The SNAPSHOT program provides current data on the allergic rhinitis burden in the adult general population of five Middle Eastern countries .A multi-country, cross-sectional, epidemiological program conducted by telephone in a random sample of the adult general population; quotas were defined per country demographics. Subjects were screened for allergic rhinitis using the Score For Allergic Rhinitis questionnaire. Current prevalence (last 12 months) was estimated. Disease severity and control were assessed using the Allergic Rhinitis and its Impact on Asthma classification and Rhinitis Control Assessment Test respectively. Quality of sleep, impact on daily activities and quality of life were measured using the Epworth Sleepiness Scale, Sheehan Disability Scale and EuroQol Five-Dimension questionnaire respectively. Multivariate logistic regression analyses were used to investigate risk factors and co-morbidities.p < 0.0001). Overall, 55% of allergic rhinitis subjects were moderate/severe and 33% were uncontrolled. Both these groups reported impaired quality of life and quality of sleep and increased impairment of daily activities compared to mild/well-controlled subjects (p < 0.0001).1808 of 33,486 subjects enrolled in the SNAPSHOT program fulfilled the case definition for allergic rhinitis. Prevalence was 3.6% [95% CI 3.2–4.0%] in Egypt, 6.4% [95% CI 5.9–6.9%] in Turkey and 6.4% [95% CI 6.0–6.9%] in the Gulf cluster. Risk factors identified were country, co-morbid asthma and income. Subjects with allergic rhinitis reported a significantly lower quality of life compared to the general population (Although the observed prevalence of allergic rhinitis in these Middle Eastern countries is low compared to western countries, its burden is considerable. Allergic rhinitis in general, and specifically uncontrolled and severe disease, results in a negative impact on quality of life, quality of sleep and daily activities. Allergic Rhinitis (AR) is a common inflammatory disorder affecting the nasal membranes characterised by symptoms such as sneezing and nasal congestion. It is a global health problem; conservative estimates suggest that approximately 500 million people worldwide are affected by the disease. The burden of AR is often under-estimated since the disease is not life-threatening. However, it can cause significant morbidity, through both the physical symptoms and its impact on quality of life and well-being . It has Large scale multinational studies that have collected data on the prevalence of AR in adults are rare and those that have taken place use different study methodologies and case definitions, and report a wide range of prevalence estimates. Several studies have been conducted in Western Europe and the United States (US). For example, one study carried out in six European countries ) using the Allergic Rhinitis and its Impact on Asthma (ARIA) definition of AR reported that overall, the prevalence of AR, confirmed by clinical examination was 22.7%. However, the prevalence varied between countries from 16.9% in Italy to 28.5% in Belgium . This ranged from 3.6% [95% CI 3.2–4.0] in Egypt, to 6.4% [95% CI 5.9–6.9] in Turkey and 6.4% [95% CI 6.0–6.9] in the Gulf cluster. After adjusting for age, gender and country there is little difference between the crude and adjusted prevalence. These results are presented in Table –5.8. Thip < 0.20) with AR: gender, age, country, health system coverage, income, BMI, having asthma or chronic obstructive pulmonary disease (COPD) and having a family history of AR. When these variables were entered into the multivariate analysis, a significant association was found between living in the Gulf cluster or Turkey and a higher risk of AR (odds ratio (OR) 2.1 and 2.0 respectively) compared to living in Egypt (p < 0.0001). In addition, a significant association (p < 0.0001) was observed between having asthma and a higher risk of AR . Income was also shown to play a role; subjects with an income above the minimum wage were associated with a higher risk of AR  compared to those with an income lower than the minimum wage. In addition, men were shown to have a lower risk of AR compared to women  (p < 0.0001). These results are shown in Fig. The following variables were tested using univariate analysis and found to have a significant association (p < 0.0001) in the AR population  compared to the non-AR population . The following co-morbidities were found to have a significant association (p < 0.20) with AR in a univariate analysis: cardiovascular, gastrointestinal, immunological, neurological, nervous, renal, respiratory, rheumatological diseases, diabetes and malignancy. These co-morbidities were entered into a multivariate analysis to investigate the relationship with AR, adjusting for age, gender and country. The co-morbidity with the highest impact was respiratory disease (OR 6.4). Detailed investigation into the conditions classified under the co-morbidity ‘respiratory disease’ revealed that this refers primarily to COPD. This was followed by nervous disease (OR 2.0), gastrointestinal disease (OR 1.9), cardiovascular disease (OR 1.7), rheumatological disease (OR 1.5), and diabetes (OR 1.3). These results are shown in Fig. The number of subjects that reported suffering from a chronic health condition was significantly higher (p < 0.0001); and Egypt reported a significantly lower proportion of subjects with well-controlled symptoms  compared to Turkey  and the Gulf cluster  (p < 0.0001). Overall, approximately 39% of subjects reported that AR symptoms had disrupted their work/school, social/leisure and family/home life. More subjects in Egypt reported disruption to their daily activities compared to the Gulf cluster and Turkey. These results are presented in Table More than half the subjects with AR had moderate/severe disease (55.9%) as defined by the ARIA classification. However, the majority (67.0%) reported their symptoms as well-controlled (RCAT ≥ 21) and most (59.3%) reported low levels of daytime sleepiness (ESS 0–5). At a country level, significantly more patients reported mild disease in Turkey  compared to Egypt 23.4%; n = 34) and the Gulf cluster   mean EQ-5D-3L utility score (0.78 ± 0.32) than the general population (0.90 ± 0.21). This relationship was observed for all participating countries or cluster of countries . The overall impact and the country-level results are presented in Fig. Overall, subjects with AR reported a significantly lower (p < 0.0001). This trend was the same across each country and the cluster of countries studied. Those subjects with moderate/severe AR reported significantly lower EQ-5D-3L utility values (0.74 ± 0.32) and EQ-VAS scores (68.4 ± 20.6) than those with mild disease (0.87 ± 0.24 0 and 74.8 ± 17.8 respectively) (p < 0.0001). This trend was the same across each country and the cluster of countries studied.Subjects with AR whose symptoms were well-controlled reported a significantly higher EQ-5D-3L utility value (0.9 ± 0.2) and EQ-VAS score (73.4 ± 19.3) compared to those with symptoms that were not well-controlled (0.7 ± 0.4 and 67.1 ± 19.6) (p < 0.0001); see Fig. p < 0.0001); see Fig. Across all dimensions of daily living a higher proportion of those with moderate/severe disease reported an impact compared to those with mild disease (p < 0.0001); see Fig. A higher proportion of subjects with uncontrolled symptoms (27.3%) reported severe daytime sleepiness (ESS 16–24) compared to those with well-controlled symptoms (16.3%)  completed the detailed questionnaire which collected data on disease management and were termed the AR responders. Demographically, there were few major differences between the AR responders and the overall AR population. The distribution in terms of age, gender, BMI, and smoking status is similar between the two populations. However, differences are apparent with respect to health system coverage. A higher proportion of subjects in the AR responders group are covered by social security and a lower proportion are uninsured. This difference is probably due to the relative contribution of the three countries/cluster involved, which have significant differences in terms of the healthcare coverage available. In Turkey, most of the population (90%) has healthcare coverage through a social security system, while in the Gulf cluster