGS among patients. NGS protocols were adjusted on the basis of total input, but for patients with low input the lack of genetic alteration detection was considered indeterminate as opposed to negative. In addition, mutations in such genes as TP53 and ATM detected in cfDNA at low allelic frequencies may be false positives as a result of clonal hematopoiesis.60 Corresponding tissue was not available for all samples to confirm TP53 status, and future studies will examine both prostate tumor tissue and blood leukocytes for genetic alterations. A final limitation was our inability to evaluate AR splice variants, including AR-V7, because of the requirement of circulating tumor cells or whole-blood RNA. The presence of AR-V7 is certainly another established mechanism of primary and acquired resistance to next-generation hormonal therapies.23 Future studies should aim to simultaneously analyze the full complement of AR aberrations, including gene mutations, amplifications, genomic structural rearrangements, and mRNA splice variants, from a single liquid biopsy.Several limitations to our study exist. Of note, the small sample size precluded multivariable analyses that incorporated more than two variables and analyses by therapy subgroup. Consistent with other reports, patients with prior exposure to abiraterone and enzalutamide experienced worse outcomes.AR status with outcomes was not robust and will need additional prospective validation. TP53 loss, especially in the context of concurrent RB1 defects, and PI3K pathway defects were associated with worse OS. These studies provide the rationale for larger prospective multi-institutional studies to additionally assess the clinical utility of integrating genetic alterations detected in cfDNA for the optimal management of metastatic prostate cancer.In summary, our findings indicate ctDNA burden was highly associated with worse outcomes to enzalutamide and abiraterone. Association of


The catalysts have been thoroughly characterized using N2 adsorption, scanning electron microscopy (SEM), X-ray diffraction (XRD), UV-visible diffuse-reflectance spectroscopy (UV-vis DRS) and X-ray photoelectron spectroscopy (XPS), and have been tested in the aqueous phase decomposition of acetic acid and in the gas phase oxidation of propene, using an irradiation source of 365 nm in both cases. The photocatalytic activity of the four metal-containing catalysts varies with the nature of the metallic species and follows a similar trend in the two tested reactions. The effect of the nature of the added metallic species is mainly based on the electrochemical properties of the supported species, being Cu/P25 (the sample that contains copper) the best performing catalyst. In the photodecomposition of acetic acid, all the metal-containing samples are more active than bare P25, while in the gas phase oxidation of propene, bare P25 is more active. This has been explained considering that the rate-determining steps are different in gas and liquid media. The commercial P25 titania has been modified with transition metallic species , added by impregnation with aqueous solutions of the corresponding nitrates. The preparation procedure also includes a heat treatment (500 °C) in argon to decompose the nitrates, remove impurities and to strengthen the metal–TiO In particular, heterogeneous photocatalysis is a process of great potential for pollutants abatement in gas and liquid phases. This method has considerable advantages over some existing technologies: it destroys pollutants rather than transferring them to another phase; it usually leads to complete mineralization of organic pollutants into COications ,2.4 and CO2, and H2 in minor concentration) [In this work, heterogeneous photocatalysis, a promising advanced oxidation technology for removing contaminants at trace levels , is seletration) . In thistration)  and is otration) . Hence, tration) . 2 has emerged as one of the most promising semiconductor photocatalysts, as it is stable also in aqueous media, tolerant to both acidic and alkaline solutions, it is recyclable and relatively simple to prepare, and shows good ability for air and water purification [2 is that the photogenerated e−/h+ pairs undergo a rapid recombination, therefore decreasing the photocatalytic efficiency of the system. Besides, TiO2 has a relatively wide band gap (3.0 eV for rutile and 3.2 eV for anatase phase [2 [TiOfication . Howeverse phase ), that lphase [2 .2 catalysts has shown to be useful to reduce the photogenerated e−/h+ recombination rate and, also, to improve the photoresponse to the visible light [2 are known to be modified when transition metallic ions replace Ti (IV) centers , occupy interstitial sites  or form aggregates on the TiO2 surface [2 lattice causes local distortion of the crystal structure, thus altering the crystallinity degree and phase transformation. Doping  can also generate trap levels, which modify the band gap and allow the capture of the photo-induced e− in the conduction band (CB) of TiO2, leaving the h+ in the valence band (VB) of TiO2. This hinders the e−/h+ pairs recombination and, thus, promotes the photocatalytic efficiency [2 can act as electrons scavengers, also preventing the recombination of h+/e− pairs. On the other hand, when a metal with a certain work function  is put um level . The abium level ,23,24. C2 [2 performance. For example, transition metals, such as Cu, Co, Ni, Cr, Fe, Mn and V [2 band gap and to extend its photo-response to the visible region.Although noble metals  have proved to be effective to enhance the photocatalytic activity of TiO2 , they arMn and V ,16,26 ha2 systems for the photocatalytic degradation of organic pollutants in the liquid and gas phase has been described by several authors [2 synthesis [2 [2 photoactivity when it contains Co, Cr, Cu, Fe, Mo and V ions added by impregnation. In contrast, Tayade et al. [2 in the degradation of acetophenone and nitrobenzene in aqueous solution was higher than that of bare TiO2, while the presence of Co or Cu led to a lower photoactivity.The use of non-noble transition metals/TiO authors ,19,20,27ynthesis ,16,26,282 photocatalysts  by impregnation of the inexpensive, versatile and commercial P25 titania (from Degussa). These new materials were characterized by N2 adsorption isotherms, scanning electron microscopy (SEM), X-ray diffraction (XRD), UV-visible diffuse-reflectance spectroscopy (UV-vis DRS) and X-ray photoelectron spectroscopy (XPS) analysis, and their photocatalytic activity was investigated in the photo-degradation of aqueous acetic acid into CO2, CH4 and H2 and in the gas phase photo-oxidation of propene. The overall performances were discussed in terms of activity and selectivity as a function of the properties of the photocatalysts. All these considerations prompted us to prepare a series of M/TiO2 has been used as a raw material because of its good activity in many photocatalytic reactions. Cr, Co, Ni, and Cu were incorporated to P25 by impregnation as follows: P25 (2 g) was put in contact with an aqueous solution (5 mL) of Cr(NO3)3·9H2O, Co(NO3)2·6H2O, Ni(NO3)2·6H2O or Cu(NO3)2·3H2O of the appropriate concentration to obtain 1 wt.