Harmony Search (HS) and Teaching-Learning-Based Optimization (TLBO) as new swarm intelligent optimization algorithms have received much attention in recent years. Both of them have shown outstanding performance for solving NP-Hard optimization problems. However, they also suffer dramatic performance degradation for some complex high-dimensional optimization problems. Through a lot of experiments, we find that the HS and TLBO have strong complementarity each other. The HS has strong global exploration power but low convergence speed. Reversely, the TLBO has much fast convergence speed but it is easily trapped into local search. In this work, we propose a hybrid search algorithm named HSTLBO that merges the two algorithms together for synergistically solving complex optimization problems using a self-adaptive selection strategy. In the HSTLBO, both HS and TLBO are modified with the aim of balancing the global exploration and exploitation abilities, where the HS aims mainly to explore the unknown regions and the TLBO aims to rapidly exploit high-precision solutions in the known regions. Our experimental results demonstrate better performance and faster speed than five state-of-the-art HS variants and show better exploration power than five good TLBO variants with similar run time, which illustrates that our method is promising in solving complex high-dimensional optimization problems. The experiment on portfolio optimization problems also demonstrate that the HSTLBO is effective in solving complex read-world application. With the scientific and social progress, new complex problems are more and more encountered in the fields of science and engineering. Especially, many high-dimensional optimization problems in engineering design, production scheduling and scientific calculation need urgently to be solved with high performance and high efficiency, for which there are three challenges: the first one is the very large search space owing to the very high dimensional problems , which makes the enormous computation burden; the second one is the large number of modals (multi-extremum points), which makes the search algorithm be easily trapped into local search; the third one is the particularity of optimization problem that may be discontinuous, non-differentiable and even have no objective function, for which the traditional mathematical optimization algorithms are powerless due to the requiring of substantial gradient information. Therefore, it is of great challenge to discover the globally optimal solution in an efficient time for solving a complex multimodal optimization problem with more than 1000 dimensions, possibly infinite number of local minima and non-differentiable.To address complex optimization problems, the swarm intelligent algorithms, mimicking the collective behavior of decentralized, self-organized systems, natural or artificial, have received much attention in recent years, most of which are nature inspired, such as genetic algorithm (GA) inspiredBoth Harmony Search (HS) –7 and TeIn the case, several HS variants –25 and mCommonly, to solve multimodal optimization problems that have one globally optimal solution and many local minimum (maximum) values by employing swarm intelligent algorithm, the overarching goal is to effectively implement the balance between exploration power and exploitation power, where the mission of exploration is to discover the unexplored regions at the early stage of search process, and the exploitation aims to obtain high precision optimal solution in a known region that has been found in the exploration stage. As a consequence, balancing the exploration and the exploitation is very important for solving a high-dimensional optimization with multimodality. Generally, the exploration power is strongly required before discovering the region in which the globally optimal solution is contained inside, however, after the globally optimal region has been found, the exploitation power should be intensified immediately and the exploration power is degraded gradually.To address the balance, in this study, we propose a hybrid optimization algorithm (HSTLBO) based on HS and TLBO, in which a self-adaptive selection strategy is designed to balance the exploration power and the exploitation power. At the early stage of search, the HS algorithm obtains higher probability for exploring the region that contains the globally optimal solution. When the globally optimal region might have been located, the TLBO algorithm begins to obtain higher probability at the later stage of search process for intensifying the local search and exploiting high precision solution. In the HSTLBO, a self-adaptive selection probability is used to choose HS or TLBO in terms of the population diversity and update-success-rate. The update-success-rate denotes the proportion that the new generated solutions are superior to the old ones, which mean the rate that a new generated solution can successfully replace the old one in one generation.The rest of this paper is organized as follows: Section 2 introduces the HS and TLBO algorithm. Hybrid HSTLBO algorithm is proposed and self-adaption selection strategy is analyzed in Section 3. In Section 4, the numerical experiments on twenty complex benchmark test functions are performed, the results are analyzed by comparing and statistical test. And the convergence of HSTLBO is also investigated. In Section 5, HSTLBO is used to solve portfolio optimization problem. Section 6 concludes this work.Some symbols are explained as NP  --The population size which is equal to HMS.max --The maximum evaluation times of objective function.Tt   --The current iteration times.SR  --the selection rate that new generated harmony insteads of the worst harmony.T   --The cycle length for recalculating the SR.r   --An uniform distributed random number between 0 and 1.th iteration.c1/ c2 --The times of updating old solutions successfully of HS/ TLBO in the tX consists of D decision variables , D denotes the dimensionality (the number of decision variables) of the optimization problem, xi  represents the ith decision variable. xi.HS algorithm mimics the process of improvising a musical harmony, in which X denotes the harmony, xi indicates the note of harmony, D is the number of notes in a harmony. Harmony memory (HM) contains HMS harmonies: {X1, X2, …, XHMS}, where In standard HS, three operators  are employed to optimize the harmonies in HM (population), which are good at exploring new region of search space . HoweverX1, X2,…, XNP} is composed of one teacher and some learners, where j = 1, 2,…, NP)  see  denotes Xj,new and Xj,old denote the ith learner’s learning status after and before learning from teacher Xteacher, TF is teaching factor, r is a uniformly distributed random vector in the range . M denotes the mean knowledge level of all learners.In the teacher phase, the learners increase their knowledge depending on the teacher who tries to improve the mean ability of all learners. The teaching operator is as follow,r(r ≠ j) is a random integer in the range .In the learner phase, each learner increases knowledge depending on communicating with other learners. The learning operator is as follow,To balance the exploration power and the exploitation power during the searching process, we propose a complementary HSTLBO algorithm.The flow chart of HSTLBO algorithm is as In the HSTLBO algorithm, we merge HS and TLBO together to compensate for each other’s deficiencies, where the HS is mainly used for exploring the search space, and the TLBO aims to speed up the exploitation process. In the search process of HSTLBO, the HS and the TLBO compete for the opportunity in each iteration according to a self-adaptive selection rate (SR) see . The selFor a new unknown problem, exploring the unknown space in the beginning stage is the first consideration for the HSTLBO algorithm, for which the HS is a good choice. As a consequence, a high selection rate SR (≥ 0.95) is assigned to HS algorithm for exploring unknown regions in the first half of search. In the remaining time, the value of SR will continue to adapt the population status. If the global region has not been found before or the space distribution of population is still extensive, the HS might be still able to obtain a high SR for exploring the unknown areas. However, if the HS obtains no or very low selection rate, , the diversity of population might be lost quickly for high-dimensional optimization problems owing to the quick convergence of TLBO. Therefore, to keep the diversity of population in a certain level, in the second half of stage, the HS is also given more than 0.3 probabilities to run.In the hybrid HSTLBO algorithm, both HS and TLBO are modified as follows.In the modified HS algorithm see , all theSP for adjusting. The formula of SP is expressed in SP is adjusted dynamically according to current iteration t, which is introduced in DIHS [Standard HS algorithm produces a new harmony in which each element (decision variable) is generated based on three HS rules . In our proposed algorithm, the producing process of new harmony is similar to that of DIHS . Dynamic in DIHS .PAR and fw are constant values. The modified HSTLBO algorithm adopts dynamic strategies to change PAR and fw for balancing the exploration power and the exploitation power ))PAR and w in IHS . The ove(see) is as follows.The primary difference between standard TLBO and modified TLBO see is as foM is equal to the mean value of learners X. In modified TLBO, it is a combination vector in which each subject M(i) is randomly chosen from the ith subject of all learners.In standard TLBO, In each iteration. TLBO performs teacher phase and learner phase, respectively. The modified TLBO only randomly chooses either teacher phase or learner phase to perform.Xnew are produced by learning from teacher or other one learner. Whereas, in modified TLBO, only a portion of dimensions of Xnew are generated by learning from teacher or other one learner, and other dimensions inherit from Xold directly, which is because an excellent learner is also imperfect on some subjects, selective learning on parts of subjects is more effective for improving knowledge level of learner than learning all subjects from one learner. The selection probability SP is shown in In standard TLBO, all dimensions of As we known, in our real lives, selective learning from multiple excellent learners on some subjects is more effective for improving our knowledge level than learning all subjects only from one excellent learner. As a consequence, in the modified TLBO, the learner on each subject will select one other learner from population for better learning new knowledge.To investigate the performance of proposed HSTLBO algorithm, numerical simulation experiments on twenty benchmark functions –35 are tTwenty well-known functions are listed in 1-F6, F8, F12-F20) and 4 complex uni-modal problems , 4 hybrid functions (F17-F20) and 10 shift functions (F9-F18).In the simulation experiment, all the test programs were performed on Windows XP 32 system with Intel(R) Core(TM) i3-2120 CPU@3.30 GHz and 4 GB RAM, and all the program codes were written in MATLAB R2014b.f(Xbest)−f(X*)|) (Prec), standard deviation of the precision (Std dev) and mean run time (Mtime) for each function are calculated over 20 independent runs, where Xbest is the best solution in population when the terminate condition is meet, and X* is the global optimal solution.In our simulation experiments, the dimension of test functions is set to 1000 and each function is run independently 20 times with 5E+6 function evaluations (FEs) as the termination condition. The precision  are employed to investigate the balance between the exploration power and the exploitation. During the search process, we trace the changes of population diversity of each compared algorithm.Three complex multimodal optimization problems (FThe http://en.wikipedia.org/wiki/Nikkei_225) and compare the test results of HSTLBO with four intelligent algorithms [To further investigate the performance of HSTLBO algorithm, complex portfolio optimization problem are employed to test the ability of solving real-world application. The portfolio optimization aims to choose the optimal proportions of various assets for obtaining maximum portfolio return with minimum risk. In this work, we apply HSTLBO algorithm to choose the optimal portfolio proportions for Nikkei 225 stock index that maps companies on the Tokyo Stock Exchange (TSE) –40. We e, TS, SA),41.http://people.brunel.ac.uk/~mastjjb/jeb/orlib/portinfo.html and the experiments are performed on two conditions:The test data is from Unconstraint. The portfolio proportion and desired number of investment assets are not constrained.Constraint. The portfolio proportion of each asset K = 10.In this work, we employ the same method of constraint handle as the literature  which caThe test results on three evaluation indexes  are presented in Both Harmony Search and Teaching-Learning-Based Optimization are new swarm intelligent optimization algorithms, which have got much attention in recent years. In this work, in order to improve the performance of HS and TLBO, a hybrid HSTLBO algorithm is presented.In the HSTLBO, both HS and TLBO are improved to enhance the global search ability. A self-adaptive selection strategy is presented to balance the exploration power and the exploitation power. At the early stage of search process, the HS algorithm gets a higher opportunity than TLBO, which aims to explore the unknown regions and avoid lose the globally optimal solution. With the increasing number of iteration, the opportunity of TLBO is raised step by step. At the later stage of search, when the population has gathered into one region which maybe contains the global optimal solution, the TLBO has obtained much more opportunity for exploiting high precision optimal solution.The experimental results also demonstrate that our method is a promising optimization algorithm in solving large scale and complex optimization problems.