healthcare coverage is expanding through medical insurance as increasingly more individuals receive healthcare in a reimbursed manner. In Egypt, for much of the population, healthcare is paid by patients out of pocket. The response rate in the three countries/cluster differed. There is a higher percentage of Turkish subjects in the AR responder population (56%) compared to the overall AR population (34%), which is reflected in the higher proportion of AR responders covered by social security compared to the overall AR population. Given the potential impact of this on the parameters associated with disease management outcomes, such as quality of life, severity and control, these results have been presented by country.The SFAR questionnaire is a validated self-report measure, which considers most features of the disease  to screen for AR, and is considered to have a higher level of sensitivity and specificity than many other questionnaires used to screen for the disease, such as that used in the European Community Respiratory Health Survey (ECHRS). However, it is possible that in this study, the first question of the SFAR questionnaire excluded many subjects due to unawareness of AR as a disease in the Middle East. Many subjects, particularly those with mild symptoms, in the absence of a physician diagnosis of AR, might interpret their recurrent nasal symptoms as being due to a cold or sinus disease and answer no to the first SFAR question and be excluded from the rest of the questionnaire. In line with this, almost 60% of AR subjects in SNAPSHOT have ARIA defined moderate/severe disease (approximately three quarters of subjects in Egypt and the Gulf cluster) suggesting that many subjects with mild disease may have been missed due to unawareness of the disease. In other countries in Europe and North America, where the SFAR questionnaire has been used widely, and awareness of AR is higher, the first question is necessary to eliminate subjects who would otherwise be wrongly classified. Moving forward, a modified version of the SFAR, may be needed in the Middle East to account for the cultural differences in the region and avoid missing subjects who would otherwise screen positive for the disease. It should also be noted that the SFAR can assess AR when the disease is not symptomatic. It is possible to reach a score of six or seven points from questions other than those on nasal symptoms. Although this does not happen frequently, it has been observed and can reduce the possibility of misclassification of subjects. Although the reported prevalence estimates from SNAPSHOT are low, the individuals identified are likely to represent the more severe end of the spectrum where the impact of AR is greatest and where medical intervention is most needed. In addition, some subjects may already be taking medications for AR. These subjects may not have experienced AR nasal symptoms in the past 12 months due to an effective treatment strategy, and therefore answered no to question one of the SFAR. Thus, although question eight asks about a previous physician diagnosis of AR, some subjects with well-treated physician diagnosed AR and therefore not experiencing bothersome symptoms, may have been excluded from the AR population, contributing to the lower prevalence estimate observed. Therefore, the estimates provided on AR prevalence and its symptoms in the Middle East in the SNAPSHOT study can be considered as conservative. For future studies on AR in the Middle East, it could be considered to first ask questions about the physician confirmed AR diagnosis and medication usage since the dates of diagnosis, followed up by questions about the AR symptoms. This will enable a chronological view of the AR diagnosis, disease management, and patient journey to be obtained which can help in better understanding the level of AR prevalence and symptoms.Limited data is available on the prevalence of AR in adults across the Middle East, and prevalence estimates vary widely both between countries and within countries. For example, one study carried out across all seven Emirates of the UAE used the ECRHS to screen for AR, and reported that at least 7% of the study population had the disease . AnotherAIMES is the only multi-country study to have been conducted in the Middle East, which was carried out in five countries  and reported the overall prevalence of physician-diagnosed AR to be 10%, ranging from 8% in Lebanon to 11% in Egypt . The preA variety of studies have been conducted in adults in Turkey. Two studies have been conducted using the ECRHS questionnaire. In Manisa, the reported AR prevalence in adults was 14.5% . In a seControl of AR is essentially seen as absence of symptoms and is an important measure which few previous studies in the Middle East have addressed. The AIMES survey reported that less than half (40%) of respondents felt their symptoms were completely or well-controlled, and 15% described their symptoms as poorly or not controlled, despite the majority taking medication to treat their symptoms . In contIt is well established that AR has a profound effect on quality of life, having a significant impact on activities such as sleep , 30, worThe impact of AR on daily activities was assessed using the SDS. The SDS was modified for use in this program from its usual 0 to 10 point discretized analogue layout anchored both numerically, verbally descriptively and visually spatially into 5 response option categories . Accordingly, data analysis was discussed with the scale author to ensure correct interpretation of the results. The use of the 12-month time frame in this study enables the data to be more relevant for governments and healthcare bodies to aid in developing healthcare policy. The questionnaire has been validated for use in face-to-face interviews, but was administered by telephone in SNAPSHOT, which should not affect the validity of the data collected.It has frequently been reported that AR results in increased daytime sleepiness. For example, a clinical trial comparing 25 healthy individuals with 25 people with seasonal AR found a significant increase in daytime sleepiness reported by those with AR and these patients also reported significant impairment to their quality of life. The impact was related to the severity of the disease . InteresAs with all studies, limitations do exist. These include the fact the survey was telephone based, which could introduce a sampling bias if certain groups do not have access to a telephone, such as those in more remote, rural areas. However, the SNAPSHOT program considered both mobile and fixed landline telephone numbers to be eligible, and the emergence of mobile telephones means that most households in these countries have access to a telephone. The survey was conducted by trained lay interviewers; and the case definition is based on perceived symptoms with no physician confirmation. However, the clinical definition of AR is difficult to implement in an epidemiological setting where large populations are studied, since it is not possible to obtain laboratory evidence of an immune response. As is the case for all studies that require participants to recall data, there is a risk of inaccuracy in the data collected. In addition, as explained in detail earlier, the interpretation of the first question of the SFAR in this region is likely to have excluded a number of subjects with AR from the program resulting in a potential underestimation of the prevalence of AR in the countries studied. It is important to collect data on symptoms of AR in such countries, since awareness of AR as a disease is low in the region.This study provides an updated prevalence estimate for AR and information on the disease burden within the adult general population of five countries in the Middle East region, using an identical methodology across all countries studied. Although the observed prevalence of AR in these Middle Eastern countries is lower than that reported in western countries, its burden is considerable. AR, and specifically uncontrolled and severe disease results in a negative impact on quality of life, quality of sleep and daily activities. The results from this study can contribute to informed decision making when setting priorities for public health policy and strategy to manage AR in these countries.