% metal loading. The mixture was stirred (2 h) and sonicated (30 min) and, then, the solvent excess was removed .Degussa Aeroxide P25 TiO2 interaction. The Ar atmosphere was selected according to the previous work [All the samples were heat-treated in argon atmosphere  to decompose the nitrates, remove impurities and to strengthen the metal–TiOous work . The use2 adsorption–desorption at −196 °C ) was measured in a Quantachrome Autosorb-6B equipment . The specific BET surface area (SBET) and the micropore volume (VN2) were determined by applying, respectively, the Brunauer–Emmett–Teller (BET) or Dubinin–Radushkevich equations to the N2 adsorption isotherms. These determinations have an associated error of 2%. Mesopore volume (Vmeso) was estimated by the difference in the volume of N2 adsorbed as liquid at P/P0 = 0.9 and P/P0 = 0.2. The total pore volume (VT) was determined by the nitrogen adsorption volume at a P/P0 (relative pressure) of 0.99. For such calculation, the density of liquid nitrogen at −196 °C was used. The average pore size (Ø) was calculated from the nitrogen adsorption isotherms by the Barret-Joyner-Halenda (BJH) method [Porosity  of the photocatalysts has been charaterized by physical adsorption of gases. N) method ,31,32.SEM characterization was performed using the JSM-840 JEOL equipment  and the scanning electron microscopy with energy dispersive X-ray (SEM-EDX) mapping was conducted with a Hitachi S3000N microscope  and the Bruker XFlash 3001 X-ray energy dispersive spectrometer . Cryst) and rutile (RCryst) has been calculated using the equations proposed by Jensen et al. [Anatase (101), ARutile(110) and ACaF2(220) are the peak areas determined in XRD spectra measured for a 50/50 (w/w) TiO2/CaF2 mixture, and 1.25 and 0.90 are the ratios between areas of the same peaks when a 100% anatase or 100% rutile crystalline TiO2 sample (respectively) is used. The percentage of crystalline (WCryst) and amorphous (WAm) phases present in the photocatalysts were determined as indicated in Equations (4) and (5), respectively.XRD analysis using Cu Kα (1.54 Å) radiation was performed in a SEIFERT 2002 equipment , in the angular 2θ range 6–80° and with a scanning velocity of 2°/min. The experimental errors are within ± 0.1°. The average crystallite size, referred to as crystal size, was calculated by Scherrer’s equation (Equation (1)) : (1)B=Kλn et al. :(2)ACrys4 as the reference sample. The spectra were recorded at room temperature in air, in the 200–800 nm range. The absorption edge wavelength was estimated from the intercept at zero absorbance of the high slope portion of each spectrum in the 200–800 nm range (absorbance method). The band gap energy was calculated using three different methods: absorbance, indirect and direct ones. In the absorbance method, the band gap can be calculated using Equation (6), where Eg is the band gap energy (eV) and λ is the edge wavelength (nm) [Diffuse reflectance spectra (DRS) were determined by means of a Jasco V-670 UV-vis spectrophotometer , using BaSOgth (nm) .(6)Eg=12 vs. hυ or (F(R)hυ)0.5 vs. hυ [In the direct and indirect methods, the band gap values are obtained from the Kubelka-Munk function of the reflectance, plotting, respectively, (F(R)hυ)5 vs. hυ .−9 mbar analysis chamber pressure and detection in constant energy mode with pass energy of 200 eV for the survey spectrum, and of 50 eV for the sweep in each individual region. Data analysis was performed with the peak-fit software of the spectrometer  using Gaussian functions with 20% Lorentzian component, and binding energy values were adjusted to the C1s transition (284.6 eV). This process has an associated error of ± 0.2 eV [XPS spectra were obtained using a K-Alpha spectrophotometer  with a high-resolution monochromator and the following specifications: Al anode (1486.6 eV) X-ray source, 5 × 10± 0.2 eV .Details of the photocatalytic activity measurements in the two studied reactions are presented next. max = 365 nm, Hanau, Germany) located in the center of the vessel and surrounded by a water-circulating jacket. Gas inlet and outlet are in the upper part of the reactor. One molar solution of acetic acid (0.35 L) and the photocatalyst (0.35 g) were introduced in the reactor. A stream of He (60 mL/min) was bubbled through the reactor for 2 h to purge lines and remove dissolved oxygen and, then, the cooling system and the UV lamp were turned on. The process was conducted for 12 h under continuous magnetic stirring. Some experiments were repeated twice to check reproducibility. A blank experiment  was also performed in the same conditions to measure the photolysis effect. Analysis of the outlet gas stream was performed by mass spectrometry . Calibrated cylinders of 2000, 2000 and 500 ppmv in helium of CH4, CO2 and H2, respectively, were used to quantify the products. The photocatalytic activity was expressed as indicated in Equation (7).4, CO2 or H2.Photocatalytic tests were performed in a cylindrical quartz reactor  with a medium pressure mercury vapor lamp  and a 365 nm Philips UV-A lamp placed parallel to the quartz reactor at about 1 cm. The assembly quartz reactor lamp is surrounded by a cylinder covered by tinfoil. A scheme of this system is shown elsewhere . initialC3H6 is the initial propene concentration, 100 ppmv, and CstationaryC3H6 is the stationary propene concentration reached after a certain irradiation time. The photocatalyst (0.11 g) was placed on a quartz wool plug inside the reactor and then, after purging with helium, a stream of 100 ppmv propene in air was fed to the reactor at 25 °C, being the outlet gas continuously analyzed by mass spectrometry . Once the propene concentration is stable (after about 3 h), the lamp is switched on and kept working until a stationary propene signal is achieved . All the tests were carried out with 30 and 60 mL/min propene flow. A blank experiment  was also performed in the same conditions to measure the photolysis effect. The experiments were repeated at least twice to check reproducibility. Propene conversion was calculated using Equation (8):2 was quantified using a calibrated cylinder (300 ppmv CO2 in helium). Water evolution was also followed by MS and a mass scan was carried out to determine if additional oxidation compounds were present in the outlet stream. An example of the type of information obtained in this type of experiment has been shown in a previous publication [The produced COlication .2 adsorption–desorption isotherms of M/P25-Ar and P25-Ar samples. It can be observed that the adsorption isotherms are type IV, according to the IUPAC classification [fication , with a The parameters calculated from the adsorption isotherms  are coll2  values of the photocatalysts have been calculated by the absorbance method, considering the direct allowed transitions (direct method) and the indirect allowed transitions (indirect method); they are presented in g values of samples M/P25-Ar are not significant, and lower than that of P25-Ar.The band gap  these new energy levels would be located below the lower conduction band edge of TiO2, decreasing the TiO2 band gap and shifting the absorption band to the visible part of the spectrum; and (ii) these energy levels would also act as e− traps, increasing the h+ lifetime and decreasing the e−/h+ recombination rate [As reported in the literature ,20,27, tion rate .3/2 and O 1s XPS spectra. The binding energy of Ti 2p3/2, about 458.6 eV  metal loading is 1 wt.