The global burden of depression and stress-related mental disorders is substantial, and constitutes a major need for effective rehabilitation. Can nature-based rehabilitation help people return to work? Objective: To study if the length of a nature-based rehabilitation program affects the outcome with regard to return to work one year after the onset of the program, in a group of patients with long-term reactions to severe stress and/or depression. Methods: A prospective, quasi-experimental study comparing results from 8-, 12-, and 24-week periods of rehabilitation. The rehabilitation of 106 participants was carried out by a multimodal rehabilitation team in a specially designed rehabilitation garden. Return to work data were collected before the intervention and one year after the start of rehabilitation. In addition, data were collected regarding self-assessed occupational competence, personal control, and sense of coherence. As many as 68% of the participants returned to work or participated in job training or work-oriented measures, full- or part-time, after one year. Participants with a longer period of rehabilitation reported better results on occupational competence, and were more likely to participate in paid work, full-time or part-time, one year after rehabilitation. Study outcomes indicate that a longer rehabilitation period in a rehabilitation garden increases the possibility of a return to paid work. The global burden of mental disorders is substantial ,2. In SwThere is increasing scientific evidence that the natural environment can relieve symptoms of stress and improve recovery from mental disorders ,12,13,14There is a long tradition of the use of nature-based therapy and rehabilitation in healthcare; horticultural therapy (HT) is one example . Since tThere are a number of theories available that offer an explanation of why nature-based interventions are good for mental health. For a long time, the health effects of staying in natural landscapes and urban parks were linked to daylight, fresh air, and physical activity. Such theories are still current . AttentiThe Supportive Environment Theory (SET)  is a bioA number of nature-based interventions have been designed specifically for the rehabilitation of stress-related mental disorders and/or depression, and the results have generally been shown to be good concerning outcomes related to stress-reduction, the ability to focus attention ,36,37,38The Alnarp Rehabilitation Garden at the Swedish University of Agricultural Sciences (SLU) campus outside Malmö, in southern Sweden, has been specially designed to offer rehabilitation programs for people with stress-related psychiatric disorders and/or depression . A signiThe program at the Alnarp Rehabilitation Garden was designed according to the Supportive Environment Theory . Our maiThus, the main aim of this study was to examine return-to-work rates one year after participating in a rehabilitation program with different durations at the Alnarp Rehabilitation Garden. In addition, we examined self-assessed occupational competence, personal control, and sense of coherence before and after nature-based vocational rehabilitation.The Alnarp Rehabilitation Garden has a framework agreement with the Swedish Social Insurance Agency stipulating a fixed cost per treatment day. This agreement made it possible to offer three different lengths of rehabilitation programs: 8 weeks, 12 weeks, and 24 weeks. If any participant needed additional psychotherapy or physical therapy after this time, this could be arranged at the Rehabilitation Garden. Since this type of intervention is defined as vocational rehabilitation, decisions on program length were made by the social insurance officers who recommended patients for the program, rather than those in the health care system who were otherwise responsible for the patients’ medical treatment. A social insurance officer is an official who manages the payment of sickness compensation from the Swedish Social Insurance Agency. The official is not medically trained. Some officials decided that their patients should receive 8 weeks of rehabilitation in the garden, while others opted for periods of 12 or 24 weeks. The duration of the program was not determined by the level of sickness of the participants. Everyone was considered healthy enough to participate in a vocational rehabilitation. The rehabilitation program was provided in addition to any possible ongoing treatment with medication.The social insurance officers referred patients who were on long-term sick leave and had a diagnosis of reaction to severe stress and/or a depressive episode. Each participant took part in rehabilitation for the amount of time ordered. No randomization was performed. This type of study can be compared to a natural experiment ,43. The Participants who met the following inclusion and exclusion criteria were considered eligible for the study, and could be referred through the framework agreement: (1) being on sick leave for at least three months due to a diagnosis of reaction to severe stress and/or a depressive episode, defined as having an International Statistical Classification of Diseases  within tA total of 118 participants were referred to the rehabilitation program. All participants were on 100% sick leave. Most of them (87 participants) had not been able to work for more than two years. Before entering the rehabilitation program, the participants received written and oral information about the study, and had an opportunity to ask questions about the study procedure before signing informed consent. The participants could withdraw from the study at any point without this influencing their relationship with their therapist. The study was approved by the Research Ethics Committee at the Medical Faculty of Lund University (LU 107-02 and EPN Lund 2011/31).A psychiatrist who is an expert in stress-related psychiatric disorders and works part time in the Alnarp Rehabilitation Garden Program examined all participants in the rehabilitation program in order to reconsider or confirm their diagnoses before they were included in the study . This waOf the 110 participants who fulfilled the inclusion criteria, 4 were excluded from further analysis and comparisons since they exceeded retirement age (65 years) prior to the 1-year follow-up, and it was therefore not possible to analyze them in terms of return to work. This left us with a total of 106 participants (96 women/10 men). Participants who were excluded from the study could continue the rehabilitation in the garden even though they were not included in the study.Participants were aged 45.7 years on average (range 22–63), and had been suffering from their disorder for an average of 3 years and 7 months (CI 3.3–4.1 years). Sixty-four percent were mid-level officials who had received education at the university-level or equivalent, such as teachers and nurses, while eleven percent were high-ranking officials or employees with even greater levels of academic education, such as medical doctors and lawyers.The study was conducted in the following steps : all parAll participants were followed during the period prior to the one-year follow-up. This included meetings at Alnarp every three months. At the final meeting, the participants were asked to answer a set of questions concerning their rehabilitation. All self-assessment questionnaires were filled out one week before the start of the intervention and one week after its completion, with the exception of the return-to-work questionnaire, which participants answered one year after the start of rehabilitation. All participants returned the questionnaires relating to background issues.Questions during final meeting: Some of these questions related to the participants’ possible needs for additional rehabilitation, such as consultations with psychotherapists or physical therapists, before the one-year follow-up. Twenty-one of the participants sought and received additional rehabilitation. All of these additional consultations took place at the Alnarp Rehabilitation Garden. For two of the participants, this rehabilitation involved psychotherapy only, and for three it involved both psychotherapy and physical therapy. All five psychotherapy consultations involved a single visit to the psychotherapist. Sixteen participants took part in physical therapy only. There were usually five visits to the physical therapist before the one-year follow-up (range 1–12).The Alnarp Rehabilitation Garden is a clinical entity within the Swedish University of Agricultural Sciences (SLU) that is dedicated to the research and development of nature-based rehabilitation interventions. This clinical care unit only accepts patients participating in SLU-related research projects. The 2-hectare garden  is locatThe garden design is based on the different theories mentioned above , as well as on earlier environmental psychological theories, such as the theory of Savannah-like environments , the ProA psychiatrist met with the participants in connection with program registration, again after a couple of weeks’ participation, and at the end of the intervention. An individual rehabilitation plan was designed for each participant. Rehabilitation took place 4 days a week—Monday through Thursday—for 3½ hours per day. The treatment was based on gardening and horticulture activities, with the occupational therapist and the landscape engineer supervising participants. Activities were changed with regard to season and weather, and we have not found any noticeable differences regarding the effects of the treatment in relation to the seasons . Each reSelf-rated return to work (RTW). The participants were asked to complete a form regarding their work situation at the start, and one year after the start, of rehabilitation. A questionnaire was mailed to them, and was followed up with two reminders. This questionnaire was designed at SLU Alnarp, and has been used previously by Palsdottir et al. . The socThe question read: “What was your work situation on xx? (xx being one year from the date he/she started rehabilitation). As of this date, it is exactly one year since you started the rehabilitation program at Alnarp”.Paid work: “Paid work as employee”, “Self-employed”, “Actively applying for paid work as employee”, or “Student/studying”.Work training: “Vocational training in the workplace”.Vocational action: “Work-oriented measures by (or supported by) the Swedish Social Insurance Agency”.Still sick: “Sick-listed”.The participants were asked to answer according to their actual work situation in percent of full time  on the given date. The grades of occupational activities were further divided into the following categories:We used paid work as the outcome variable to compare with other variables , since paid work sets higher demands than other forms of occupation.Occupational competence: The Occupational Self-Assessment protocol (OSA) derives from the model of human occupation (MOHO), which has become standard among occupational therapists ever since it was first presented in 1980 . It is aPersonal control: The Mastery Scale measures how participants experience their personal control, or how they master situations in everyday life ,58. The Sense of coherence: The original version of the Sense of Coherence scale (SoC-29) was used in this study. SoC-29 is considered to be reliable and valid . Accordit-test analysis was used.To analyze variations regarding return to work rates between different rehabilitation programs, we used the Mantel–Haenszel Chi-Square test. To analyze changes regarding return to work and self-rated occupational competence, personal control, and sense of coherence before and after nature-based rehabilitation, a paired p-values < 0.20, were inserted into the logistic regression analysis [To analyze how different factors  affected the outcome of the intervention with regard to return to work, we used a stepwise binary logistic regression analysis to find the best explanatory model. Variables which in a bivariate Spearman correlation analysis were associated with return to work, with analysis ,66. In tdifferences between means, using Hedges’ g , where mdegree of association between binary variables, using odds ratio (OR) , where mWe calculated the effect size  based onForty-two participants (44%) had returned to full-time or part-time paid work after one year . Of thesFifty-three participants had not returned to paid work. Of these, 23 participants took part in some kind of job training in a workplace or other forms of work-oriented measures, such as customized job training. Thirty participants did not participate in any work-related activity one year after the intervention . After op < 0.05) percentage of full-time