Hyperglycaemia is a common side effect of steroid and L-asparaginase combinations, occurring most often during acute lymphoblastic leukemia (ALL) induction phase. To date in Indonesia, it has not been obtained data on the incidence of hyperglycemia in children with ALL in the induction phase and how the role of combinations of L-asparaginase and different type of steroid used. The purpose of this study is to determine the incidence of hyperglycemia in children ALL induction phase, knowing the difference between prednisone and dexamethasone (in combination with L-asparaginase) in causing hyperglycemia in children with ALL and determine the relationship of other factors related to hyperglycaemia. rd (pretest), 4th, 5th and 6th week of protocol (post-test).This was a prospective analytic study with a pre- and post-test design, conducted in three hospitals . Patient’s blood glucose levels (BGL) were checked at the 3Of the 57 patients, 5.2% had hyperglycemia. The patients’ age ranged from 1.4 years old to 15.8 years old (6.7 years old). There was no relationship between age, central nervous system (CNS) infiltration, leukocytosis, Down syndrome, nutritional status, family history of diabetes, infections and ALL stratification with hyperglycemia (p>0.05). Dexamethasone has more chance of obtaining higher mean rate of change in BGL compared to prednisone. .The incidence of hyperglycemia in this study is 5.2%. Dexamethasone, in combination with L-asparaginase, despite having no difference in causing hyperglycemia, has an increased risk of changing BGL compared to prednisone.  Leukemia is a heterogeneous group of hematological malignancies which derived from abnormal or poorly differentiated hematopoietic stem cells . It is responsible for 8% of all cases of cancer, including all age groups and imposes large costs regarding diagnosis and therapy . Acute lymphoblastic leukemia (ALL) is the most frequent type of leukemia in children. Globally, around 1-4.75 per 100,000 children suffers from ALL, with the peak incidence rate between age 2-6 years old . Mostert et al., (2006) reported that, out of all of the children suffering from ALL in Yogyakarta, 35% refused medication, 23% died of medication-associated causes, 22% had their conditionworsened or experienced relapse and 20% had 5-years event-free survival (EFS). Regardless of significantly better prognosis, EFS, and overall survival (OS) with risk-based stratification chemotherapy, the risk of relapse, complications during chemotherapy, and toxicity-associated death is still high . Failure to receive full chemotherapy due to its toxicity has been associated with rather undesirable outcomes in children with ALL .Hyperglycemia is one of the most common side effects of steroid and L-asparaginase combination during the induction phase of chemotherapy. However, reports have variable results. Taking the incidence of hyperglycemia into consideration . Zhang et al., (2014) study has shown association between the incidence of hyperglycemia 5-year overall survival rate and the relapse-free survival rate. To date, there is still no official report available on the incidence rate of hyperglycemia in children with ALL receiving induction phase of chemotherapy in Indonesia. Additionally, the role of combining L-asparaginase with different kinds of steroids used in the events of hyperglycemia is still unclear. The importance of obtaining that data could not be underestimated as it is crucial to know how high or low the incidence of hyperglycemia is, and to see whether a routine check on blood glucose level (BGL)- especially in that particular group of patientsis needed or not.This research is an analytic prospective study with pre-post test design in order to assess the role of corticosteroid (dexamethasone and prednisone), and L-asparaginase exposure to children with phase induction ALL. The research was conducted in the Hematology-Oncology Clinic and Ward of Pediatrics at the following hospitals: Cipto Mangunkusumo Hospital, Dharmais Cancer Hospital, and Gatot Soebroto Hospital, taking time from September 2016 to June 2017. The inclusion criteria used in this research are the all ALL patients undergoing the induction phase of chemotherapy, and all patients aged 0-18 years old. Meanwhile, patients receiving high-dose and long-term steroid treatment for other diseases besides ALL, patients with hyperglycemia or previous history of diabetes mellitus, and patients whose parents or guardians are not willing to participate in this research, were excluded from the studyThe minimum of sample was calculated using the estimated proportion formula. Based on these calculations, the minimum of samples needed is 52 subjcets. Samples were chosen consecutively from ALL patients undergoing the induction phase of chemotherapy following the Indonesian Protocol of Acute Lymphoblastic Leukemia 2013. The BGL of patients with ALL undergoing the induction phase of chemotherapy were checked in the third week (before they were given L-asparaginase), and once every week up to the sixth week of protocol. Characteristics of the subjects included the origin of the hospital, gender, age (<10 years old or >10 years old), body mass index (BMI) category , CNS infiltration status (Yes or No), leukocyte level >50.000/μl (Yes or No), ALL stratification (standard risk or high risk), Down syndrome (Yes or No), family history of diabetes mellitus (Yes or No), and whether there was infection during the induction phase (Yes or No).Statistical AnalysisThe data were analyzed through Statistical Package for the Social Sciences (SPSS) version 21 software. A descriptive analysis of population was compiled using percentage (%) for categorical variables and mean, median, standard deviation, and maximum and minimum for continuous variables. Fisher exact test was used to determine the association of risk factors with the incidence of hyperglycemia, meanwhile to compare the proportion of post-test result from the prednisone and L-asparaginase group and the dexamethasone and L-asparaginase group were analyzed through Chi-square Test. Paired t-test was used to compare the average of ALL patients before and after chemotherapy. p-value less than 0.05 were considered statistically significant. The dependent variable of this study is hyperglycaemia, while the independent variable is combination between steroid and L-asparaginase.As many as 57 patients from three hospitals were collected. Most of them are from Cipto Mangunkusumo Hospital 57.9% patients. The mean age of the patients was 6.7 years (SD = 3.98). th week and 6th week. On the 4th week p value was 0.614 and on the 6th week p value was 0.373. In rd week and 4th week, 3rd week and 5th week, and 3rd week and 6th week. There is no correlation between ALL stratification with hyperglycemia on both 4rd delta (6th week BGL minus 3rd week BGL). A recode was then conducted based on the mean of 15. With an X2 test, analysis of the influence of different kinds of corticosteroid (dexamethasone and prednisone) towards the mean of 3rd delta was performed , a result of 15,14 were obtained for the 3rmed see . ResultsIn our research, the age interval of the patients is between 1,4 years old up to 15,8 years old, with a mean value of 6.7 years old. Most of the patients come from Greater Jakarta (76.4%) and male patients are more common than female patients (68.4% vs 31.6%). Patients were taken from three different hospitals in Jakarta; Cipto Mangunkusumo Hospital (57.9%), Dharmais Cancer Hospital (24.6%), and Gatot Soebroto Hospital (17.5%).The incidence of hyperglycemia in this research is 3/57 . This value is lower compared to previous research, such as that of Pui et al., (1981) , Baillargeon et al., (2005) (11%), Robertson et al., (2008) (16%), Lowas et al., (2009) (20.4%), Sonabend et al., (2009) (34%), and Koltin et al., (2012) (15.6%). This is due to the age of the patients in our research, who are mostly below 10 years of age (71.9%), with normal BMI value (87.7%), and no history of diabetes mellitus in the family (64.9%). These three factors are most often associated with the incidence of hyperglycemia.Our research did not find a correlation between overweight and the incidence of hyperglycemia. This result may be due to the significantly lower amount of overweight patients as compared to patients with normal weight in our research . On the contrary, Sonabend et al., (2008) had a high amount of patients with obesity (36%), and therefore reported a high prevalence of hyperglycemia in the induction phase.The measurements of BGL did not produce normally distributed data due to a high level of variation within several different outliers. This can be seen in Another big difference can also be seen in the measurement of BGL in week 4. This can be associated with the incidence of hyperglycemia that occurred in a patient in week 4 and 2 patients in week 6. The hyperglycemia incident in week 4 was a transient hyperglycemia. Meanwhile the other two incidences that occurred in week 6 were septic shock with hyperglycemia.In our research, only 1/57 (1.7%) subjects had transient hyperglycemia, while Baillargeon et al., (2005) had 11% had transient hyperglycemia. The difference in the average age of the patients may be the reason. Baillargeon et al., (2005) reported that their patients were between 13-18 years of age. Pui et al., (1981) found that children aged 10 years old or older were more susceptible to hyperglycemia, due to an increase in insulin resistance associated with gonadotropic steroid hormone in puberty. In our research, only 28.1% of subjects were above 10 years of age.None of the patients in our research had diabetic ketoacidosis (DKA). This result is in line with that of Robertson et al., (2008), whose study only had 6 out of 797 patients (0.8%) with DKA, even though they reported a high incidence of hyperglycemia. In our research, there were 2 patients who had hyperglycemia in week 6 with sepsis who then passed away due to septic shock.Secondary hyperglycemia due to acute stress, which increases the production of glucose and insulin resistance, is not unheard of. However, this research did not find any statistically significant correlations between infection and hyperglycemia. This result is different compared to Sonabend et al., (2008), who reported that patients with hyperglycemia had an increased risk of bacteremia/fungemia up to 4.2 fold. This can be caused by a difference in follow-up time, where Sonabend’s research was retrospective and observed for any infection for one year. Another plausible cause is the different characteristics of the subjects, where other contributing factors such as obesity and older age, were scarce in our subjects.A study conducted by Dare et al., (2013) found correlations between hyperglycemia, risk of infection, and early deaths during the induction phase of ALL. However, the incidence of hyperglycemia was also high (36%). The results obtained from our research, although statistically insignificant, were clinically relevant, which is reflected by the two severe infection cases that arose during our research.We found no correlation between ALL stratification and the incidence of hyperglycemia in week 4 (p = 0.614) and week 6 (p = 0.373). There were also no significant correlations between the incidence of hyperglycemia see  and otheOur research has some limitations. The small incidence of hyperglycemia (5.2%) in this research makes it difficult to determine the effect of risk factors on hyperglycemia. Another limitation is the presence of participant bias - patients were informed of the objectives and advantages of this research beforehand, which may have affected their diet during the induction phase of chemotherapy. Different laboratory standards among hospitals, such as measurement tools for BGL may also influence the quality of the data obtained, which should be evaluated later.