%, this reveals that the samples show a certain surface enrichment in metallic species on the more external surface of the catalyst.The results obtained in the study of the acetic acid photodecomposition in the liquid phase and the photooxidation of propene at low concentration in the gas phase are presented and discussed next.4, CO2 and H2 (in mmol per mol of acetic acid (HAc)) generated during 12 h in the different catalytic tests performed. Because the H2 production is very low, the comments on the obtained results will be based on the production of CH4 and CO2. It can be observed that the produced amounts of CH4 and CO2 by photolysis are small compared to those produced in the presence of the photocatalysts.4 and CO2 [4/CO2 molar ratio equal to 1. However, it is interesting to note that in the obtained results, the amount of CO2 produced is higher than the amount of CH4, being the CH4/CO2 ratio lower than one for all the tested catalysts. This means that some other reactions, besides the photo-Kolbe one, have to be taken into consideration. Mozia, et al. [2 could be originated by the oxidation of acetic acid with photogenerated O2, as shown in Equation (10): Methane formation during the photocatalytic decomposition of HAc is supposed to follow the so-called photo-Kolbe reaction (Equation (9)), and CO2 ,48,49,504/CO2 ratio would be 0.30. However, the measured CH4/CO2 ratio ranges between 0.47 and 0.77, meaning that with the tested catalysts and conditions, the photo-Kolbe reaction is predominant, prevaling acetic acid degradation over its oxidation.Thus, it could be assumed that both the photo-Kolbe (Equation (9)) and the oxidation reaction (Equation (10)) occur simultaneously in the experiments carried out. If both reactions would occur up to the same extent, the theorethical CH4 and CO2 than P25-Ar, confirming a clearly positive effect of the metal. Data of 2 is the only oxidation product. Also, quantification of the produced CO2 has allowed to perform a carbon balance which confirms that total mineralization of propene takes place, according to the following reaction (Equation (11)), and in agreement with the literature [terature ,51.2 C3Photodegradation of propene by photolysis is negligible compared with the photocatalytic oxidation. The activity order of the investigated photocatalysts follows the same trend in both flow-rates tested (P25-Ar > Cu/P25-Ar > Ni/P25-Ar > Cr/P25-Ar ≈ Co/P25-Ar), which can be considered as a proof of reproducibility. As expected, propene conversion is higher when the used flow is 30 mL/min. 2 phase distribution . All the other metals must be located either in the network interstices or as aggregates on the TiO2 surface. The latter is the most plausible option since no significant changes in the crystalline structure of these M/P25-Ar samples compared to P25-Ar have been observed. The ionic radius of Cr (III) is similar to that of Ti (IV), and this would let Cr (III) replace Ti (IV) in the TiO 500 °C, . Moreove2 to achieve an efficient photocatalytic behaviour. The metallic species can, thus, behave as efficient traps for photogenerated electrons, preventing the e−/h+ recombination and, consequently, improving the photocatalytic activity [As indicated in the introduction, electron transfer can occur from lower work function materials to higher work function ones. In the case of the M/P25-Ar samples, the metal work function must be higher than that of TiOactivity .2O and CuO species are less negative than that of TiO2, while the valence band (VB) edges of Cu2O and CuO are less positive than that of TiO2. This implies that in the Cu-P25-Ar sample, Cu2O and CuO introduce energy levels close to both the CB and VB of TiO2, which can act as effective e− and h+ traps. Besides, the coexistence of the two Cu species generates electron transfer in cascade, from more positive energy values to less positive ones. This way of electron transfer may result in a better e−/h+ separation, associated to higher photoactivity.The activity results obtained show that Cr/P25-Ar and Co/P25-Ar are the samples with the lowest photocatalytic activity in both reactions. In the case of the Co catalyst, it can be due to the lower work function of Co (II) ions with respect to that of Ti (IV). This would not allow the Co (II) ions to capture electrons in an efficient way ,22,53. A2 systems in abatement of pollutants in liquid and gas phase and found that the photocatalysts’ behaviour was different in the two phases [2 exposed surface) and this is also influenced, as mentioned above, by the metal’s nature. In the liquid phase reaction, the diffusion limitations likely make the whole reaction slower and, thus, the pathway in which the supported metallic species are involved is not limiting the reaction. In this case, the positive effect of the metallic species as electron traps prevails. In the photodecomposition of acetic acid, Cu/P25-Ar is clearly more active than the rest of M/P25-Ar photocatalysts. Besides, as mentioned above, all the M/P25-Ar samples are more active than P25-Ar. On the other hand, in the gas phase oxidation of propene, Ni/P25-Ar and Cu/P25-Ar show a close behavior, being bare P25-Ar more active than the M/P25-Ar photocatalysts. This indicates that the course of the two reactions seems to depend differently on the catalysts properties. Previous works compared the photoactivity of TiOo phases ,58. Thiso phases ,60,61,62o phases . Probabl2 photocatalysts  were prepared by impregnation of the commercial P25 titania followed by heat treatment in Ar at 500 °C. In the present work, a series of M/TiO2 surface. The crystal size of anatase and rutile was very similar in all M/P25-Ar samples, although in Cr/P25-Ar the average crystal size of rutile was the lowest. The band gap energies of the M/P25-Ar samples were a bit narrower than that for bare P25-Ar. The XPS analysis indicated that excepting Co, the rest of the metals are present in more than one oxidation state. The Ti 2p and O 1s spectra of Cr/P25-Ar show, compared to the rest of samples, a shift to lower binding energies, which suggests that Cr could have partially been incorporated in the TiO2 structure in this sample, leading to some defects. This is possible because the atomic radii of Cr (III) and Cr (VI) ions are smaller than that of Ti (IV), and it could explain the differences in the textural properties and crystal size of Cr/P25 compared to the rest of M/P25 photocatalysts. The characterization results have shown that the textural properties of the M/P25-Ar photocatalysts are very close to those of bare P25-Ar, and only sample Cr/P25-Ar shows a slightly higher surface area. The crystalline structure of P25-Ar is, as well, almost not affected by the metal incorporation, and XRD patterns due to metal oxides have not been detected, which allows us to conclude that the metallic species are highly dispersed on the TiO2 lattice, which results in lattice defects and oxygen vacancies that would facilitate recombination of electrons and holes. The low activity of Co/P25-Ar has been explained by the low work function of Co (II) compared to that of Ti (IV). The Cu/P25-Ar photocatalyst showed the best photocatalytic performance among the M/P25-Ar samples because Cu ions dispersed on TiO2, present as Cu (II) and Cu (I), have suitable energy levels of CB and VB edge to trap e− and h+ and carry out an effective electronic transfer. The photoactivity of the M/P25-Ar photocatalysts follows the same trend in the two studied reactions: Cu/P25-Ar > Ni/P25-Ar > Cr/P25-Ar ≈ Co/P25-Ar. To explain the differences between catalysts, the work functions and the ionic radii of the incorporated metallic ions have been considered to have a role. It has been concluded that the low activity of Cr/P25-Ar sample is related with the partial incorporation of Cr species in the TiO2 surface, and the supported metallic species reduce the exposed titania surface, decreasing the photocatalytic efficiency. In contrast, in the liquid phase, as the diffusion processes make the reaction slower, the positive effect of the metallic species as electron traps can be better identified.Although the relative activity order of the metal-containing photocatalysts is the same in both reactions, P25-Ar shows better activity than Cu/P25-Ar in propene oxidation, whereas Cu/P25-Ar is more active than P25-Ar for acetic acid degradation. This can be explained considering that the rate-determining steps are significantly different in gas and liquid media. In the gas phase, propene oxidation is a rapid process that happens mainly on the TiO