work (44%) one year after beginning the rehabilitation period compared to those participating in the 12-week (37%) and 8-week (20%) programs (p < 0.05).Patients participating in the 24-week program reported a significantly higher  including length of intervention  and OSA . The Hosmer and Lemeshow Test showed that there was no evidence of a lack of fit in any of these models.The analysis yielded a significant model . According to our main hypothesis, which is derived from the Supportive Environment Theory (SET), a longer nature-based rehabilitation program would increase the ability to return to work. This, in turn, would be due to improvements in occupational competence, personal control, and sense of coherence.Our results show that forty-four percent of the participants had returned to paid work, most of them part-time, and just under 15% were able to go back to full-time paid work. An additional 24% participated in some kind of job training or customized job training on a full-time or part-time basis one year after the nature-based rehabilitation program. Thus, a total of 68% of the entire group were back to some work-place-related activities. It should be noted that the participants had not worked or studied for an average of 3 years and 7 months.The effect of different intervention time periods was examined, and it was found that the longer the rehabilitation period, the higher the rate of return to the labor market. In the regression analysis, the length of the rehabilitation program was found to be the significant and decisive factor in predicting return to work. Sense of coherence, personal control, occupational competence, and background factors such as age and socioeconomic status did not predict return to work. These results indicate that the length of rehabilitation period does matter in a dose response manner and that eight weeks of therapy may be insufficient for most participants, which is consistent with SET.Recently, Shanahan et al.  reportedAlready in the 1990s, studies showed that high stress levels swiftly, within minutes, decreased when subjects experienced natural environments with restorative qualities . This woThe results show that the effect size increased the longer the rehabilitation program lasted as regards return to work and occupational competence (OSA). A vital part of the OSA protocol is the ability to focus attention, be able to plan, implement what has been planned to and decided to do, and maintain the structure of everyday life; that is, abilities associated with individuals’ level of executive function. Executive function is used, for example, to focus attention, inhibit distractions, prioritize and organize information, plan everyday life, and carry out what was planned . A suffiThe “Attention Restoration Theory” claims that human beings effectively can recover their ability to focus attention in natural environments . SeveralWhen the garden was laid out, the goals were to increase the participants’ occupational competence and opportunities to return to work. This was to be achieved by carrying out activities in a natural/garden environment together with therapists and a limited number of participants who were in a similar situation ,33. AccoThe results from this study show that the effect size increased the longer the rehabilitation program lasted as regards return to work and occupational competence (OSA). Self-assessed abilities in the OSA protocol deal with physical energy and capacity, executive functions, as well as with being able to socialize with other people and other coping capabilities. The result could be interpreted as follows: by staying regularly in Alnarp Rehabilitation Garden, participants can lower stress levels and increase their capacity of directed attention over a period of time. With the activities carried out in this natural/garden environment, the physical strength and fitness of the participants can be developed. Executive function is adversely affected by stress, lack of sleep, sadness, loneliness, and/or lack of physical exercise . ConsequA number of studies show that nature-based interventions have a positive impact on stress-related mental health and/or depression ,35,40,41Over 75% of those who received care at Alnarp had university educations, with the majority working in the health care or education sectors as nurses, teachers, psychologists, medical doctors, etc. These professions all require emotional commitment, which has proved to involve a higher risk of burnout and exhaustion . As suchThe study was not randomized, which is a limitation. In 1991, however, Andrews pointed out that the randomized controlled trial design is challenging to use in rehabilitation research, and that other strategies can provide equally safe or safer results . Graham The response rate on distributed forms was high. In general, the forms were returned to us. The study included 106 participants, and only 11 did not reply at one year follow-up. The self-assessment forms we received were completely or almost completely filled in. The number of participants in the Tables varies somewhat depending on which self-assessment protocol is reported. Using imputations would have increased the response rate, but we chose not to do this.The rehabilitation which is described in this article is not too different from horticultural therapy in the United States, UK, Germany, Japan, Korea, and several other countries. In Materials and Methods, the reader can find references that in great detail describe activities, programs, and the design of the garden. From this, others can carry out similar activities. One strength of this study is that the three groups were similar in terms of gender, marital status, age, socioeconomic status, primary diagnosis, length of illness, and baseline values for outcome measures.The outcomes from this study indicate that nature-based rehabilitation increases the likelihood of returning to work. In order to achieve a strong effect, however, the results suggest that the rehabilitation needs to be carried out over a longer period of time.


Thus, we hypothesize that the AR challenge may activate these inflammatory mediators in the hippocampus, which in turn contribute to the abnormal behavior and neurological deficits associated with AR.Allergic rhinitis (AR) is a major concern in personal and public health, which negatively affects emotions and behavior, leading to cognitive deficits, memory decline, poor school performance, anxiety, and depression. Several cellular and molecular mediators are released in the inflammatory process of AR and activate common neuroimmune mechanisms, involving emotionally relevant circuits and the induction of anxiety. Responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis to allergic processes have been reported, which may also include responsiveness of the hippocampus, cortex, and other brain regions. Here, we have used an optimized rat model of AR to explore whether the disease has a relationship with inflammatory responses in the hippocampus. AR was established in adult rats by ovalbumin sensitization, and the expression of various inflammatory substances in the hippocampus was measured by specific assays. Comparison between experimental and various control groups of animals revealed an association of AR with significant upregulation of substance P, microglia surface antigen (CD11b), glial fibrillary acid protein (GFAP), tumor necrosis factor- Allergic rhinitis (AR) is an IgE antibody-mediated immune reaction, in which expansion of the type 2 (TH2) subset of T cells , 2 causeβ in hypersensitivity reactions, which activates the hypothalamic-pituitary-adrenal (HPA) axis, stimulating the release of cortisol that in turn modifies serotonin release, leading to mood disturbances . Nasal mucosa tissues were examined by means of routine HE staining and light microscopy. Results showed that in the AR group of rats, the structural integrity of nasal mucosa tissue was impaired and the ciliated epithelium was irregular with disordered arrangement, exhibiting various degrees of fall-off and edema, and mixed with eosinophils and lymph cells  = 5.46, P < 0.05, P < 0.01, α expression was higher in the AR group compared to the control group  analysis, we wanted to determine whether inflammation in the hippocampus relates to AR. As shown , the expThe results presented here document an inflammatory response of the hippocampus in AR. Simultaneously, we observed higher levels of several proinflammatory mediators, which may relate to behavioral changes. Thus, we postulate that in an AR condition, the activated inflammatory response of the hippocampus may involve emotionally relevant circuits, which would then translate into abnormal behavior and negative emotions, mentioned earlier.α, and CD11b. The OX42-reactive CD11b surface marker is characteristic of brain-specific immune cells, specifically the microglia, and thus, its increased expression in AR rats confirms an immune response. In fact, along with SP and IL-6, the CD11b+ microglia population exhibited the most significant increase in the AR group compared to the control group. Moreover, there is a related report that allergy led to a reduced microglia presence and activity and to an elevated level of neurogenesis, such as those of DCX(+) cells and BrdU(+) cells in the hippocampus of allergic mice [To our knowledge, this is the first study to examine the inflammation mechanisms of hippocampus activity in nasal hypersensitivity of a live animal AR model. In this model, we demonstrated upregulation of a significant cohort of proinflammatory molecular markers that included both cytokines as well as glial surface antigen, namely, GFAP, SP, IL-6, TNF-gic mice .Nevertheless, previous studies reported that other brain regions such as the amygdala and hypothalamus are also responsive to respiratory allergies as measured by the expression of c-fos , 31. In α, IL-6, GFAP, and SP, together with microglial activation, follows the pattern of a Th2 cell-dependent allergic reaction, the exact mechanism of their increase or function in the hippocampus is unknown at this time. It is tempting to speculate that increases in these cytokines may provide the link between the hippocampus and behavioral changes. The recognition that AR is a clinical condition, capable of influencing behavioral responses, has far-reaching significance in preventing AR development. If AR is exacerbated or not controlled properly, it may cause brain cognitive and memory dysfunctions or other adverse behaviors such as attention deficit and mental disorders, to name a few. Thus, our study suggests that it is important to reduce the occurrence of AR and manage patients with AR for pathological as well as social and economic reasons.While the expression of TNF-Although this study provides important new information about the inflammatory response of the hippocampus in the AR process, it has some limitations. First, we did not evaluate emotion-related behavior in view of a lack of objective scores but rather used external manifestations in the AR rats, such as fur color, physical energy, activity, and food intake, none of which may accurately reflect emotion-related behavior. Second, the exact mechanisms underlying the observed hippocampus inflammatory response in the AR rats are likely to be more complex, and possibly other neuroimmune mediators involved in emotional changes remain to be elucidated. In the future, more elaborate and sophisticated experiments need to be considered and designed, such as the use of 24-hour video surveillance and objective assessment methods to monitor AR behavior, in order to further investigate the relationship between the hippocampus and AR. Objective behavioral assessments may include emotional behavior evaluations, including anxiety scores and depression scores. In clinical experiments, we can use multiple detection methods such as functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) to investigate the hippocampus of AR patient-related behavior, for instance, the potential depression- and anxiety-related disorders, memory loss, and cognitive impairment. Nevertheless, our findings open the door to novel insights into the inflammatory response of the hippocampus to allergic rhinitis in the rat model. Additional studies will certainly elucidate the complex neuroimmune relationship between the hippocampus and diverse emotional reactivity disorders. Lastly, our findings emphasize the importance of molecular behavioral studies for comprehensive awareness and prevention of AR. The impact of mental health disorders on society is substantial, especially in children with multiple allergic diseases, and implementing primary prevention activities may be warranted.