The role of molecular changes in the androgen receptor (AR) as AR variants (AR-Vs) is not clear in the pathophysiology of benign prostatic hyperplasia (BPH) and hormone-naïve PCa. The aim of the current work was to identify the presence of AR isoforms in benign tissue and primary PCa, and to evaluate the possible association with tumor aggressiveness and biochemical recurrence in primary PCa. The mRNA levels of full length AR (AR-FL) and AR-Vs  were measured using RT-qPCR. The protein expression of AR-FL (AR-CTD and AR-NTD) and AR-V7 were evaluated by the H-Score in immunohistochemistry (IHC). All investigated mRNA targets were expressed both in BPH and PCa. AR-FL mRNA levels were similar in both groups. AR-V4 mRNA expression showed higher levels in BPH, and AR-V1 and AR-V7 mRNA expression were higher in PCa. The AR-V7 protein showed a similar H-Score in both groups, while AR-CTD and AR-NTD were higher in nuclei of epithelial cells from BPH. These results support the assumption that these constitutively active isoforms of AR are involved in the pathophysiology of primary PCa and BPH. The role of AR-Vs and their possible modulation by steroid tissue levels in distinct types of prostate tumors needs to be elucidated to help guide the best clinical management of these diseases. Prostate cancer (PCa) is the most common neoplasia among Brazilian men, with 68,220 new cases estimated for the year 2018, corresponding to an estimated risk of 66.12 new cases per 100 thousand men, only behind non-melanoma skin cancer , 4. It iAR gene is composed of eight exons encoding a 110 kD protein. The AR protein comprises different domains: an N-terminal domain (NTD), encoded by exon 1; a DNA-binding domain (DBD), encoded by exons 2 and 3; and the ligand-binding domain (LBD), encoded by exons 4 to 8, which is the region where hormone binding occurs [The g occurs , 6. Wheng occurs . Howeverg occurs , 8–12. Ag occurs , 9, 10. g occurs . Some aug occurs ; howeverg occurs , 9, 12. g occurs , 9. In ag occurs . As demog occurs , AR-V7 hTissue samples from patients with primary PCa (n = 61) or BPH (n = 128) were obtained from radical prostatectomy, prostatectomy or transurethral ressection (TURP). For mRNA analysis, 25 samples of primary PCa and 30 of BPH were used. For the immunohistochemical analysis, 6 samples of each group were used. The pathological diagnosis of the tissues was confirmed by the Pathology Service. This study was approved by the Research Ethics Committee of the Hospital de Clínicas de Porto Alegre (HCPA). Informed consent was obtained from all individual participants included in the study. Clinical data for each patient were collected from the hospital electronic records and any of the authors had access to any information which could potentially identify any individual patients.B2M) was used as a reference gene for the stabilization of gene expression levels among the different samples. For amplification of B2M and AR, inventoried assays from Life Technologies™ were used . It is noteworthy that the assay used for AR amplified transcripts 1 and 2 of the receptor (AR-FL). For the design of primers and probes specific for isoforms, the mRNA sequences described by Guo et al. [TP53, MDM2, CDKNA and apoptosis-related genes BAX and BCL2 to study their possible association with AR-Vs and to help towards a better understanding of the molecular mechanisms of AR-Vs. The sequences of primers and probes are shown in Molecular analysis was performed by quantitative PCR (qPCR) following reverse transcription (RT), with a TaqMan® probe detection system (Life Technologies™) using 10-fold diluted samples. The gene for beta-2-microglobulin .We chose to use three different antibodies to investigate AR expression. The PG21 antibody recognizes the N-terminal domain (NTD), which is encoded by AR-FL and AR-Vs. Because of this, antibodies recognizing AR-NTD detect both AR-FL and AR-Vs. The C-19 antibody recognizes the C-terminal domain (CTD), and since LBD-truncated AR-Vs lacks the CTD region, they are consequently not recognized by the C-19 antibody. An alternative approach to detect the presence of AR-Vs is to combine data obtained using antibodies recognizing AR-NTD and AR-CTD, respectively.IHC was performed on 4-μm sections derived from FFPE blocks using the mouse AR-V7 monoclonal antibody (Precision Antibody), diluted 1:200; the rabbit polyclonal AR (C-19) (Santa Cruz), diluted 1:400; and the rabbit polyclonal AR (PG21) (Millipore), diluted 1:400. Antigen retrieval was achieved by heating in a water bath the slides in citrate buffer (pH 6.0) for 1 hour at 95°C. Endogenous peroxidase was blocked using 3% H2O2 solution in methanol for 30 minutes. The reaction was visualized using DAB chromogen  followed by counterstaining with Harris hematoxylin. Cases were scored blind by two evaluators as clinical data using the modified H-score  method, a semiquantitative assessment of staining intensity that reflects antigen concentration , 16. Brit-test or Mann-Whitney U test, respectively. In order to verify the association between the results of isoform gene expression and the other continuous variables, a Spearman’s rank correlation was performed. All P values were two-sided and P<0.05 was considered significant. All statistical analyses were performed using SPSS 20.0 program. Graphs were constructed using SPSS or GraphPad.Data are reported as mean (standard deviation) when parametric, and as median values with interquartile range (IQR) when non-parametric, and analyzed by Student’s Clinical characteristics of patients are described in P = 0.331) (P = 0.041) (P = 0.001) (P<0.001)  . PCa sam= 0.041) . Convers= 0.001) . Using tP<0.001) . When PCBAX, BCL2, TP53, CDKN1A and MDM2, all of these were more highly expressed in the PCa group  than in the BPH group   was quantified separately in the nucleus and cytoplasm of epithelial and stromal compartments . AR-NTD We observed positive staining for the three antibodies in all epithelial samples, which is remarkable since benign tumors (like BPH) and primary cancer are not expected to express AR-Vs, which are supposed to arise only after androgen deprivation therapy. Nuclear stromal staining was positive only for AR-NTD in both groups. Although AR-CTD was detectable in the BPH group, the score was so minimal that it was considered negative. AR-CTD in PCa samples and AR-V7 in both nuclear stromal groups were negative.BCL2 , suggesting that with the increase of tumoral aggressivity, there was an increase of AR-V4 expression.Using Spearman’s rank correlation analysis, we found a positive correlation among AR-Vs . In the P = 0.048), AR-FL/AR-V4  and AR-FL/AR-V4V7  in the BPH group. These associations between ratios and age were not found in the PCa group, but in this group, the ratios AR-FL/AR-V4 and AR-V7rv were positively correlated with BAX .Age at surgery was negatively correlated with the ratios AR-FL/AR-V1  and apoptosis-related genes  are presented in Besides the correlation between cell cycle-related and apoptosis-related genes and AR-Vs and clinical features, these genes were correlated among themselves. The correlations of cell cycle-related genes , represented by the Kaplan-Meyer curve  and ≥7(4+3), high-grade patients were older (P = 0.045) and BLC2/BAX was associated with protection of biochemical recurrence . These associations remained when these variables were tested together in a multivariable model, .In a Cox regression univariable model Table in , the ratWe investigated the AR-FL expression using an assay specific for transcripts 1 and 2 (GI:349501065 and GI:349510166), which prevents untrue conclusions about differences in AR-FL and AR-V expression. We also analyzed AR-V mRNA, and importantly, we detected the expression of all isoforms in samples from hyperplastic tissue and primary PCa. Truncated AR-Vs were previously assumed to be expressed primarily in CRPC, where, at least for AR-V7, their presence was associated with resistance to therapy , 18. Thiin vitro by Watson et al. [Together, our findings suggest that these constitutively active isoforms participate in different proliferative events in different types of prostatic tumors, not only as the main driver of castration resistance. During tumor progression, selection for cells that express AR-Vs could occur, via selection of more malignant or castration-resistant subclones or even via the hormone-dependant regulation of AR isoforms . It has n et al.  that AR-When the PCa group was stratified into Gleason ≤7(3+4) and ≥7(4+3), age was higher in patients with Gleason ≥7(4+3). This indicates that older patients tend to present more aggressive PCa than younger patients. Despite the relatively small cohort number, a greater AR-V expression was seen in PCa samples with a high-grade Gleason score compared to a low-grade Gleason score. AR-V4 and AR-V7rv levels were higher in the high-grade ones. Even without statistical significance, AR-V1 showed a higher distribution in high-grade tumors. In line with these results, we found an inverse correlation between the AR-FL/AR-V4 ratio and the Gleason score, which indicate that AR-V expression increases with higher Gleason scores.et al. [When we stratified PCa cases by whether biochemical relapse occurred or not, we observed a similar expression of all mRNAs analyzed between those patients who had biochemical recurrence and in those who did not present recurrence, which is in concordance with the results reported by Zhao et al. . They shet al. .et al. [Some studies have reported a positive correlation between AR-V7 and AR-V1 in PCa samples , 19, in et al.  observedet al. . The higet al. . AR-V1 het