AR gene as the cause of 46,XY disorder in our population.The aim of this study was the molecular characterization of the We studied 41, non related, 46,XY disorder of sexual differentiation index cases, having characteristics consistent with androgen insensivity syndrome (AIS). Genomic DNA was isolated from peripheral blood leukocytes of all patients and 25 family members from 17 non-related families.AR gene analysis revealed an abnormal sequence in 58.5% of the index patients. All of the complete AIS (CAIS) cases were genetically confirmed, while in the partial form (PAIS) a mutation in AR was detected in only 13 (43.3%). Molecular studies revealed other affected or carrier relatives in 87% of the index cases. The AR mutations were found spread along the whole coding sequence, with a higher prevalence in the ligand binding domain. Nine out of 23 (39%) AR mutations were novel. In 17% of patients with detected AR mutations, somatic mosaicism was detected in leucocyte DNA. In our cohort, long-term follow up gender dysphoria, raised as male or female, was not found. Finally, in suspected PAIS, the identification of AR mutation occurred significantly less than in CAIS patients.The AR complex and signaling network might contribute to long term outcome and genetic counseling in AIS patients.Improved knowledge of the components of the  What is already known on this topic?Androgen insensitivity syndromes (AIS) the most frequent known monogenic cause of 46,XY disorder of sexual differentiation. Mutations of variable severity in androgen receptor gene are associated with a wide phenotypic spectrum, ranging from complete AIS to a partial form or a mild form.What this study adds?Characterization of the clinical phenotype, long term follow up, in particular gender identityand the contribution of the AR gene to the molecular cause of 46,XY disorder of sexual differentiation in a single tertiary pediatric center of Buenos Aires, Argentina are reported. Nine novel AR mutations are described.AR)  , located on exon 1, a DNA-binding domain (DBD), located on exons 2 and 3 containing two zinc fingers, a hinge region connecting the ligand-binding domain (LBD) to the DBD and a C-terminal LBD, located on exons 4-8  and dihydrotestosterone (DHT), exert their effects via a single intracellular receptor protein, the androgen receptor (AR) . AR-medifunction . The AR to VIII) . The AR AR gene are associated with a wide phenotypic spectrum, ranging from complete AIS (CAIS) to a partial form (PAIS) or a mild form (MAIS). Patients who present with CAIS exhibit female external genitalia, testes located in the inguinal or abdominal area, and complete breast development with sparse to absent axillary and pubic hair. Patients with PAIS present with a predominantly male phenotype with hypospadias or a predominantly female phenotype with cliteromegaly and/or posterior labial fusion, ambiguous genitalia and variable degrees of gynecomastia at puberty. Patients with MAIS present with normal external male genitalia associated with infertility  is the most frequent known monogenic cause of 46,XY disorders of sex development (DSD) and is an X-linked recessive condition. Mutations of variable severity in the ertility .AR gene to the molecular cause of 46,XY DSD in our population.The aim of this study was to characterize the clinical phenotype and the contribution of the AR gene was the first candidate for molecular analysis.We studied 41 unrelated 46,XY DSD patients with clinical and hormonal characteristics consistent with AIS. CAIS was suspected in 11 of the patients and PAIS in 30. All patients presented with female or ambiguous external genitalia, adequate T production without evidence of steroidogenic blockade and no Müllerian structures evident on abdominal ultrasound. Patients with hormonal determinations previous to gonadal biopsy or gonadectomy presented no biochemical evidence of gonadal dysgenesis and had normal male follicle-stimulating hormone (FSH) levels. In these individuals, the Informed consent for the genetic study was obtained from all of the patients or their parents guardians after full explanation of the purpose and nature of all procedures.The study was approved by the Independent Ethics Committee “Prof. Dr. J. P. Garrahan Pediatric Hospital” (reference number: 971).® Assay System ; assay sensitivity was 0.17 nmol/L; interassay coefficient of variation ranged from 7.4 to 13%.Serum luteinising hormone (LH) and FSH levels were determined by the IMX systems ; assay sensitivity was 0.3 IU/L for LH and 0.2 IU/L for FSH; interassay coefficient of variation ranged from 3.1-8.7% for LH and from 3.8-12% for FSH. Serum anti-Müllerian hormone (AMH) levels were determined by ELISA; assay sensitivity was 0.5 pmol/L and assay limit of quantification 1.2 pmol/L serum T was determined by a DPC ImmuliteAR gene were polymerase chain reaction (PCR) amplified and sequenced by automated analysers  and 25 family members from 17 families according to standard procedures. There were seven families in which family members were not available for molecular studies. The entire coding region (exons 1-8) and splice sites in flanking intronic regions of nalysers .® 3130 Genetic Analyzer capillary DNA sequencer . The primers used for sequencing were the same as those used for PCR. Previously reported intronic mutations were also analysed . The nucleotide sequences obtained were compared with those from Genebank accession number: NG_009014.2. Nucleotide changes were reconfirmed in each sample DNA by antisense sequence and resequencing after a new PCR product was produced from the original DNA extract.After PCR, the products were assessed by electrophoresis on a 1% agarose gel stained with ethidium bromide and showed a single band with expected size. The PCR products were purified  and sequenced using a BigDye Terminator version 3.1 cycle sequencing kit  on an ABI PRISMNonsense and frameshift mutations which implicate a premature stop codon and a truncated protein were considered deleterious.The sequence homology-based tool, , version 2.0.6, the structure-based tool PolyPhen-2  and Mutation Taster (http://www.mutationtaster.org/) were used to predict the pathogenicity of the previously undescribed missense variants using default settings. To evaluate the implication of a novel synonymous mutation, we used The Berkeley Drosophila Genome Project (http://www.fruitfly.org/) as a splice site prediction program.The SIFT algorithm predicts the functional importance of the substitutions based