It is well known that β-adrenoceptors (β-ARs) play a critical role in emotional arousal and stressful events, but the specific contributions of the β2-AR subtype to the psychological disorders are largely unknown. To investigate whether β2-AR are involved in anxiety-like behavior and reward to addictive drugs, we conducted a series of behavioral tests on β2-AR knock-out (KO) mice. β2-AR KO mice exhibited increased preference for the dark compartment and closed arm in tests of Light/Dark box and elevated plus maze, indicating that β2-AR deletion elevates level of anxiety or innate fear. β2-AR KO mice also showed decreased immobility in tail suspension test (TST), suggesting that β2-AR deletion inhibits depression-like behavior. Interestingly, β2-AR ablation did not change basal locomotion but significantly increased locomotor activity induced by acute cocaine administration. β2-AR KO mice showed enhanced place preference for cocaine, which could be attenuated by β1-selective AR antagonist betaxolol. Consistently, β2-AR agonist suppressed cocaine-conditioned place preference (CPP). These data indicate that β2-AR deletion enhances acute response and reward to cocaine. Our results suggest that β2-AR regulates anxiety level, depression-like behavior and hedonic properties of cocaine, implicating that β2-AR are the potential targets for the treatment of emotional disorders and cocaine addiction. Anxiety and depression are the common types of psychiatric disorders, with high incidence and life-long prevalence  resulted in increased anxiety in the light-dark box and elevated plus maze tasks, decreased depression-like behavior in tail-suspension test (TST), and elevated cocaine-induced rewarding effects in locomotion and conditioned placed preference tasks. Our data suggest the necessity of β2-AR in control of anxiety and response to cocaine.ad libitum. Treatments were strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Animal Care and Use Committee of Shanghai Medical College of Fudan University. The male mice with 8–10 weeks’ age were used for all behavioral tests. The behavioral tests were performed in the following orders: open field (OF), light/dark box and elevated plus maze tasks were carried out with the first cohort of mice . TST and forced swimming test (FST) were taken out with the second cohort . The third cohort  were submitted to locomotion tests induced by acute cocaine administration and the forth cohort  were submitted to cocaine conditioned place preference  and backcrossed onto a C57BL/6J background. Experiments were carried out in wild type (WT), HET and KO littermates. All animals were housed with a reversed 12 h light/dark cycle and with access to food and water available Cocaine hydrochloride  was dissolved in 0.9% saline at 2 mg/ml for mouse CPP, 4 mg/ml for locomotor response test. Betaxolol , a selective β1-AR antagonist, was dissolved in saline at 2 mg/ml and administered at a dose of 10 mg/kg . The protein concentration was determined by BCA assay . Equal amounts of total protein (30 μg) isolated from cell membrane of the hippocampus and mPFC from WT and β2-AR KO mice were loaded on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred to polyvinylidene difluoride membranes . Then the membranes were incubated in primary antibody for β1-AR , β2-AR  or β-tubulin  at 4°C overnight. The membranes were then incubated with corresponding secondary antibody  for 2 h after washing in TBST for three times. Protein bands were visualized using odyssey (LI-COR Biosciences). The immunoblots were analyzed with Image-Pro Plus to measure the optical density of the bands of β1-AR and β2-AR. Data of β-AR protein levels for WT and KO mice were expressed as percentage of the averaged values of β-tubulin.Mice brains were removed on ice, hippocampus and medial prefrontal cortex (mPFC) were rapidly dissected and the tissues were prepared as following. Briefly, brain samples were homogenized in ice-cold 0.32 M sucrose, 5 mM HEPES (pH 7.4), 0.1 mM EDTA, and protease inhibitors mixture in a glass homogenizer. Homogenates were centrifuged , and the supernatants were spun at 14,000× g for 30 min in a centrifuge at 4°C. Then the pellets were resuspended in 200 μL of 1× P buffer  and were allowed to explore the arena freely for 30 min. Total distance, distance and duration in the center area, entrance times to the center area were analyzed using OF Activity Software package v. 4.0 . Testing was conducted with 25 Lux illuminance in the chamber. In the test for locomotor activity induced by acute cocaine administration, mice were placed in the OF chamber for 30 min followed by another 60 min after an injection of cocaine (20 mg/kg i.p.).Three days prior to locomotor activity assessments, mice were habituated to the testing room for 30 min each day. During the test, mice were released from the center of an OF test chamber (20 × 20 cmThe light/dark box (46 × 27 × 30 cm) is composed of two compartments: the dark compartment (one third of the box) and the light compartment (two thirds of the box). Testing was conducted with 25 Lux illuminance in the light box  with adhesive tape from a horizontal bar and were positioned such that the base of their tails was vertical to the bottom of the bar. The tasks were taped by a digital video camera for 5 min. Immobility was analyzed with the Tail Suspension Software (CleverSys). Mice were considered immobile only when they hung passively and completely motionless.FST is another widely used model for assessing the antidepressant-like activity in mice. Mice were gently released into a transparent Plexiglas cylinder  filled with water  for a 5 min session (under <500 lux ambient light). Immobility  was scored with the ForcedSwimScan software (CleverSys). Each mouse was judged to be immobile when it ceased struggling and remained floating motionless in the water, making only those movements necessary to keep its head above water as previous reported  ANOVA followed by the Bonferroni’s p < 0.001, β1-AR: p = 0.732; Figure p = 0.024, β1-AR: p = 0.614, t-test).In general, β2-AR KO mice showed no genotype-dependent differences in body weight, no obvious developmental abnormalities, or deficits in touch, vision and hearing compared with their WT littermates. To verify the deletion of β2-AR, the hippocampus and mPFC were dissected from β2-AR KO mice and WT mice for Western blot analysis Figures . The resF = 10.638, p < 0.001, one-way ANOVA; Figure F = 7.322, p = 0.001, one-way ANOVA). The results from both tests consistently showed that the anxious state and innate fear were increased by deletion of one or two copies of Adrb2 and sufficient β2-AR expression might have the anxiolytic effects.In the L/D box and EPM tasks, both β2-AR KO and HET mice spent significantly less amounts of time in the light compartment and the open arms than WT mice  = 6.315, p = 0.004, one-way ANOVA), however, immobility time during each 5-min were indistinguishable of the test  = 1.344, p = 0.255, two-way RM ANOVA). In FST, immobility duration in total or each 5 min was comparable in β2-AR KO, HET and WT mice  = 1.137, p = 0.33, one-way ANOVA; Figure Fgenotype × time = 1.356, p = 0.219, two-way RM ANOVA). These results suggest that behavioral despair is suppressed by β2-AR ablation in TST.Next, the performance of β2-AR KO mice was assessed in TST and FST, two behavioral assays widely used to examine behavioral despair in mice  = 1.506, p = 0.229, one-way ANOVA; Figure Fgenotype × time = 1.506, p = 0.229, RM two-way ANOVA). β2-AR KO mice did not show difference in time spent in the center of the chamber and distance traveled, or number of entries into the center of the OF chamber from HET and WT mice  = 2.857, p = 0.064; F = 0.680, p = 0.510; F = 0.863, p = 0.462; one-way ANOVA). These results indicate that β2-AR KO does not change locomotor activity.To assess the locomotor activity in β2-AR KO mice, the OF task was performed. The data showed that the total distances traveled by β2-AR KO mice, HET and WT littermates during both the entire test period and each 5-min were indistinguishable  = 7.604, p = 0.003, two-way RM ANOVA) during a 60-min period following cocaine injection in the OF test. Two-way RM ANOVA revealed a significant genotype by time interaction and Bonferroni’s post hoc revealed that acute cocaine treatment induced an enhanced locomotor activity in β2-AR KO mice, the increase in locomotor activity primarily took place during the first 35 min of the test  = 4.132, p < 0.001). The results indicate that ablation of β2-AR enhances response to acute cocaine administration.Acute cocaine  administration significantly increased total distance traveled in WT, HET and β2-AR KO mice, but β2-AR KO mice displayed significantly higher locomotor activity than WT and HET mice  = 3.827, p = 0.03), and Bonferroni’s post hoc revealed a significant higher preference for the cocaine-paired side developed in β2-AR KO and HET mice compared with WT mice, indicating β2-AR KO increased rewarding properties of cocaine. To further verify the role of β2-AR in cocaine rewarding effect, clenbuterol (5 mg/kg), was administrated 30 min before each cocaine conditioning session  = 7.439, p = 0.012, two-way RM ANOVA). With regard to the normal expression level of β1-AR in β2-AR KO mice, the role of β1-AR in cocaine-CPP was tested. Betaxolol (10 mg/kg) was given 30 min before cocaine conditioning daily  = 4.176, p = 0.048, two-way RM ANOVA; Figure Ftreatment × session = 4.667, p = 0.039, two-way RM ANOVA). The above results indicate that β2-AR might suppress rewarding effects induced by cocaine, producing effects opposite to the function of β1-AR.In the task of cocaine-CPP, all groups showed preference for cocaine-paired side under the dose of 10 mg/kg cocaine Figure . Two-wayn Figure . CPP scoy Figure . We founTo investigate the role of β2-AR in stressful or emotional behaviors, a battery of behavioral tests was performed on β2-AR KO mice. In this study, the β2-AR KO mice showed increased preference for the dark compartment and closed arm in L/D and EPM tests. β2-AR KO mice also showed decreased immobility in TST. Interestingly, β2-AR deletion did not change basal locomotor activity, but significantly increased locomotor activity induced by acute cocaine administration and greatly enhanced preference for cocaine, which was significantly attenuated by betaxolol, the selective β1-AR antagonist. Consistently, the β2-AR agonist disrupted preference for cocaine. Our results suggest that β2-AR play pivotal roles in regulating anxiety, depression-like behaviors and reward induced by cocaine.