al. , which wet al. , 20, 23.BCL2 with AR-FL and AR-V7rv in PCa suggests an association between the AR-mediated pathways and the antiapoptotic process involved in tumor development. Elevation in the level of the BCL2 protein has been shown to provide protection from apoptosis, and the BCL2 gene family is implicated in the development of CRPC and resistance to therapy since its expression increases during progression of prostate cancers [MDM2 was also positively correlated with AR-FL in PCa, and with AR-V1 in PCa and BPH. However, the interactions among AR, AR-Vs and MDM2 are poorly described. One study suggests that MDM2 induces AR ubiquitination, promoting its degradation [The correlation between  cancers , 25. Theradation . TherefoWe used IHC to investigate protein expression in FFPE samples from BPH and primary PCa. This technique is reliable and widely used in clinical diagnosis. Our approach to detect protein expression was based on three antibodies: AR-NTD (which recognizes AR-FL and AR-Vs), AR-CTD (which recognizes AR-FL) and AR-V7 (specific to this isoform). Using two antibodies against different regions of the AR protein (AR-NTD and AR-CTD), it is possible to successfully show the overall frequency of C-terminal truncated AR-Vs . We obseWe observed considerable heterogeneity in AR staining within samples. Epithelial cells showed positive staining for the three antibodies in both groups. AR-NTD and AR-CTD had a higher H-score (HS) in epithelial nuclei of BPH. AR-NTD was stained less intensively and more heterogeneously in epithelial nuclei and stromal cells of PCa than in BPH, and this is consistent with the report of Miyamoto . AR-V7 sAR-V7 protein expression was similar between the analyzed groups. Our results differ from Guo , whose iFor AR action, nuclear import is an essential step. Different AR-Vs have different abilities to translocate to the nucleus. For example, AR-V7 is expressed predominantly in the nucleus when alone, but other isoforms, like AR-V1 and AR-V4, mainly localize in the cytoplasm . As is aThe use of AR-V7 expression has been proposed as a biomarker for PCa relapse , 20 and However, it is noteworthy that AR-Vs are also expressed in benign tissue from BPH and in primary PCa. All samples used in our research were obtained from patients who had never undergone ADT, so our data do not support the suggestion of variants arising only after therapy, as a response to the treatment. The current idea is that the presence of AR-Vs is also biologically relevant in BPH and primary PCa. In addition, studies to help elucidate the role of AR-Vs in benign tissue and hormone-naïve PCa are needed to better clarify the involvement of these isoforms in the pathophysiology of prostatic tumors.These results support the assumption that the constitutively active AR-Vs are involved in the pathophysiology of prostatic tumors. Our data shows that the AR-Vs participate in different proliferative events in prostate cells, yet the exact role of these isoforms in benign tissue and primary cancer needs to be elucidated. The identification and functional characterization of differentially expressed molecules between normal and tumoral tissues are fundamental steps to achieving a better understanding not only of the carcinogenic process and the development of new anti-tumoral strategies, but also a better understanding of the processes that govern proliferation in benign tissues such as BPH. Additional studies aiming to elucidate in vitro hormonal modulation of AR-Vs may contribute to the understanding of their role in prostate physiology and tumor development and progress.S1 FigP = 0.818).Distribution of the ratio AR-NTD/AR-CTD in epithelial nuclei of samples from BPH and PCa ((TIF)Click here for additional data file.S2 FigP = 0.004).PCa samples were stratified for Gleason score ≤7(3+4) and ≥7(4+3). Samples with Gleason ≥7(4+3), time to recurrence was significantly lower (31 months) than samples with Gleason ≤7(3+4) (65 months) ((PDF)Click here for additional data file.S1 TableP = 0.045) and BLC2/BAX was associated with protection of biochemical recurrence . In the multivariable model these associations remained when these variables were tested together .Ratio AR-V1/AR-FL was associated with a higher risk of biochemical recurrence Click here for additional data file.


Rickettsia asembonensis, the most well-characterized rickettsia of the Rickettsia felis-like organisms (RFLO), is relatively unknown within the vector-borne diseases research community. The agent was initially identified in peri-domestic fleas from Asembo, Kenya in an area in which R. felis was associated with fever patients. Local fleas collected from domestic animals and within homes were predominately infected with R. asembonensis with < 10% infected with R. felis. Since the identification of R. asembonensis in Kenya, it has been reported in other locations within Africa, Asia, the Middle East, Europe, North America, and South America. With the description of R. asembonensis-like genotypes across the globe, a need exists to isolate these R. asembonensis genotypes in cell culture, conduct microscopic, and biological analysis, as well as whole genome sequencing to ascertain whether they are the same species. Additionally, interest has been building on the potential of R. asembonensis in infecting vertebrate hosts including humans, non-human primates, dogs, and other animals. The current knowledge of the presence, prevalence, and distribution of R. asembonensis worldwide, as well as its arthropod hosts and potential as a pathogen are discussed in this manuscript. Rickettsia asembonensis is a Gram negative, obligate intracellular bacteria of the order Rickettsiales and family Rickettsiaceae . The RFLOs are genetically related to R. felis but consist of a unique group of rickettsiae that are associated with various arthropods including fleas, ticks, mites, and tsetse flies for which limited knowledge of their biology and pathogenicity is available  and characterized.tsiaceae . Among Ris Table  . R. typhi is the causative agent of murine typhus, a febrile disease that is found throughout the world. R. typhi is vectored by various flea species-especially X. cheopis, but also other Xenopsylla species such as X. astia and X. brazilliensis  that genetically clusters within the transitional group of rickettsiae  had been detected previously in C. felis near the Thailand-Myanmar border (gltA gene sequence) between Rickettsia sp. RF31 and the latter is notable . The first agent, referred to as Rickettsia sp. RF2125, was detected in Ctenocephalides canis in western Thailand near the Myanmar border  from the original Rickettsia sp. RF2125 DNA preparation was obtained at that same time as the gltA but was not reported in the original article  collected from various domestic animals  and houses (by light traps) in Asembo, Kisumu, in western Kenya during an epidemiologic surveillance study (R. felis was identified in 7.2% of febrile patients (R. asembonensis agent was accomplished utilizing a multilocus sequence typing (MLST) algorithm (R. felis (8.3%)  .Rickettsia asembonensis NMRCii, was successfully cultured from a pool of five individual flea triturate cultures isolated from C. canis and C. felis fleas obtained from domestic dogs. The cultures were initially grown in S2 and subsequently in C6/36 cell lines at 25°C  in the cytoplasm of the host cells, with normal rickettsial size . A cell wall membrane, defined periplasmic space, and cytoplasmic membrane were observed, as well as the electron lucent “halo”  (The culture of croscopy . The R. –45 days . Rickett–45 days . The new–45 days . Transmie layer) . Intranue layer) .R. asembonensis NMRCii by MLST using rickettsial genes rrs, gltA, ompA, ompB, and sca4; plasmid analysis; and whole genome sequencing confirmed that the new agent was indeed a unique Rickettsia species , which is 1.49 Mb in size and contains 1,400 protein-coding genes, 33 tRNA genes, and three rrn operons. Of the R. felis proteins, 1,157 (83%) have homologs in R. asembonensis  and other recognized Rickettsia species was determined  and the name officially changed  from “Candidatus Rickettsia asemboensis” to Rickettsia asembonensis ; Xenopsylla ; Archaeopsylla (A. erinacei); Echidnophaga ; Pulex (P. irritans); and Synosternus . In the family Ceratophyllidae, R. asembonensis has been detected in three genera: Ceratopsyllus (C. fasciatus); Orchopeas (O. howardi); and Nosopsyllus (N. laeviceps) and in one genus in the family Coptopsyllidae: Coptopsylla (C. lamellifer) (as Table . It has ellifer) .R. asembonensis have been reported in C. felis and C. canis (sympatric species), S. pallidus, X. ramesis, and X. gerbilli with up to 95, 95, 91.4, 100, and 33.3% of the fleas positive for R. asembonensis, respectively  in Egypt   , 25–27.R. felis DNA was detected in 3.7 and 7.2% of fever patients' blood  of the patient's blood identified R. sp. RF2125 (R. asembonensis was detected by NGS (gltA sequence similarity) of R. sp. RF2125/”Ca. R. asemboensis” , and its potential for interference with other rickettisal flea-borne pathogens (R. felis and R. typhi), as well as non-rickettsial pathogens such as Yersinia pestis, will be crucial to fully defining its pathogenicity and probability as a public health concern/nuisance across the world.All authors contributed to the conception and design of the review. AM wrote the first draft of the manuscript. JJ, AL-F, HS, CF, and AR wrote revisions of the manuscript. All authors contributed to the manuscript's final version, and read and approved the submitted version.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.


Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by polyglutamine (polyQ) expansion in the androgen receptor (AR). Prior studies have highlighted the importance of AR nuclear localization in SBMA pathogenesis; therefore, in this study, we sought to determine the role of AR nuclear export in the pathological manifestations of SBMA. We demonstrate here that the nuclear export of polyQ-expanded AR is impaired, even prior to the formation of intranuclear inclusions of aggregated AR. Additionally, we find that promoting AR export with an exogenous nuclear export signal substantially reduces its aggregation and blocks hormone-induced toxicity. Moreover, we show that these protective effects are conferred by destabilization of the mutant protein due to an increase in proteasomal degradation of the cytoplasmic AR. Despite a growing body of evidence that global disruption of nucleo/cytoplasmic transport occurs in ALS and HD, our data suggest that no such global disruption occurs in models of SBMA; rather, AR-specific mechanisms, including reduced phosphorylation at Serine 650, are likely responsible for the impaired nuclear export of polyQ-expanded AR. SBMA affects approximately 1 in 40,000 males2, with onset of neurologic symptoms typically occurring between 30 and 50 years of age4. As in other CAG trinucleotide repeat disorders, such as Huntington’s Disease (HD), longer CAG repeat tracts are associated with earlier disease onset6.Spinal and bulbar muscular atrophy (SBMA) is an X-linked neuromuscular disease caused by a polyglutamine-encoding CAG trinucleotide repeat expansion in the first exon of the androgen receptor gene7, causes weakness and cramping of the muscles, dysphagia, dysarthria, and immobility11. Additionally, progressive loss of the bulbar musculature predisposes patients to potentially fatal aspiration-induced pneumonia. While SBMA patients show signs of androgen insensitivity, including gynecomastia and reduced fertility, the neurologic symptoms of SBMA are not caused by a loss of AR function, as neuromuscular symptoms are not observed in patients with complete androgen insensitivity syndrome12. Rather, SBMA is caused by a toxic property of polyQ-expanded AR, as further evidenced by the fact that expression of polyQ-expanded AR in the presence of ligand is sufficient to cause neuromuscular symptoms in mouse models16 and toxicity in cultured cells17. Moreover, while evidence from a Drosophila model of SBMA suggests that SBMA arises from a gain of native transcriptional activities18, findings from a knock-in mouse model of SBMA suggest that enhancing AR transcriptional activity can be protective19.SBMA is characterized by progressive degeneration of lower motor neurons in the brain stem and spinal cord, which, along with a primary myopathy21. Upon binding of testosterone or 5α-dihydrotestosterone (DHT), the AR undergoes a conformational change, inducing its nuclear localization and transcriptional regulation of target genes. There is evidence that the AR is exported from the nucleus and degraded in the cytoplasm by the ubiquitin-proteasome system26.The AR is a steroid hormone receptor that resides in the cytoplasm, in the absence of ligand, in an inactive aporeceptor complex that contains chaperones , p23, and immunophilins 27. The putative nuclear export signal (NES) contained within this region is Leptomycin B-insensitive, suggesting that this regulatory motif is not a candidate for chromosomal maintenance 1 (CRM1) – mediated nuclear export upon hormone withdrawal28. However, a putative CRM1 binding site was identified near the C-terminus of the AR, and the rapid nuclear export of AR that occurs upon inhibition of GSK-3β in prostate cancer cells is blocked by Leptomycin B30. Thus, while AR undergoes slow, Leptomycin B-insensitive export upon hormone withdrawal, it may undergo rapid, CRM1-mediated nuclear export depending on the physiological conditions of the cell29. Interestingly, a similar phenomenon has been observed in the nuclear export of the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily with a high degree of homology to the AR. While GR export induced by ligand withdrawal is slow and Leptomycin B-insensitive, GR undergoes rapid, Leptomycin-B sensitive nuclear export regulated by c-JUN N-terminal kinase (JNK) upon UV exposure31.In contrast with other aspects of AR metabolism, little is known about the mechanism of AR nuclear export. Mutagenesis experiments identified a 75-amino acid region (residues 743–817) in the AR ligand binding domain (LBD) that is both necessary and sufficient for AR nuclear export in a PC3 prostate cancer cell model33. Additionally, phosphorylation of the AR at serine 650 has been shown to regulate nuclear export of the AR35. In COS-7 and LNCaP cells, phosphorylation of S650 is mediated by the MAPK kinase 4/JNK and MAPK kinase 6/p38 stress kinase signaling pathways. Inhibition of JNK and p38 reduced the nuclear export of wildtype AR in COS-7 cells to an equivalent extent as a phospho-null (S650A) mutation but had no effect on AR with a phospho-mimic (S650D) mutation34. Consistent with these findings, knockdown of MAP3k11, an upstream regulator of JNK activity, decreased S650 phosphorylation in LNCaP and C2-4B cells36. Additionally, inhibition of protein phosphatase 1 alpha (PP1α) by tautomycin led to an increase in S650 phosphorylation and a decrease in AR nuclear accumulation in LNCaP cells35. Notably, increasing AR nuclear export was associated with a decrease in its transcriptional activity, while blocking export increased AR transcriptional activity35.In addition to the NES identified in the AR LBD, residues in both the DNA-binding domain (DBD) and the hinge region also appear to regulate AR nuclear export. A double mutation in the DBD  is sufficient to block nuclear export; however, DNA binding itself is not required for AR nuclear export, as the DNA-binding mutant V581F does not affect export37. While the extent to which toxicity in each of these diseases is mediated by nuclear versus cytoplasmic aggregation is not fully understood, targeting polyQ proteins to the nucleus or cytoplasm can modulate aggregation and/or toxicity in several cell and animal models of polyQ disease42. In some polyQ diseases, there is already evidence for defective nuclear export of the polyQ disease protein. For example, polyQ-expanded N-terminal huntingtin fragments exhibited a reduced interaction with the nuclear pore protein translocated promoter region (Tpr), increasing their nuclear accumulation43. Additionally, nuclear export of both mutant ataxin-3 and mutant ataxin-7 was reduced compared with the respective wildtype proteins44, while nuclear export of mutant ataxin-1 was completely blocked45. Thus, nuclear localization appears to play a key role in the pathogenesis of polyQ disease, with evidence for the therapeutic benefit of decreasing nuclear accumulation of some polyQ disease proteins.Nuclear accumulation and aggregation of polyQ disease proteins is a common pathological feature of all nine CAG trinucleotide repeat disorders, highlighting its importance for further study46 and the nuclear localization46 of mutant AR are necessary for toxicity and neuromuscular pathology, with the formation of intranuclear inclusions of aggregated AR in neuronal and non-neuronal tissues a hallmark of the disease state48. Given the requirement for nuclear localization in disease-mediated toxicity, we sought to determine if the nuclear export of polyQ-expanded AR is disrupted and whether enhancing the nuclear export of polyQ-expanded AR would be protective in cell models of SBMA. The experiments presented here represent the first investigation of the role of AR nuclear export in SBMA pathogenesis and offer new insights into the effect of a polyQ-expansion on this important process.In cell and animal