on the alignment of orthologous and/or paralogous protein sequences. The PolyPhen-2 algorithm predicts the functional effects of amino acid changes by considering conservation, physicochemical differences and the proximity of the substitution to the predicted functional domains. Unlike SIFT or PolyPhen which handle only single amino acid substitutions, MutationTaster works on the DNA level and allows insertions and deletions up to 12 base pairs.The original sequence of the protein was obtained from the Ensembl and UniProt/Swiss-Prot databases.This study describes the genotype and clinical phenotype of patients with AIS. A statistical analysis was not necessary.AR gene analysis revealed an abnormal sequence in 24 individuals . All of the CAIS cases (n=11) were genetically confirmed, while in PAIS (n=30) a mutation in AR was detected in only 13 patients (43.3%).In our study De novoAR mutations were found in three  out of 13 mothers analyzed. In two non-related index cases (P12 A and P15), two 46,XY affected siblings raised as female were detected. Interestingly, even though in P12A PAIS was established, normal external female genitalia, in the affected sister, was observed (P12 B). As shown in AR mutations were detected. The AR mutations were found spread along the whole coding sequence, with a higher prevalence in LBD: 8.3% were located in NTD; 16.6% in the DBD; 70.8% in the LBD and 4.3% were gross deletions (Family studies were performed in 25 family members from 17 families. The molecular studies and affected family members are shown in AR mutations were novel. Two novel mutations were located in the NTD domain (P1 and P16). They were both out of frame deletions that ultimately created a nonsense stop codon and premature truncation of the protein. The others, located in the LBD, were:four missense mutations, a nonsense mutation together with a 2bp deletion and a duplication of 7bp that produce a frameshift with a premature stop codon. Three patients  harboured somatic mosaicisms: a nonsense mutation, a 7bp duplication and a 20bp deletion which result in a truncating frameshift mutation. One missense mutation was located in the DBD. All novel mutations were predicted to be pernicious by all in silico tools.Nine out of 23  In four individuals , 17% of AR-mutated gene patients, somatic mosaicism of mutant and wild type alleles was detected in DNA derived from blood leukocytes. Of the 17 individuals without a defect in the AR, two patients were finally diagnosed  with 5-alpha reductase deficiency. In the others, diagnosis remains unknown. The clinical phenotype and follow-up of the genetically confirmed patients is shown in AR gene mutations are the main cause of 46,XY DSD. To date, the AR gene mutations database (http://www.mcgill.ca/androgendb/) has reported more than 800 different AR mutations from patients with AIS. We describe a series of unrelated patients affected by different degrees of AIS. AR mutations responsible for the phenotype were identified. However, similar to other cohorts, in PAIS phenotype cases, AR mutations were identified in only 38%. Overall, in our series of 41 index patients, the AR gene proved to be abnormal in 58.5%, confirming the diagnosis. Similarly, Boehmer et al  should also be taken into consideration. Several cofactors, such as coactivators steroid receptor coactivator 1 (SRC1), transcriptional mediators/intermediary factor 2, SRC3 and corepressors nuclear receptor-interacting protein 1, nuclear receptor subfamily 0 group B member 1, are actively involved in the regulation of AR-mediated transcription, and might play an important role in AIS etiopathogenesis (APOD(apolipoprotein D) gene in cultured genital fibroblasts (APOD-assay). However, the usefulness of this APOD assay needs to be confirmed in a large cohort.In all CAIS cases, er et al  and Audier et al  report aer et al  and Akcaer et al  describeogenesis ,13,14,15ogenesis  developeAR gene are distributed throughout the sequence with a preponderance (70.8%) located in the LBD  a severe mutation was detected but presenting with a PAIS phenotype. It is noteworthy that detection of somatic mosaicism in AR has a great impact for patients with AIS because further virilization is possible after birth and this is an important consideration for genetic counseling (Somatic mosaicism has been reported. Interestingly, even though the patients carried severe unseling .No gender dysphoria was observed in our cohort, even though systematic assessment was not available in all cases.AMH gene expression in Sertoli cells is inhibited via the AR receptor pathway (AR expression in Sertoli cells during mini puberty could explain our findings, suggesting that other as yet unidentified factors might be involved in the regulation of AMH synthesis (In contrast to previous reports, in this cohort, AMH serum concentrations during the neonatal period were within the normal male reference range in the only two PAIS cases in whom it was assessed ,23. AMH  pathway . The lacynthesis . In agreement with previous reports, normal gonadotropin levels were the most frequent finding .in silico tools predicted the novel mutations to be damaging for protein structure and function, functional assays should be performed to confirm pathogenicity.Even though all In summary, we report a series of 41 46,XY DSD index patients in whom AR was the candidate gene. Molecular diagnosis is useful for genetic counseling of the families. However, similar to other series, the percentage of suspected casesin whom an AR mutation was found wasonly around 60%.Emerging technological advances might contribute to an increase in the accuracy of determining the etiology in suspected AIS cases.


CH) caused by severe left ventricle (LV) volume overload (VO) from chronic aortic valve regurgitation (AR) in male rats was influenced by androgens. We studied Wistar rats with/without orchiectomy (Ocx) either sham‐operated (S) or with severe AR for 26 weeks. Loss of testosterone induced by Ocx decreased general body growth. Cardiac hypertrophy resulting from AR was relatively more important in intact (non‐Ocx) animals than in Ocx ones compared to their respective S group . The intact AR group had more LV dilation, end‐diastolic LV diameter being increased by 37% over S group and by 17% in AROcx rats (P < 0.0001). Fractional shortening (an index of systolic function) decreased only by 15% in AROcx compared to 26% for intact AR animals (P = 0.029). Changes in LV gene expression resulting from CH were more marked in intact rats than in AROcx animals, especially for genes linked to extracellular matrix remodeling and energy metabolism. The ratio of hydroxyacyl‐Coenzyme A dehydrogenase activity over hexokinase activity, an index of the shift of myocardial substrate use toward glucose from the preferred fatty acids, was significantly decreased in the AR group but not in AROcx. Finally, pJnk2 LV protein content was more abundant in AR than in AROcx rats, indicating decreased activation of this stress pathway in the absence of androgens. In summary, testosterone deficiency in rats with severe LV VO resulted in less CH and a normalization of the LV gene expression profile.The aim of the study was to characterize if the development of cardiac hypertrophy ( Cardiovascular diseases are among the leading causes of mortality in both men and women . Significant pressure overload  or volume overload  will induce left ventricle (LV) remodeling characterized by changes in wall thickness, chamber diameter, extracellular matrix organization, gene transcription, cell signaling, and energy metabolism  caused by severe aortic valve regurgitation (AR), females developed as much if not more CH than males. However, male showed more LV dilation but lost more contractile function than females , Ocx sham‐operated , AR; (n = 13) and Ocx AR . AR was induced 2 weeks later at age 10 weeks as previously described by perforation of one or two aortic valve leaflets using a catheter via the right carotid and under echocardiographic guidance   were studied. Twenty‐two of which were purchased after Ocx at age 8 weeks. Experimental groups were as followed: Sham‐operated  was converted to cDNA using the QuantiTect® Reverse Transcription kit (Qiagen), a procedure which included a genomic DNA elimination step. The cDNA obtained was further diluted 11‐fold with water prior to amplification . Five‐microliter of diluted cDNA were amplified in duplicate by Q‐PCR in a Rotor‐Gene™ thermal cycler , using optimized specific primer pairs , enoyl‐CoA hydratase, short chain 1 , natriuretic peptide precursor type A , natriuretic peptide precursor type B , and Cyclophilin A .LV gene expression was quantified for six animals per group by quantitative RT‐PCR as described elsewhere  for hexokinase (HK), hydroxyacyl‐Coenzyme A dehydrogenase (HADH), succinate dehydrogenase (SDH), citrate synthase (CS), and creatine kinase (CK) were determined as previously described , and β‐mercaptoethanol (30 mmol/L). The homogenate was then sonicated. NADH formation was monitored by UV spectroscopy at 340 nm as an indication of BDH activity. The samples were tested in duplicates.For the beta‐hydroxybutyrate dehydrogenase (BDH) enzyme assay, LV tissue was homogenized with 40 volumes of an extracting solution. The extracting solution was as follows: triethanolamine , EDTA (1 mmol/L), MgClProtein content was estimated by Western blotting as described previously . LV pieces fixed in paraffin were cut and deparaffinized. Tissue sections were then rehydrated and then colored for 1 h in 0.1% Sirius red diluted in saturated picric acid. Sections were then washed in 1% acetic acid, dehydrated three times in 100% ethanol for 5 min and finally washed in xylene. A picture of each mid‐LV section was acquired with a Zeiss optical microscope and Zen software (Germany) with a size of 280 equal area tiles.Picrosirius red staining was done in order to measure mid‐LV sections size and fibrosis content . Two‐way ANOVA was performed and Holm‐Sidak post‐test was used for comparison between the groups . A Student's P = 0.052) . There were no significant changes in lungs weight (a marker of heart failure) between the S and the AR and between the SOcx and the AROcx groups  . AR severity was similar between the groups  (Table P = 0.056)  dimensions Fig. . Inter‐v1) Table . A large1) Table B. As exp56) Fig. C. Heart 56) Fig. .We measured diastolic flow parameters through the mitral valve. E wave, representing LV rapid filling, was significantly higher in AR animals Fig. D. LikewiAnp gene expression, this being significantly stronger in intact animals  remodeling markers. In intact AR animals, ats Fig. C. Gene e3α1 Fig. .A metabolic transition from fatty acid to glucose substrate use is a feature of pathological CH  that only tended to be reduced compared to sham rats. This downregulation was also present in the AROcx animals when compared to their respective controls. However, no significant difference was observed for the AROcx group for Gabpa and Nduf4 genes. Nduf4 encodes for a subunit of the first complex of the respiratory chain. Cox5b1, which encodes for a subunit of cytochrome c (the fourth complex of the respiratory chain), was significantly more expressed in SOcx rats compared to S , an enzyme implicated in the oxidation of medium chain fatty acid, was not significantly different between the groups although it tended be lowered in the AR group Fig. A.The hexokinase (HK) enzymatic activity determination was performed to measure myocardial glucose use capacity. HK activity was increased in both AR groups Fig. B. Moreovβ‐hydroxybutyrate dehydrogenase (BDH) activity in order to measure ketone bodies use in the hypertrophied myocardium. AR reduced BDH activity in intact animals only. Ocx also decreased BDH activity in both sham and AR rats  activity was not different between the groups, except for AR, which tended to decrease it Fig. D. AR or ats Fig. G.Mitogen‐activated protein kinases (MAPK) pathways are implicated in cell growth, cardiac myocytes hypertrophy, and response to stress (Heineke and Molkentin Protein kinase B (Akt) is implicated in both hypertrophic and survival signaling pathways  and implicated in the regulation of protein synthesis and cell growth, was then analyzed. Phospho‐S6 was decreased by Ocx, but total form levels were constant between the groups Fig. . Loss ofOur results suggest that androgens are important drivers of pathological cardiac hypertrophy development caused by severe left ventricle overload from chronic aortic regurgitation. Testosterone deficiency in AR rats resulted in reduced cardiac hypertrophy, less LV dilation, better function and a strong tendency for better survival of the animals. Changes in LV expression for an important number of genes associated with pathological myocardial remodeling were less marked in AROcx animals than in intact AR. In fact, the LV gene expression pattern we observed in AROcx rats had similarities with the one we reported recently in a study comparing AR females to males . Physiological heart growth and maladaptive hypertrophy development share and have distinctive signaling pathways. Activation of the Akt/mTOR pathway is associated with pathological CH (even in AR) although normal cardiac growth also relies upon this pathway . This suggests that androgens may be implicated in the myocardial response to mechanical wall stress. Interestingly, FAK , a mediator of integrin activation, is strongly downregulated in Ocx myocardium regardless of the presence or not of a hemodynamic stress.Hypertrophic markers gene expression results showed that the myocardium of AR animals responded to the increased LV wall stress, as expected. Interestingly, some of those genes had lower mRNA levels in the LV of AROcx rats  had higher levels of pAkt and thus seemed to have enhanced Akt activity, as the pGsk3 content was similarly increased. Androgens action can be mediated by delayed genomic effect (classic androgen receptor) and a rapid non‐genomic effect via G‐coupled receptors such as GPCR6A or ZIP9. Testosterone has been shown to be pro‐hypertrophic in cultured neonatal rat cardiac myocytes  impairs heart function and adaptation to stress by enhancing fibrosis  and DHT levels are increased in the hypertrophic heart, which could enhance DHT negative effects  extracellular matrix remodeling (B) markers.Figure S2. Comparison of SOcx with S with real‐time quantitative RT‐PCR of the LV mRNA levels of genes encoding for glucose uptake and glycolysis (A), fatty acid oxidation (B), and transcription factor (C) markers.Figure S3. Comparison of SOcx with S with real‐time quantitative RT‐PCR of the LV mRNA levels of genes encoding for mitochondrial function markers.Figure S4. LV protein contents of S6, Fak, and Pkd. Representative blots for each signaling molecules (A).Click here for additional data file.