β2-AR are widely distributed in the brain, including frontal and piriform cortices, the medial septal nuclei, the olfactory tubercle, the hippocampus and the midbrain  and not sensitive for examining the innate anxiety level in mice.In this study, β2-AR KO and HET mice showed decreased duration in the light compartment and open arms in L/D box and EPM tasks, indicating an increased anxiety or innate fear by β2-AR deletion. Considering that β1-AR antagonism attenuates the anxiety-like behavior during early withdrawal from chronic cocaine administration in rats, and β1-AR antagonist infusion in the basolateral amygdala facilitated anxiolytic effect , and Ministry of Science and Technology .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The androgen receptor (AR) has long been the primary target for the treatment of prostate cancer (PC). Despite continuous efforts to block AR activity through ligand depletion, AR antagonism, AR depletion and combinations thereof, advanced PC tumors remain resilient. Herein, we evaluate two galeterone analogs, VNPT-178 and VNLG-74A, in PC cell models of diverse androgen and AR dependence attempting to delineate their mechanisms of action and potential clinical utility.Employing basic biochemical techniques, we determined that both analogs have improved antiproliferative and anti-AR activities compared to FDA-approved abiraterone and enzalutamide. However, induction of apoptosis in these models is independent of the AR and its truncated variant, AR-V7, and instead likely results from sustained endoplasmic reticulum stress and deregulated calcium homeostasis. Using in silico molecular docking, we predict VNPT-178 and VNLG-74A bind the ATPase domain of BiP/Grp78 and Hsp70-1A with greater affinity than the AR.Disruption of 70 kDa heat shock protein function may be the underlying mechanism of action for these galeterone analogs. Therefore, despite simultaneously antagonizing AR activity, AR and/or AR-V7 expression alone may inadequately predict a patient’s response to treatment with VNPT-178 or VNLG-74A. Future studies evaluating the context-specific limitations of these compounds may provide clarity for their clinical application. New approaches are therefore necessary to combat the progression of these lesions to the lethal phenotype referred to as castration-resistant prostate cancer (CRPC).Prostate cancer (PC) is among the most frequently diagnosed and lethal malignancies in American men . While iThe androgen receptor (AR) is a transcription factor belonging to the nuclear hormone family of receptors and continues as the primary target for PC drug development as its role in the maintenance of PC tumors in all stages of progression is well-documented . AR spliConsequently, many research efforts have focused on the evaluation of natural and synthetic small molecules capable of promoting the depletion of the AR protein, including AR-Vs, in addition to disruption of its activity. Although some of these AR depleting agents (ARDAs) have demonstrated promising results in preclinical models of CRPC, the Phase 3 clinical failure of galeterone versus enzalutamide NCT02438007] reinforces our need for improved understanding of their mechanisms of action as well as potential context-specific limitations of their use 38007 rei. Indeed,In eukaryotes, the 26S proteasome functions as the primary machinery for the recycling of soluble proteins but it requires that these targets be specifically marked by K48-linked polyubiquitination; ergo, the ubiquitin-proteasome pathway is largely selective of proteins that are in excess, misfolded, or generally nonfunctioning. It has been shown that galeterone is capable of directly binding the AR-LBD and comparative studies have indicated treatments of galeterone and the proteasome inhibitor MG132 both promote the accumulation of polyubiquitinated AR proteins , 8. HoweEarly studies of galeterone action in AR-negative PC cell lines also revealed its ability to activate the unfolded protein response (UPR)/endoplasmic reticulum stress response (ERSR) . The endWhile previous galeterone studies demonstrated its activation of the UPR and the proteasome-mediated depletion of the AR/AR-V7, no additional targets have been identified that could help to explain these separate biological responses. We believe these phenomena are linked, if not by a common mediator, by a motif or domain shared by distinct proteins with critical roles in maintaining AR stability and ER homeostasis. Therefore, herein we evaluate the biological activities of two galeterone analogs that have demonstrated improved potential in our preliminary studies. Using basic biochemical assays and optimized experimental models, we conclude that VNPT-178 and VNLG-74A antagonize the full-length AR (fAR), promote its post-translational depletion and initiate and sustain activation of the UPR leading to induction of apoptosis. In addition to our functional analyses, in silico molecular docking reveals the 70 kDa heat shock protein chaperones BiP/Grp78 and Hsp70-1A may be primary targets for VNPT-178 and VNLG-74A. To our knowledge, these data are the first to reconcile two of the divergent cellular responses to ARDA treatments and may provide additional insight to potential advantages and caveats associated with studies of galeterone analogs.plots) with 50 and 90% growth inhibitory (GI50 and GI90) concentrations estimated in the low micromolar range . As with galeterone, both VNPT-178 and VNLG-74A elicit similar responses in all three cell lines while the AR-negative PC-3 cells are markedly resistant to abiraterone and enzalutamide.Our first objective was to evaluate the antiproliferative activity of galeterone analogs VNPT-178 and VNLG-74A Figure  in widel50 values  versus vehicle-treated controls from at least three independent experiments.t test and the significance level was determined by the largest p-value within each treatment group.22Rv1 cells were seeded in 6-well plates at a density of 500 cells per well in 2 mL culture media (refreshed 2x weekly) containing vehicle or compound (1 or 5 μM) and adherent colony formation was evaluated on day 15 using 0.05% (w/v) Crystal Violet Staining Solution (10 mM PBS containing 1% (v/v) Formaldehyde and 1% (v/v) Methanol; pH 7.4). Images of the 6-well plates were generated using a digital scanner. Colonies were manually counted (>30 cells) and total colony area was calculated using ImageJ software with “Colony Counter” and “ColonyArea” plugins. Representative images and plots of the mean ± standard deviation of treated groups versus vehicle controls are shown. Statistical analysis was performed using Student’s coli (JM109) were transformed by heat-shock to express either the firefly or Renilla luciferase reporters and grown on ampicillin-containing agar plates. The firefly luciferase gene is fused to an ARR2-modified rat Probasin promoter  while the Renilla luciferase reporter is pRL-null . A single bacterial colony containing each reporter was selected and grown in 40 mL LB broth overnight . Plasmid DNA was purified using an EndoFree Plasmid Maxi Kit  and quantified using a BioPhotometer Plus . LNCaP and 22Rv1 cells were seeded in 12-well tissue culture-treated plates and grown overnight in phenol red-free RPMI-1640 supplemented with 10% charcoal/dextran-treated FBS and 1% penicillin/streptomycin (C/D Media) to reduce androgen availability. The cells were co-transfected with the firefly and Renilla luciferase reporters using Effectene (#301425) transfection reagents in accordance with the manufacturer’s suggested protocol  for six hours in C/D media. After transfection, media was aspirated and replaced with C/D media containing vehicle or 10 μM of each compound for a four-hour pre-treatment before the addition of DHT . Cells were incubated for an additional 20 hours at 37°C and 5% CO2. Media was aspirated from each well and 200 μL of 2.5X Passive Lysis Buffer (Promega) was added. Plates were agitated on automated plate shakers for four hours at 4°C to complete cell lysis. Full-length AR transcriptional activation was analyzed using a Dual-Luciferase Reporter Assay System (E1980) in accordance with the manufacturer’s recommendations . Luciferase activity was measured in white 96-well flat bottom plates (Corning) using a Tecan M1000 plate reader. For three independent experiments (n=4), mean relative luciferase units (RLU) and standard errors were calculated using Excel and presented as fold-change versus vehicle-treated positive control.Competent E. Short hairpin RNAs (shRNA) specific for the human androgen receptor (AR) and AR-V7 (aka AR3) were packaged into the pLKO.1-puro vector for the transient transfection of 22Rv1 cells. The oligo sequences used for plasmid construction are as follows: fAR 5’-TGC ACT GCT ACT CTT CAG CAT TCA AGA GAT GCT GAA GAG TAG CAG TGC TTT TTT C-3’ and 5’-TC AGA AAA AAG CAC TGC TAC TCT TCA GCA TCT CTT GAA TGC TGA AGA GTA GCA GTG CA-3’; AR-V7 5’-TGT AAT AGT GGT TAC CAC TCT TCA AGA GAG AGT GGT AAC CAC TAT TAC TTT TTT TTC-3’ and 5’-TCG AGA AAA AAA AGT AAT AGT GGT TAC CAC TCT CTC TTG AAG AGT GGT AAC CAC TAT TAC A-3’.22Rv1 cells were seeded in 6 well plates at a density of 150,000 cells per well and incubated overnight. The following day, the media was aspirated and refreshed containing an optimal dosage of mature lentivirus . 22Rv1 cells were cultured in the presence of the lentivirus for 48 hours before the media was aspirated and refreshed with media containing vehicle or VNPT-178 for another 24 hours.Representative LNCaP, 22Rv1, and PC-3 cell images were taken at 200x total magnification using a Fisher Scientific Micromaster inverted digital microscope and the Micron software. Images shown are a 300x300 pixel crop (600x600 for vehicle controls) of the original 3MP (2048x1536) image for clarity purposes.Antibodies (primary 1:500) purchased from Santa Cruz Biotechnology  include: AR N20 . Antibodies  purchased from Cell Signaling Technology  include: PSA/KLK3 , PARP , Caspase-3 , β-Tubulin , GAPDH , CHOP , LC3-A/B , Beclin-1 , PERK , (p) eIF2α , eIF2α , ATF-4 , ATF-6 , IRE1α , XBP-1s , Calnexin , BiP/Grp78 , ERO-1Lα , PDI , anti-Rabbit IgG , anti-Mouse IgG . Antibodies (primary 1:1000) purchased from Abcam  include: (p) PERK . Whole cell lysates were collected for each condition in RIPA buffer  supplemented with 0.02% EDTA , Complete protease inhibitor cocktail , and phosphatase inhibitor cocktails 1 and 2 . Samples containing 15-30μg total protein were resolved by SDS-PAGE using 8, 10, or 12% polyacrylamide gels. Proteins were captured on PVDF membrane (#162-0177) purchased from Bio-Rad Laboratories  and HRP-conjugated secondary antibodies were detected using SuperSignal West Pico chemiluminescent substrate (#34080) purchased from Thermo Scientific  and X-Ray film.G ° ). Docking computations were performed on the AR-LBD, BiP/Grp78, and HSP70-1A crystal structures . The ligand SMILES strings were first converted to 2D MOL format by Indigo (EPAM) then to 3D MOL format by OpenBabel [The ligands VNPT-178 and VNLG-74A were docked to the AR, BiP/Grp78 and Hsp70-1A binding sites by sampling their rotational and translational degrees of freedom and the generated poses were subsequently scored by their estimated free energy of binding (ΔpenBabel . SubsequpenBabel . The VinpenBabel . The inipenBabel . Essenti