models of SBMA, both the presence of hormone55. Using PC12 cells expressing AR10Q (wildtype) or AR112Q (polyQ-expanded), we performed a heterokaryon shuttling assay, a technique previously used to measure the nuclear export of wildtype AR34. Briefly, AR expression was induced in PC12 cells for 24 hrs with doxycycline (DOX). Following DOX washout, the cells were treated for 2 hrs with DHT to induce AR nuclear translocation and with cycloheximide to block protein synthesis from any remaining AR mRNA. Separately, NIH/3T3 mouse fibroblast cells, which lack AR expression, were marked by staining with CellTrackerTM Orange CMTMR Dye, a diffuse cytoplasmic marker that converts to a membrane-impermeable form after cell uptake. These two cell populations were then fused, generating heterokaryons, i.e., cells containing both PC12 and NIH/3T3 nuclei within a shared cytoplasm. Heterokaryons were then treated with DHT and cycloheximide for an additional 4 hrs to allow nucleocytoplasmic transport to occur between the PC12 (donor) and NIH/3T3 (acceptor) nuclei. While some soluble AR oligomeric species were detected by SDS-agarose gel electrophoresis (SDS-AGE) within this timeframe 56 to the N-terminus of the AR and generated a new, stably transfected clonal PC12 cell line expressing NES-AR108Q. As shown in Fig. Having established that polyQ-expanded AR exhibits impaired nuclear export, we next wanted to determine if this aspect of AR metabolism plays a role in the aggregation and toxicity of mutant AR. Previous studies in several cell and animal models of SBMA have highlighted the requirement of AR nuclear localization for disease55. The addition of the exogenous NES reduced the frequency of cells containing intranuclear inclusions  leads to autophagic AR degradation17. Thus, we hypothesized that targeting mutant AR to the cytoplasm with an exogenous NES could increase its degradation by one or both of these pathways. As shown in Fig. In order to understand the mechanism by which increasing the nuclear export of polyQ-expanded AR reduces its aggregation and blocks hormone-induced toxicity, we assessed the stability of NES-AR108Q following DHT treatment. Degradation of wildtype AR by the ubiquitin-proteasome system in the cytoplasm has been widely reported63 – aggregates of misfolded proteins that form when the capacity of the proteasome to degrade these proteins is exceeded (such as when the proteasome is inhibited)64 64 Fig. . 3B5H10-)64 Fig. , suggest)64 Fig.  is the r)64 Fig. . Taken t34, a residue within the hinge region of the AR. Having found that polyQ-AR is deficient in its nuclear export by the heterokaryon shuttling assay, we hypothesized that changes in the phosphorylation state of S650 in polyQ-expanded AR could play a role in its impaired nuclear export. Analysis of AR phosphorylation at Serine 650 (pS650) revealed that polyQ-expanded AR exhibits markedly reduced phosphorylation at S650 compared to wildtype AR  or phospho-mimic (S650D) mutations were established and DOX doses determined to achieve equivalent AR protein levels in the absence of DHT  as well as a polyQ repeat disorder (like HD), we sought to investigate whether cell and animal models of SBMA display evidence of global disruption in nucleocytoplasmic transport, and whether this could provide a mechanism for the impaired nuclear export of polyQ-expanded AR.A number of studies have implicated disruptions in nucleocytoplasmic transport in the pathogenesis of neurodegenerative diseases such as ALS78, and deficient nuclear translocation of this protein was reported in neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSCs)74. In PC12 cells, expression of polyQ-expanded AR did not affect the nuclear/cytoplasmic distribution of NLS-tdTomato-NES . Mislocalization of NLS-tdTomato-NES has been observed in primary cortical neurons expressing mutant huntingtin or TDP-43NES Fig. . This suons Figs  and S2A.79  analysis of cells stably expressing AR10Q-Dendra2 or AR109Q-Dendra2. In the absence of stimulation by UV light, proteins tagged with Dendra2 fluoresce in the green spectrum, allowing for visualization of live cells. This FRAP analysis revealed a modest, but statistically significant, decrease in the absolute fluorescence recovery rate of AR109Q-Dendra2 compared with AR10Q-Dendra2 over time. Additionally, we found a reduction in the maximum fluorescence recovery achieved by AR109Q-Dendra2 post-photobleaching  that the nuclear export of polyQ-expanded AR is impaired, (2) that enhancing the nuclear export of polyQ-expanded AR with an exogenous NES decreases its aggregation and toxicity by promoting its proteasomal degradation, (3) that polyQ-expanded AR exhibits reduced phosphorylation at S650 and that blocking S650 phosphorylation further exacerbates its nuclear export deficiency, and (4) that global disruption of nucleo/cytoplasmic transport is not a common feature of SBMA models.45 correlates with altered protein-protein interactions that contribute to its toxicity by both gain-of-function and loss-of-function mechanisms85. An important question for future studies is whether AR nuclear export becomes acutely impaired at a polyQ tract length of 38, the threshold for fully penetrant disease in SBMA patients1. It also remains to be determined if longer polyQ tract lengths correlate with increasing impairment of AR nuclear export.Deficient nuclear export of mutant AR may contribute to its toxicity, as persistence in the nucleus could lead to aberrant protein-protein interactions, thereby disrupting normal cellular processes. Indeed, lack of nuclear export of polyQ-expanded ataxin-135. Intriguingly, polyQ-expanded AR exhibits substantially reduced pS650 compared with wildtype AR; moreover, genetic mutation to prevent phosphorylation of S650 further disrupted the nuclear export of polyQ-expanded AR. These results suggest that the reduced phosphorylation of S650 in polyQ-expanded AR may contribute to its deficient nuclear export.In order to understand why the nuclear export of mutant AR is impaired, we turned to previously published insights into the mechanism by which wildtype AR undergoes nuclear export. This led to an investigation of the status of S650 phosphorylation of polyQ-expanded AR, as phosphorylation at this residue had been shown to regulate the nuclear export and nuclear accumulation of wildtype AR86. Specifically, expression of the AR coactivator SNURF  has been shown to slow AR nuclear export and increase AR association with the nuclear matrix28. AR phosphorylation at S650 may function similarly, facilitating nuclear export by altering the interactions between AR and its binding partners, perhaps including exportins. Furthermore, changes in the AR interactome that occur upon S650 phosphorylation may also contribute to the process of AR aggregation.Phosphorylation of the AR is known to regulate numerous aspects of AR function by modulating its interactions with coactivatorsWhile reduced pS650 represents one mechanism by which the nuclear export of polyQ-expanded AR is impaired, the regulation of AR nuclear export likely depends on other signals that remain to be identified. Our data indicate that phosphorylation of S650 is not absolutely necessary for AR nuclear export in PC12 cells (as AR S650A 109Q still undergoes nuclear export), nor is a phospho-mimic S650D mutation sufficient to rescue the impaired nuclear export of polyQ-expanded AR. Nevertheless, elucidating the precise mechanism by which pS650 regulates AR nuclear export will be important for developing a better understanding of this process.in vitro studies offer evidence for the role of S650 on AR transcriptional activity, finding that a phospho-null S650A mutation decreases AR transcriptional activity to varying degrees89. In humans, an S650G mutation has been associated with partial androgen insensitivity