Neuropilin‐1 (NRP1) is a highly interactive molecule that exists as transmembrane and soluble isoforms. Measurement of circulating levels of soluble NRP1 (sNRP1) in human serum and plasma has proven to be difficult due to present matrix interferences and due to the lack of a reliable technique.We developed a highly specific and sensitive sandwich ELISA assay for total sNRP1 quantification in peripheral blood, and we validated the test according to ICH guidelines. The linear epitopes of the employed polyclonal and monoclonal anti‐human NRP1 antibodies were mapped with microarray technology. We included a sample pre‐treatment step with guanidine hydrochloride (GuHCl) to release sNRP1 from existing interferants.The ELISA assay which is calibrated with sNRP1 isoform 2 and covers a calibration range from 0.375 to 12 nmol/L detects sNRP1 in human serum and plasma . Multiple linear epitopes recognized by the polyclonal coating antibody are distributed over the whole sNRP1 sequence. The monoclonal detection antibody binds to a linear epitope which is in the N‐terminal region of the a1 domain of human sNRP1. Assay parameters like precision (intra‐assay: 6%), dilution linearity (95%‐115%), specificity (98%), and spike recovery (81%‐109%) meet the international standards of acceptance.Our novel sandwich ELISA provides a reliable tool for the quantitative determination of total human sNRP1. The assay detects free and previous ligand‐bound total NRP1. GuHClguanidine hydrochlorideNRP1neuropilin‐1sNRP1soluble neuropilin‐11165 isoform of vascular endothelial growth factor A.Neuropilin‐1 (NRP1) is a single‐pass transmembrane glycoprotein of 923 amino acids first described in 1987 by Fujisawa et al.165 antagonist that has antitumor activities.Alternative splicing or ectodomain shedding by the metalloprotease ADAM10 leads to the release of the extracellular a and b regions as soluble NRP1 (sNRP1).To study the function of NRP1 and its role as a potential biomarker in various diseases, we developed a sandwich ELISA for robust and reliable quantification in human blood. We included a sample pre‐treatment step to overcome interferences, which finally allowed us to measure free and previous ligand‐bound total NRP1. We unveiled linear epitopes of the employed monoclonal and polyclonal anti‐human NRP1 antibodies to provide best possible insight into the binding properties of the antibodies utilized in the assay. Furthermore, we validated the assay based on the principles of bioanalytical validation defined by the ICH harmonized tripartite guideline Q2 (R1).22.1Venous blood samples were obtained from apparently healthy subjects after informed consent was given. Blood collection tubes for serum or plasma  were used for sample collection. Samples were incubated 10 minutes at room temperature before centrifugation . Samples were stored at −20°C. Testing was done in an anonymized manner.2.2165  for 10 minutes, followed by a dissociation period of 3 minutes in PBS. Reference sensor 2 was incubated in parallel with PBS. Regeneration of both sensors was performed with 3 mol/L GuHCl in PBS for 5 minutes, followed by a stabilization in PBS for 2 minutes.Measurements were performed on an Octet K2 instrument  at 25°C. For amine coupling, two AR2G sensors (ForteBio) were activated for 10 minutes with a mixture of 1‐ethyl‐3‐(3‐dimethylaminopropyl carbodiimide) hydrochloride and N‐hydroxysulfosuccinimide and further loaded for 15 minutes with monomeric sNRP1 diluted in sodium acetate (pH 4). Then, sensors were deactivated for 10 minutes with 1 mol/L ethanolamine‐HCl (pH 8.5). Sensor 1 was incubated with recombinant human VEGF2.3The polyclonal sheep anti‐human NRP1 antibody was raised against and affinity‐purified with full‐length NRP1. The monoclonal mouse anti‐human NRP1 was also raised against full‐length NRP1, and purification was done via caprylic acid/ammonium sulfate precipitation.2.4Plates were coated with 150 µL polyclonal sheep anti‐human NRP1 antibody overnight at 4°C. After aspiration, wells were blocked with blocking solution for 3 hours at room temperature. Then, wells were aspirated and dried. For calibration of the assay, sNRP1 isoform 2 (0.375‐12 nmol/L), expressed in a mouse myeloma cell line, was spiked into sNRP1‐free human serum matrix, that was generated by immunoaffinity chromatography depletion with a polyclonal anti‐human NRP1 antibody. Samples, control sera, and calibrator (10 µL) were pre‐treated with the chaotrope reagent guanidine hydrochloride (GuHCl). Briefly, samples were pre‐diluted 1:2 with GuHCl with a final concentration of 3 mol/L and incubated for 30 minutes at room temperature followed by 1:10 dilution with protein‐based assay buffer. Wells were pre‐filled with 50 µL protein‐buffered assay buffer. Thereafter, 50 µL of the pre‐treated samples, controls, and calibrator were pipetted together with 50 µL of the biotinylated mouse monoclonal anti‐human NRP1 detection antibody to the coated and blocked wells. After a two hours incubation, wells were washed for five times with wash buffer and 150 µL of the conjugate solution consisting of horseradish peroxidase‐labeled streptavidin was added for 1 hour to allow complex formation with the biotinylated detection antibody. After subsequent washing, 150 µL of the substrate solution was added. The enzymatic reaction catalyzing the color change of the substrate was stopped after 30 minutes incubation in the dark by adding 50 µL stop solution. The absorbance was measured with a microplate reader (BioTec) at 450 nm with an absorbance correction at 630 nm. A dose‐response curve of the absorbance vs the concentration of the calibrator was generated with a 4PL algorithm. The concentration of samples was calculated from the dose‐response curve. Our assay has been commercialized by Biomedica (Bi‐20409).2.5The influence of varying concentrations of GuHCl on sNRP1 quantification in samples was tested by pre‐treating human sera from different human donors with a final concentration of 3 mol/L, 1 mol/L, 0.15 mol/L, 0.05 mol/l, and 0 mol/L GuHCl. Further, eight serum samples were measured without GuHCl pre‐treatment and with varying incubation times of the detection antibody. Another three sera were pre‐treated with a final concentration of 3 mol/L GuHCl before measurement with varying detection antibody incubation times. Differences of NRP1 concentrations were analyzed with Wilcoxon rank testing.2.6Validation was performed according to ICH guidelines. For the calculation of the lower limit of quantification (LLOQ), standard 2 (0.375 nmol/L) was diluted in twofold steps in protein‐based assay buffer and measured in five replicas per dilution. To calculate intra‐assay precision, seven serum samples were measured in replicates of five by one operator within one kit lot. For inter‐assay precision, seven serum samples in nine replicates within three kit lots were measured by three operators. To analyze assay specificity, samples from four different matrices  were tested with and without prior addition of a molar surplus of coating antibody acting as competitor. The accuracy of the assay was determined by spiking 6 nmol/L recombinant sNRP1 into serum, EDTA