Rana (R.) rugosa the androgen receptor (AR) gene on the W chromosome (W-AR) is barely expressed. Previously we showed that incomplete female-to-male sex-reversal occurred in Z-AR transgenic female frogs. To date, however, there is no report showing that AR with androgens can determine genetically programed male sex fate in any vertebrate species. Here, we examined whether AR together with androgens functions as a sex determinant in an amphibian species.In the Japanese frog R. rugosa frogs, we produced AR-transgenic (Tg) and -knockdown (KD) female R. rugosa frogs by the I-SceI meganuclease-mediated gene trap and CRISPR/Cas9 system, respectively. AR-Tg and -KD tadpoles were reared in water containing testosterone (T) at 0 to 7.1 ng/ml. Frozen sections were prepared from the gonads of metamorphosed frogs and immunostained for laminin, Vasa, Pat1a, CYP17 and AR. We also employed PCR analysis to examine Dmrt1, Pat1a and CYP17 expression in the gonads of KD and placebo-KD female frogs.To examine whether complete female-to-male sex-reversal occurs in AR-Tg ZW female frogs when a low dosage of T was supplied in the rearing water of tadpoles. However, no sex-reversal was observed in AR-KD ZW female frogs when the gonads were treated with dosages of T high enough to induce complete female-to-male sex-reversal even in wild type frogs.Complete female-to-male sex-reversal occurred in the R. rugosa frogs.These results suggest that AR with its androgen ligand functions as a male sex-determinant in the ZW type  R. rugosa frog possesses two sex-determining systems (the XX/XY and ZZ/ZW types) within the same species [In most vertebrate species sex is genetically determined. As in other vertebrate species, heterogametic sex chromosomes in amphibians determine the male (XX/XY) or female (ZZ/ZW) fate . Of grea species . The fro species . In addi species . To dateAR-knockout mice, males have a female-like appearance and body weight, but female-to-male sex-reversal does not occur [AR does not appear to participate in sex determination in mice, although the Sry on the Y chromosome has been identified as a male sex-determinant in this species [Androgens exert various effects in male reproductive organs, brain skeletal muscle and other target tissues. Androgenic effects are mediated by tissue-specific transcriptional control of target genes via the nuclear androgen receptor (AR) . In AR-kot occur , 6. Ther species .R. rugosa frog the AR gene is located on the sex  chromosomes, reportedly on the inverted region of the Y and W chromosomes [AR gene is undergoing a process of evolutional degradation due to the lack of recombination between the inverted and non-inverted regions of the sex chromosomes . It is known that the AR gene on the W chromosome (W-AR) is hardly expressed in R. rugosa embryos, perhaps due to variations in promoter element and cognate transcription factor interaction between the W- and Z-AR genes [W-AR is transgenically expressed in Xenopus A6 cells, W-AR proteins can trans-activate androgen-dependent transcription in reporter assays [W-AR degradation is incomplete in R. rugosa. Interestingly, up-regulation of the AR expression is observed in the male gonad of R. rugosa tadpoles prior to sex determination [R. rugosa synthesize more androgens than females [AR could be involved in male sex determination in this species. To address this issue, we produced Z-AR-transgenic (Tg) female (ZW) frogs. A subset of these frogs developed a hybrid of testis and ovary, called ovotestis. In other words, incomplete female-to-male sex-reversal took place in Tg ZW frogs. It is known that the AR protein contains an N-terminal trans-activation domain (NTD), a central DNA binding domain (DBD), and a C-terminal ligand binding domain (LBD) that binds androgens such as testosterone (T) and dihydrotestosterone [R. rugosa, we produced AR-knockdown (KD) ZW frogs, as well as Tg ZW frogs. By employing the CRISPR/Cas9 system [AR gene lacking the NTD, DBD and LBD by frame-shifting. Here we report for the first time that AR together with androgens can be a male sex-determinant in a vertebrate species.In the omosomes . Structuomosomes , 10. Thu females . In viewosterone . Thus, w9 system , we succAll the animal experiments in this study were performed with official approval from the Committee of Animal Experimentation of Waseda University (Permit Number: 2016-A076) as described in detail previously .R. rugosa was used. Unfertilized eggs were artificially ovulated and inseminated [For all experiments the ZZ/ZW type eminated . Fertilieminated  and Tayleminated . The geneminated . PhotogrTotal RNA was prepared from the gonads of frogs and then cDNA was synthesized to use for PCR analysis as previously reported .We constructed the vectors for transgenesis as reported in detail elsewhere .I-SceI meganuclease-mediated gene trap [I-SceI meganuclease  and the I-SceI-cleaved plasmid encoding Z-AR and V5 (GKPIPNPLLGLDST), using a NANOJECT II injection apparatus . We also produced the placebo-Tg (pTg) ZW frogs as control by injecting I-SceI-cleaved pDPCG vectors and the meganuclease into ZW embryos. Tg and pTg embryos and tadpoles were reared as previously described [Z-AR integration into genomic DNA, we extracted DNA from the tail tip of all Tg tadpoles at St. V, using the AllPrep DNA/RNA Micro Kit  [5’-GCGGTTTTTCCAACTTACCA-3’ and reverse, 5’-CGAGACCGAGGAGAGGGTTA-3’.We produced Tg ZW frogs, using the ene trap . Fertiliescribed . To conf, Japan) . The PCRZ-AR/V5 expression in Tg ZW gonads. Total RNA was prepared from the gonads of wild-type (Wt) and Tg frogs just after metamorphosis using ISOGEN  and cDNAs were synthesized [Z-AR/V5, Dmrt1 and CYP17) of 95°C (30 sec), 62°C (30 sec), and 72°C (1 min), ending with 7 min of extension at 72°C. DNA fragments for Z-AR/V5 (260-bp), CYP17 , and Dmrt1  cDNAs were amplified by PCR using a set of primers for each respective template. Primer sequences are given in We employed PCR analysis to examine thesized . The PCRCas9 cDNA in the pCS2+ vector was obtained from Thermo Fisher Scientific Inc. . The DNA construct was linearized with NotI and transcribed with the mMessage mMachine SP6 Kit (Thermo Fisher Scientific Inc.) to produce capped Cas9 mRNA, which was then purified with the RNeasy Mini Kit according to the RNA clean protocol (QIAGEN). To create an engineered single guide RNA (gRNA) expression vector, we placed a T7 promoter followed by two BsaI sites upstream of the recently described gRNA scaffold [AR gene of interest with the form 5’-CC-(N)20-GGG-3’. The GGG was the protospacer-adjacent motif (PAM). The resulting construct was digested with DraI and transcribed using the mMessage mMachine T7 Kit (Thermo Fisher Scientific Inc.). The gRNA was purified using the RNeasy Mini Kit (QIAGEN). We produced KD ZW frogs using the CRISPR/Cas9 system by which we have successfully disrupted the NTD, DBD and LBD of the AR gene by frame-shift.scaffold . The gRNCas9 mRNA at a dose of 500 pg per embryo together with gRNA (300 pg/embryo) targeting the AR into 2202 one-cell stage R. rugosa embryos. The MMR (Marc’s Modified Ringer) buffer  and gRNA (300 pg/embryo) were also injected into 1071 embryos as control. We designated the placebo AR-knockdown embryos as “pKD” embryos. After KD and pKD embryos were subjected to genomic PCR to amplify the target region, PCR products were inserted into a pTAC-2 vector using a TA PCR Cloning Kit  and sequenced.We injected Immunohistochemistry was performed as previously described . The priR. rugosa frogs after metamorphosis. The reaction mixture contained 4 μl of master mix, 4 μl of a 2.5 μM concentration of each forward and reverse primer  cDNAs were used as template. GA3PDH (AB284116) cDNA was used to standardize the mRNA input levels of each gene. PCR products were electophoresed on 4.5% polyacrylamide gels and stained with ethidium bromide [http://imagej.nih.gov/ij/download.html). Relative expression levels of each gene was calculated by the following formula: relative expression level = gene X (T-treated) x GA3PDH (T-untreated) / gene X (T-untreated) x GA3PDH (T-treated) [PCR analysis was performed to examine the expression of genes in the KD and pKD gonads of e primer , 1 μl ofDH AB2841 cDNA wasDH AB2841 cDNA wasData are represented as the mean±SE. Differences (P<0.05) determined using one-way ANOVA, were considered statistically significant.Images were scanned and adjusted for brightness and contrast by Adobe Photoshop CS2.R. rugosa tadpoles [The wild-type (Wt) ZW ovaries were much bigger than the Wt ZZ testes , but not in the Wt ZW gonad (ovary)  .We prepared frozen sections from the Wt and Tg gonads of frogs and stained for Vasa, Pat1a, CYP17 and AR. Vasa is a protein specific to germ cells in both the male and female gonads, but Pat1a is specific to only the immature oocytes in the ovary . CYP17 iZ-AR in the genomic DNA of R. rugosa females.Strong immuno-positive signals for Vasa were observed in all the gonads examined: Wt testis , Wt ovarAR gene by frame-shift. When we injected Cas9 mRNA together with gRNA targeting the AR into 2202 embryos, and the MMR (Marc’s Modified Ringer) buffer or gRNA alone into 1071 embryos as control, 511 and 419 embryos developed into tadpoles at St. 25-1W, respectively. Then we examined mutagenesis in 44 KD and 38 pKD frogs with normal outward appearance 1 week after metamorphosis. The mutagenesis rate of KD (ZW) frogs was 50 to 100%, whereas pKD (ZW) frogs showed no genomic mutation. Mutated sequences from one representative KD (ZW) sample injected with Cas9 mRNA and gRNA are shown in We produced KD ZW frogs using the CRISPR/Cas9 system by which we have successfully disrupted the NTD, DBD and LBD of the The Wt ZW ovaries were much bigger than the Wt ZZ testes  is involved in testis formation in R. rugosa [Dmrt1 expression was at very low levels in the Wt ZW ovary and the ZW gonad treated with 0.02 ng/ml of T (ovary), but increased in the pKD ZW gonad treated with T at 2.0 ng/ml (testis)  compared with that in the pKD treated with T at 2.0 ng/ml (testis)  ZW females of the R. rugosa frog. The AR-Tg ZW females developed a hybrid of testis and ovary, called “ovotestis.” However, the AR-Tg female frogs formed the testes when a low dosage of T was supplied in the rearing water of tadpoles. In the sex-reversed testes the expression of Dmrt1, AR and CYP17 genes required for masculinization were significantly up-regulated. Next, we produced AR-knockdown (KD) ZW female frogs by the CRISPR/Cas9 system. Interestingly, no sex-reversal was observed in AR-KD ZW female frogs when the gonads were treated with dosages of T high enough to induce complete female-to-male sex-reversal, even in wild type frogs. In the AR-KD ZW female gonads the expression of genes required for masculinization was not up-regulated. These results indicate that AR together with androgens can be a male sex-determinant in an amphibian species.Previously we showed that incomplete female-to-male sex-reversal occurred in


Alex passed away on April 15 from complications due to esophageal cancer with metastasis to the lung and liver. He was 76 years old.Alex Turner earned a PhD from the University of Oklahoma and began his career in medical physics with his entry as a post‐doctoral fellow at the University of Texas MD Anderson Hospital in the early 1970s. His contemporaries include Bernie Hranitzky, Walter Grant, Leroy Humphries, Jim Purdy, and George Oliver.Alex served for a number of years as the physics director of the Central Arkansas Radiation Therapy Center (CARTI) in Little Rock, AR. Later he served as the director of physics in various locations, including Sacramento (CA), Oklahoma City (OK), Fort Worth (TX), and eventually settled in retirement in Breckenridge, CO. His last position of which I am aware was for the San Jose, California‐based Xoft Corporation in service to their electronic brachytherapy product.He served as Chair of the American College of Medical Physics (ACMP) in 1997, and was a Fellow of both the ACMP and the AAPM.Alex, who loved skiing, was among a small group of physicists that founded what eventually became the Winter Institute of Medical Physics (WIMP). No doubt, his love of the mountains persuaded him to retire at high altitude in Colorado.Alex had many talents, first and primarily as a clinical medical physicist. He was one of the first wave who acquired the Nomos Peacock system and was a pioneer in intensity‐modulated radiation therapy (IMRT). In addition, he excelled in managing and directing large projects. He was also an inventor. I remember commissioning and using a rotating half‐beam block with half‐moon‐shaped wedges that he developed. This gadget worked beautifully in the simpler era that was the late 1980s.JACMP. In November of 1997, I approached Alex, who was at the time Chair of the ACMP. He supported the concept of an open‐access journal for clinical medical physics publications, and served on the original ACMP Task Group for a Journal of Clinical Physics. The JACMP likely would not have come into existence without Alex's active and enthusiastic support.My primary interaction with Alex occurred at the birth of the For all of Alex Turner's accomplishments and creativity in medical physics, those of us who knew him know that his real passion for living stemmed from his relationships with people, especially students and young physicists, and his love of the outdoors, particularly hiking and skiing. He leaves behind his beloved wife, Karen, and his two sons, John with his wife Jennifer, and Michael with his spouse Claire. Alex and Karen have five grandsons who were the joy of Alex's life. Alex was also well‐known internationally, with friends throughout the world.Alex's efforts in promoting the value of the medical physics profession are well known, and will be long remembered. He never missed the opportunity to educate a student or new professional. He was not prone to lecture, but rather his way was to freely share what knowledge he had. Sometimes that knowledge was technical, sometimes political or professional, and sometimes even personal. Also, he let his personal philosophy invade his work persona; his integrity, personable nature, and attitude characterized his work presence. He also knew how to separate work from his personal life and focus on life outside of physics when that's where he was. This was the way he conducted himself throughout his career, always being a man of high principle and always a true gentleman. Alex truly was a credit to our profession. He will be greatly missed.This work is licensed under a Creative Commons Attribution 3.0 Unported License.


Simultaneous thermogravimetric-differential thermal analysis coupled to mass spectrometry (TG-DTA-MS) experiments of NaH-NaOH composites reveal two principal features: Firstly, an H2 desorption event occurring between 240 and 380 °C and secondly an additional endothermic process at around 170 °C with no associated weight change. In-situ high-resolution synchrotron powder X-ray diffraction showed that NaOH appears to form a solid solution with NaH yielding a new cubic complex hydride phase below 200 °C. The Na-H-OH phase persists up to the maximum temperature of the in-situ diffraction experiment shortly before dehydrogenation occurs. The present work suggests that not only is the inter-phase synergic interaction of protic hydrogen (in NaOH) and hydridic hydrogen (in NaH) important in the dehydrogenation mechanism, but that also an intra-phase Hδ+… Hδ– interaction may be a crucial step in the desorption process.Solid-state reactions between sodium hydride and sodium hydroxide are unusual among hydride-hydroxide systems since hydrogen can be stored reversibly. In order to understand the relationship between hydrogen uptake/release properties and phase/structure evolution, the dehydrogenation and hydrogenation behavior of the Na-O-H system has been investigated in detail both  With the continued depletion of fossil fuel resources and the increasing impact of environmental pollution, renewable and clean energy sources such as wind and solar technologies have become a major priority and been the subject of increased research interest. However, many renewable energy sources are intermittent and so a means by which energy can be stored and transported is vital . One appCompared to storage of hydrogen as a compressed gas or a cryogenic liquid, solid-state hydrogen storage is more effective volumetrically and alleviates safety and cost concerns associated with high pressure and/or low temperature. For more than 10 years materials such as light metal hydrides , complexet al. found that sodium oxide, Na2O can absorb hydrogen easily at close to ambient temperature (~60 °C) to form NaH and NaOH. Further, the hydrogenated products, NaH and NaOH, can be readily converted back to Na2O by thermal treatment [Among various hydrogen storage materials, sodium based materials are very promising as hydrogen storage media for stationary applications since sodium is one of the most abundant elements on Earth (2.64 wt%), and is relatively cheap ,13,14,15reatment ,17:Na2δ+ and Hδ–, respectively, the interaction of these two hydrogen species could be responsible for the relative decrease in dehydrogenation temperature between Na-O-H and Na-H [ca. 10 mol% of NaOH could apparently be incorporated into the NaH structure and resulted in enhanced hydrogen motion in NaH above 150 °C [– can be substituted for H– within the NaH structure above 150 °C. Given the connectivity between proton conduction and hydrogen uptake/release kinetics [in-situ synchrotron X-ray powder diffraction methods to elucidate the subtle changes in composition and structure immediately prior to hydrogen release.Dehydrogenation in the NaH-NaOH system occurs at a lower temperature than that for NaH alone and given that NaOH and NaH contain Hand Na-H ,17. The and Na-H ,20,21,22e 150 °C . These pkinetics , an inve2O with temperature (at 18 bar) is shown in −1, hydrogen uptake is initiated close to room temperature (30–50 °C). Upon heating to 140 °C, the hydrogen uptake rate increases significantly and 2.5 wt% H2 can be stored under these conditions. Subsequently, hydrogenation is relatively slow and a total of 3.3 wt% hydrogen is absorbed by 400 °C. These uptake results are consistent with previous studies [2O with moisture during hydrogen uptake, but powder X-ray diffraction (PXD) patterns before and after hydrogenation suggest that the reaction of an impurity in the starting material with hydrogen may also contribute as discussed below.The time-resolved hydrogenation profile of as-milled Na studies ,17, alth2O starting material after mechanical milling (sample 1), the material after hydrogenation (sample 2) and finally after subsequent dehydrogenation (sample 3). Although Na2O was the main phase in the ball milled starting material as expected, an impurity phase of Na2O2 was also detected, and analysis of the diffraction pattern yielded a phase fraction of ca. 10 wt%. After hydrogenation at 400 °C, NaH and NaOH were formed and the diffraction peaks from Na2O and Na2O2 were no longer present. Upon dehydrogenation at 400 °C, Na2O reformed and was present as the majority phase. Overall, therefore, the material system demonstrated a reversible reaction as described by Equation 1. The presence of NaOH in the dehydrogenated product, sample 3 could suggest partial hydrolysis of Na2O, but the more likely origin of the hydroxide is from hydrogenation of the original Na2O2 impurity in the starting material (since Na2O2 + H2→2NaOH). This leads to an excess of NaOH in the hydrogenated product, sample 2  which persists during the dehydrogenation process at 400 °C However, Rietveld refinement for hydrogenated Na2O shows that the molar ratio of NaOH and NaH in the sample is 1.66:1. The phase fraction of NaOH exceeds the amount expected solely from the hydrogenation of 10 wt% Na2O2. Thus, both the hydrolysis of Na2O (and possibly of NaH) and the hydrogenation of the original Na2O2 impurity lead to the observed excess of NaOH.PXD was performed to clarify the chemical reactions that occur on hydrogenation. 