plasma, heparin plasma, and citrate plasma. Dilution linearity was assessed by diluting serum, EDTA plasma, heparin plasma and citrate plasma 1 + 1, 1 + 3 and 1 + 7 in protein‐based assay buffer. Stability of endogenous sNRP1 was determined by storing samples at different conditions . Further, freeze‐thaw stress was applied on endogenous and recombinant sNRP1 for one to five freeze‐thaw cycles.2.7® Analyzer Software based on 16‐bit tiff files. Median intensities of duplicates were averaged, and intensities with variations >40% were zeroed.The sequence of human sNRP1 was covered by 623 synthetic linear 15mer peptides with an overlap of 14 amino acids. Peptides were printed on a microarray in duplicates (Pepperprint GmbH). After blocking, the array was incubated overnight with 1 µg/mL of coating or detection antibody. Bound antibodies were traced with donkey anti‐sheep IgG or with goat anti‐mouse IgG, respectively, both labeled with DyLight680. Arrays were scanned on a LI‐COR Odyssey Imaging System. Quantification of spot intensities was performed with PepSlide2.8A three‐dimensional model of mouse sNRP1 obtained by X‐ray diffraction (PDB ID: 4GZ9) was used to visualize the domains a1a2b1b2, the linear epitopes identified by microarray analysis, and the binding sites of three NRP1‐binding molecules SEMA3A, with the2.9165 (Enzo Life Sciences).Recombinant human sNRP1 (1.5 nmol/L) spiked in sNRP1‐depleted serum matrix was tested in the novel sNRP1 ELISA with and without GuHCl sample pre‐treatment. The sample was further tested with and without prior addition of a molar surplus of the NRP1 ligands SEMA3A (R&D Systems) or VEGF‐A165 with sNRP1 binding of the monoclonal anti‐human NRP1 detection antibody employed in the sandwich ELISA were further tested in bio‐layer interferometry measurements. First, dissociation rates of these two NRP1 ligands were tested. Therefore, biotinylated sNRP1 (4 µg/mL) diluted in PBS was loaded to two streptavidin sensors (ForteBio). Sensors were then either incubated for 10 minutes with 10 µg/mL VEGF‐A165 or SEMA3A. Dissociation was performed in PBS for 15 minutes. To further investigate if SEMA3A or VEGF‐A165 binding to sNRP1 interferes with antibody binding, four additional sensors were loaded with sNRP1 as described. Two sensors were then either incubated with 10 µg/mL VEGF‐A165 or SEMA3A for 10 minutes, while two other sensors were incubated with PBS alone acting as reference. After a one‐minute stabilization phase, association of the monoclonal antibody (2 µg/mL in PBS) was performed for 10 minutes for all four sensors, followed by a 15 minutes dissociation phase.Interferences of SEMA3A or VEGF‐A2.10P < 0.05 were considered as statistically significant.Statistical analyses were carried out with GraphPad Prism 6 software. Values of 33.1165. VEGF‐A165 stably bound to sensor‐immobilized sNRP1 but was completely removed after regeneration with a high concentration (3 mol/L) of the strong chaotrope GuHCl  of GuHCl prior to testing influenced sNRP1 levels. In detail, when 3 mol/L GuHCl was used for pre‐treatment, measured sNRP1 concentrations were 6.3‐fold higher compared to sNRP1 concentrations in samples that were untreated (0 mol/L) or treated with 0.05‐1 mol/L GuHCl Figure A. Furthe3.2The human sNRP1 sandwich ELISA uses a polyclonal sheep anti‐human NRP1 antibody for capturing sNRP1 and a labeled monoclonal mouse anti‐human NRP1 antibody for detection. The calibrator sNRP1 isoform 2 defines the dynamic range of the assay between 0.375 nmol/L and 12 nmol/L. The quantification limit (LLOQ), as the lowest concentration with a percent CV of <25% and a percent backfit  between 75% and 125%, was defined as 0.09 nmol/L. The CV of intra‐assay precision ranged from 3%‐10% (mean: 6%) and from 8%‐15% (mean: 10%) for inter‐assay precision Table . To deteThe assay parameters met the international standards of acceptance according to ICH guidelines.3.3The full extracellular domain of human NRP1 is not resolved so far; therefore, a three‐dimensional X‐ray model of mouse NRP1The homology of NRP1 to other human proteins is low. According to BLAST analysis, highest sequence identity of 53% was found for NRP2 isoform 6. The non‐neuropilin hit with the highest identity of 40% was found for neuropilin and tolloid‐like protein 1 (NETO1). However, the NRP1 epitope of the monoclonal antibody ,165 with a high concentration of the strong chaotrope GuHCl completely removed VEGF‐A165 from sNRP1. To enable interference‐free quantification of sNRP1 in our ELISA, we included this finding and developed a sample pre‐treatment method based on 3 mol/L GuHCl.Interestingly, during ELISA development we found out that our assay setup was indeed affected by an unknown interferant. This interferant was shown to partially overlap with antibody binding sites as low sNRP1 concentrations could still be measured. In fact, it was the epitope of the monoclonal detection antibody that was affected as longer incubation of the detection antibody with the sample displaced the factor and released sNRP1. To simulate this disturbing factor, we used the NRP1 ligand with strongest reported affinity, VEGF‐A165, which has a higher affinity to NRP1 than SEMA3A.165 was resolved by crystallization.Although multiple NRP1 ligands are described, binding sites on NRP1 are only known for few ligands. The first NRP1 ligand that was identified was SEMA3A,165 binding to sNRP1 almost completely prohibited monoclonal antibody binding, which indicates for steric hindrance of the monoclonal antibody due to sNRP1‐VEGF‐A165 complex formation. Consequently, the majority of VEGF‐A165‐bound sNRP1 would not be detected in the sandwich ELISA without GuHCl pre‐treatment. SEMA3A, which binds to sNRP1 in close vicinity of the monoclonal antibody epitope, also inhibited antibody binding. However, the fast dissociation of SEMA3A from sNRP1 may allow the monoclonal antibody to bind and further displace SEMA3A from sNRP1, which may account for the time‐related sNRP1 increase in endogenous samples without GuHCl pre‐treatment.We could show that high‐affinity VEGF‐AIn conclusion, we could demonstrate that it is possible to release sNRP1 from a distracting factor in the sample matrix by treating the samples with the chaotrope GuHCl. The implication of this procedure into the ELISA protocol of the described sandwich ELISA now allows quantification of total sNRP1, that is, free as well as separated sNRP1 that was ligand‐bound before. Total sNRP1 measurement discriminates our assay from the R&D Systems Quantikine Human Neuropilin‐1 ELISA. The R&D assay is described by the manufacturer to be unaffected by interferences as tested molecules like NRP2, SEMA3A, SEMA3E, and Plexin A4 did not interfere with the assay performance. However, this minimal selection of competitors did not even include the main NRP1 ligand VEGF‐A, which makes a general exclusion of interferences impossible.Beyond this present example, interferences are a frequent problem in the quantification of ligands that circulate with concentrations in a low molar range. The described method may therefore help to overcome such obstacles also in the development of other immunoassays.