2O. By comparison, only NaH and NaOH were detected in sample 4. The absence of the NaOH·H2O impurity in sample 4 is attributed to dehydration during ball milling.The dehydrogenation process in the NaH-NaOH system was investigated further. ca. 360 °C, which can be assigned to the decomposition of NaH to Na metal and hydrogen. For NaOH, the DTA profile shows two endothermic peaks at 299 and 319 °C, which can be assigned to the α-β (orthorhombic-monoclinic) phase transition and the melting point, respectively [2O and hydrogen. The results further confirmed that the dehydrogenation pathway of the NaH-NaOH mixture is entirely different to that observed for NaH. The first endothermic event is not observed in either NaH or NaOH. It would appear therefore that the synergic interaction of hydrogen species in NaH and NaOH not only contributes to the lower dehydrogenation temperature vs. NaH itself, but also leads to likely reaction in the solid state and structural changes during the heating period prior to dehydrogenation. To clarify the structural changes that occur before dehydrogenation, variable temperature, in-situ synchrotron PXD experiments were conducted on the NaH-NaOH mixture and are discussed below.The thermal decomposition behavior of sample 4 compared to NaH and NaOH was investigated by DTA, as shown in ectively ,26. By cEx-situ XRD characterization was performed on several dehydrogenated hydride-hydroxide mixtures of varying molar ratio after heating each to 400 °C (2O was the main phase in the dehydrogenated NaH-NaOH (1:1) sample, but some residual NaOH remains also detected. In this case, the presence of residual NaOH can be attributed to the fact that the NaH and NaOH starting materials contain low levels of NaOH and NaOH·H2O impurities, respectively. Hence the NaH:NaOH ratio departs from the ideal stoichiometric 1:1 value in the mixture. In order to obtain single phase Na2O as a dehydrogenation product, we optimized the quantity of NaH and NaOH in the starting mixture. As shown in 2O with minimal impurities (sample 5).o 400 °C . ClearlyThe thermal behavior of sample 5 was investigated by TG-DTA-MS . TG resuIn-situ synchrotron PXD methods were used to clarify the reaction mechanism underpinning the first endothermic event at ca. 170 °C by heating sample 5 from room temperature to 260 °C. in-situ diffraction patterns for sample 5, the relevant peak indices and a plot of the resulting cell volume changes of the NaH and NaOH phases, respectively, as a function of temperature. Structure refinements performed against the synchrotron PXD data were conducted for each data set (see supplementary information). A fundamental parameter (FP) approach was employed in TOPAS to perform whole-pattern profile fitting of the diffraction data collected in transmission mode. The diffraction background was fitted with Chebychev functions. Structural data from the ICDD PDF4 (2014) database for NaH (02-0809) and α-NaOH (078-0188) were used as starting models in TOPAS. At room temperature NaH crystallizes in cubic space group Fm3m  Å), while α-NaOH is orthorhombic  Å, b = 3.4011(1) Å, c = 11.3901(1) Å).ca. 116.4 Å3 to ca. 118.5 Å3 over this temperature range, corresponding to a volume expansion of approximately 1.8%. Over the same temperature range, the cell volume for NaOH increases by approximately 0.99% and the phase fractions of NaH and NaOH remain effectively constant, indicating that no reaction occurs between the hydride and hydroxide.Before heating, room temperature PXD data show that sample 5 consists only of NaH (44 wt%) and NaOH (56 wt%). There are no impurity diffraction peaks observed in the PXD pattern. As shown in From 110 °C, all the NaH peaks become increasingly asymmetric with some evidence of reflections emerging at slightly higher 2θ values to the main peaks as manifested by a broadening tail to all NaH reflections. Meanwhile, the NaH peaks shift continuously to lower 2θ angles and the hydride cell volume increases significantly such that at 180 °C the value is approximately 7.2% larger compared to that at room temperature e. Most s2O over the entire temperature range of the experiment. The cell volume of the hydride at 240 °C is 127.1 Å3, which is approximately 9.2% larger as compared to the room temperature NaH structure. The results suggest that a structural change begins from 110 °C and that by 190 °C NaOH reacts appreciably with NaH in the solid state to form a complex hydride with an NaH-type structure. As the temperature increases further, a single composition of the NaH-like phase is formed. Thus we propose that an NaH-NaOH solid solution forms in which up to 50% of the hydride is replaced by hydroxide; NaH1-x(OH)x where x ≤ 0.5. Hence, one might reasonably speculate that the formation of the hydride-hydroxide is the vital precursor to an intraphase Hδ… Hδ– interaction and the hydrogen evolution step during desorption.By 240 °C there is no evidence of NaOH in the diffraction patterns and the NaH-like phase reflections become more symmetric and sharper, indicative of a single phase. Also noteworthy is that there is no evidence for the formation of NaThe process could thus be represented by the modified version of the reaction equation below:AH-AOH(AOH)2 systems , similar solid solution behavior has only been observed previously in the K-O-H system [2O, but rather KH decomposes independently while KOH remains to 500 °C [2O, no solid solution phases are reported prior to oxide formation upon heating, which might be a consequence of the relatively low dehydrogenation temperature [2 system prior to dehydrogenation [δ+… Hδ– interaction may be a crucial step in the desorption process. Furthermore, the ensuing lattice expansion and anion disorder of the sodium hydride hydroxide could play a significant role in the diffusion of hydrogen in the solid state either or both as protons and hydride. The mobility of one or both of these species is likely to be key to mediating and controlling the hydrogen uptake and release kinetics in this and similar systems. Strategies involving nanostructuring, additives and catalysts are likely to be crucial in the development of cheap, abundant materials systems such as hydroxides into potentially useful hydrogen stores.When compared with other H system . On heato 500 °C . By contperature ,27,28. Tgenation . Na-O-H 2O , NaH , and (α-)NaOH  were used as received. Before the hydrogenation or dehydrogenation experiments, Na2O and NaH-NaOH mixtures (employing different NaH:NaOH molar ratios as indicated elsewhere in the text), respectively, were milled using a 50 mL stainless steel milling jar under argon atmosphere in a Retsch PM100 planetary ball mill. Milling was performed at 400 rpm for 1 min in one direction, paused for 1 min and then reversed to give a total milling duration of 1 h. A ball:powder mass ratio of approximately 50:1 was employed throughout. All manipulations were performed in an Ar-filled recirculating glovebox .Naex-situ powder X-ray diffraction (PXD) experiments were conducted with a Bruker D8 powder diffractometer in transmission geometry with spinning sealed capillaries. Diffraction data for phase identification were typically collected over 5° ≤ 2θ ≤ 85° with a 0.017° step size and scan times of 1 or 10 h.Room temperature in situ high temperature measurements were conducted with a flow cell under an atmosphere of 1 bar argon (99.99%) using a Cyberstar hot air blower to heat the quartz capillary from 50 °C to 260 °C at a constant heating rate of 10 °C min–1. Data were collected with an exposure time of 150 s at every 10 °C step. Data analysis was performed using the TOPAS 4.2 software package [Synchrotron PXD data were collected using incident radiation with λ = 0.9533 Å using a Mythen-II detector at the powder diffraction beamline, Australian Synchrotron. Time-resolved  package .–1. The variation in sample mass and temperature with time was recorded.Hydrogen absorption experiments were performed on an intelligent gravimetric analyzer  with samples of ∼50 mg contained in stainless steel sample holders. Hydrogen gas  at 18 bar was introduced into the reaction chamber and absorption was performed between 20 and 400 °C with a fixed ramp rate of 3 °C min2O, 1 ppm O2). Samples of ca. 20 mg were loaded into alumina pans and heated to 673 K under a flow of Ar gas at a rate of 5 °C min–1 in order to achieve complete decomposition of the samples.Thermal behavior of NaH-NaOH samples was analyzed via simultaneous thermogravimetric-differential thermal analysis with coupled mass spectrometry  in an Ar-filled recirculating glovebox  under a relatively low hydrogen pressure (18 bar). The hydrogenated products, NaH and NaOH, release hydrogen at a lower temperature than the binary hydride NaH. Moreover, differential thermal analysis shows an endothermic event near 170 °C in the NaH-NaOH system that cannot be associated with the dehydrogenation reaction of the mixture or with phase transitions of either of the individual components. In-situ synchrotron PXD results show that NaOH forms a solid solution with NaH that persists until 240 °C. The NaH and Na cubic phases demonstrate a large volume expansion in the temperature range of 160−240 °C. The resulting predominantly intraphase interaction of protic hydrogen and hydridic hydrogen provides a likely driving force for the subsequent dehydrogenation in the hydride-hydroxide system.In summary, the Na-O-H reversible hydrogen storage system has been examined in detail. The (de)hydrogenation behavior of the Na-O-H system was investigated by means of IGA measurements, simultaneous TG-DTA-MS, XRD, and


Goals of this study were: i) to define a possible association between Estrogen Receptor (ER), Progesterone Receptor (PR), Androgen Receptor (AR), human EGF receptor 2 (HER2) and brain progression in EOC patients, and ii) to identify differences in ER, PR, AR and HER2 protein expression from primary EOC and its matched resected brain metastasis. A retrospective series of 11 EOC with matched brain metastasis surgically removed was collected. For comparison, a “Control dataset” of 22 patients, without evidence of brain involvement after an adequate follow up was matched. ER, PR, AR and HER2 status was significantly associated with brain localization, both considered as discrete variable  and as continuous one (p=0.035). Multivariate analysis confirmed this trend (p=0.053). When considered as continuous variables, ER and AR showed greater expression in primary tumors in comparison with brain metastases .Univariate analysis showed that AR In our series, AR predicts brain involvement, with a 9.5 times higher propensity for AR-negative EOC. Moreover, brain dissemination is probably the result of progressive dedifferentiation of primary tumor, shown by reduction of ER and AR expression in metastases. Further studies are required, in order to anticipate and improve multimodal treatment of brain metastases. Epithelial ovarian cancer (EOC) is the first cause of death among women with gynaecological malignancies . In its status  and Epidermal growth factor receptor 2 (HER2) status have been widely investigated as potential prognostic parameters in EOC patients, but reported results are controversial .Two observers, who were blinded to clinical data, independently evaluated the staining results. Hormone receptors were considered as follows: i) continue variable (number of positive neoplastic cells considered as a percentage ranging from 0 to 100%), ii) discrete variable using cut-off of 1% , 31, 37 All statistical analyses were performed using SPSS software for Windows . Quantitative variables were initially compared with Pearson Chi-square test, but due to the small number of cases results are not reliable. Therefore Fisher exact test has been considered for the further statistical analyses. Qualitative variables were compared using analysis of variance (ANOVA) or dependent T test for paired samples. Multivariate analysis was performed using regression analysis. P values <0.05 were considered significant, and all tests were two-tailed.