syndrome90, and alterations in AR phosphorylation at S650 have been reported in breast cancer tumors91. Taken together, S650 appears to regulate several AR functions relevant to SBMA, making it an intriguing subject for further study.The relationship between the role of S650 in regulating AR nuclear export and its regulation of other aspects of AR metabolism could offer new insights into how these pathways converge in SBMA. In the present study, we found that the S650D mutation increased the frequency of cells with intranuclear inclusions of polyQ-expanded AR, demonstrating a role for this residue in AR aggregation. A pressing question is whether facilitation of the slow, CRM1-independent nuclear export mechanism for AR actually contributes to its misfolding and aggregation, as suggested by this observation; resolving this question will be the subject of future investigation. Additionally, 65. We were particularly intrigued by the possibility of pharmacologically directing AR export through CRM1, as this would mirror the mechanism by which the exogenous NES tag we utilized in our experiments directs nuclear export56. In our cell models, however, GSK-3β inhibition had no effect on the subcellular localization of AR. This result, along with our finding that S650 phosphorylation did not affect wildtype AR nuclear export in PC12 cells, suggests that previously reported mechanisms of AR export may not be strong regulators of polyQ-expanded AR nuclear export. This highlights the need for further investigation into the molecular mechanisms underlying the deficient nuclear export of polyQ-expanded AR.Another previously reported mechanism of AR nuclear export that we examined in our cell models is the CRM1-dependent nuclear export of AR that occurs in several prostate cancer cell lines upon inhibition of GSK-3β92. While cytoplasmic AR aggregates have been reported in SBMA patient tissue 94, nuclear aggregates are more frequently observed and are considered to be a hallmark of disease pathology. Our data support the theory that nuclear aggregates do not disrupt nucleo/cytoplasmic transport machinery, highlighting an important difference between the pathology of SBMA and related neurodegenerative disorders.Despite a growing body of evidence that cell-wide disruption of nucleo/cytoplasmic transport is a common pathological hallmark of ALS, HD, and other neurodegenerative disorders, we found no evidence for such disruptions in the SBMA models examined in this study. While we cannot rule out the possibility that nucleo/cytoplasmic transport and/or nuclear membrane structure is perturbed in SBMA patient tissue, in the SBMA models investigated here, which display numerous aspects of AR aggregation and toxicity characteristic of the disease, no global disruption was observed. This finding indicates that, in these models, a general disruption of nucleo/cytoplasmic transport is not present, and thus not responsible for AR nuclear retention and toxicity. Notably, even severely symptomatic transgenic PrP-AR112Q mice exhibit no mislocalization of RanGap1, suggesting that the toxicity caused by expression of polyQ-expanded AR is not mediated by defects in nuclear trafficking. Indeed, our observation that nucleo/cytoplasmic transport is not disrupted in SBMA models agrees with a recent study in which aggregation-prone artificial β-sheet proteins targeted to the cytoplasm, but not to the nucleus, interfered with nucleo/cytoplasmic transport machinery of transfected HEK293T cellsThis study offers the first evidence that the nuclear export of polyQ-expanded AR is impaired, highlighting a novel aspect of AR metabolism that may contribute to the pathogenesis of SBMA. Furthermore, our data support a model in which the mutant AR is rapidly degraded by the proteasome once exported from the nucleus, thereby decreasing its stability, aggregation, and toxicity. Although additional work will be required to fully elucidate the mechanism underlying deficient nuclear export of polyQ-expanded AR, our results provide a new direction for investigations into therapeutic manipulation of the mutant, polyQ-expanded AR in SBMA.2. Experiments were performed utilizing charcoal-stripped serum. For the analysis of AR degradation, cells were treated with 20 μM MG132 (to inhibit the proteasome) or 5 mM 3-MA (to inhibit autophagy) for 24 hrs. Cells were lysed and analyzed by western blot as described below.PC12 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum, 2mM L-glutamine, 100 units/mL penicillin/streptomycin, 200 μg/mL hygromycin, and 100 μg/mL G418 Sulfate. HEK293 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2mM L-glutamine, 100 units/mL penicillin/streptomycin, 15 μg/mL blasticidin S, and 100 μg/mL hygromycin. NIH/3T3 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2mM L-glutamine, and 100 units/mL penicillin/streptomycin. All cells were maintained at 37 °C, 10% COet al., 2001. Stable Tet-On PC12 cells expressing AR10Q or AR112Q were treated with 500 ng/mL doxycycline for 24 hrs to induce AR expression. Separately, NIH/3T3 mouse fibroblast cells were plated in complete media. Prior to fusion, actively growing NIH/3T3 cells were stained with 1 μM CellTrackerTM Orange CMTMR Dye in Optimem for 45 min, then incubated for an additional 60 min in complete media. Concurrently, PC12 cells were washed of doxycycline and treated for 2 hrs with 10 nM DHT  and 10 μg/mL cycloheximide (to inhibit protein synthesis).The heterokaryon shuttling assay for analysis of AR nuclear export was carried out using modifications of the protocol of Black PC12 cells and stained NIH/3T3 cells were trypsinized, mixed in Optimem at a 1:1 ratio, and then pelleted. The cells were fused in suspension with 100 μL 50% PEG-1500 (w/v) added slowly over 2 min. The cell/PEG-1500 mixture was then diluted with 5 mL Optimem, added slowly during an additional 3 min. The fused cells were pelleted and re-plated onto poly-D-lysine coated coverslips in the presence of 10 nM DHT and 10 μg/mL cycloheximide for 4 hrs prior to fixation and immunostaining.et al., 2009 following 20 min fixation in 4% paraformaldehyde. Spinal cord tissue was embedded in OCT and cryosectioned (7μm) prior to fixation. All animal procedures were performed following the guidelines of the Office of Laboratory Animal Welfare and with the approval of the Thomas Jefferson University Institutional Animal Care and Use Committee.Immunostaining was carried out as described by Montie Antibodies used include: AR , AR , AR pS650 , RanGAP1 , Ran , Lamin B1 , HDAC6 , HSP70 , neurofilament-heavy chain , and 3B5H10 . Immunostained cells were visualized using a Leica DMR Fluorescence microscope  and imaged using iVision Mac® or ProgRes® software.In PC12 cells, the percentage of inclusions was determined by counting at least 300 cells in triplicate wells. Experiments were repeated at least three times.Protein extracts were prepared by lysing cells in Triton-DOC lysis buffer . All lysates were sonicated at least three times for 10 s using a Branson cup sonifier. Protein concentration was determined by DC protein assay (Bio-Rad). Lysates were electrophoresed by SDS-PAGE and transferred to 0.45 μm PVDF membrane (Immobilon-P). Blots were incubated with primary antibodies diluted in 5% nonfat milk in TBST  for 60 min at room temperature or overnight at 4 °C and with HRP-conjugated secondary antibodies  for 60 min at room temperature. Detection was performed using ECL substrate (Thermo Fisher) with analysis on a ChemiDoc MP (Bio-Rad).Antibodies used include: AR , AR pS650 , α-tubulin , β-catenin , Lamin B1 , p62 , and GAPDH .Stable Tet-On PC12 cells expressing AR112Q or NES-AR108Q were treated with 500 ng/mL doxycycline  in the presence and absence of 10 nM DHT for 12 days. The percentage of dead cells was determined by counting the percentage of Trypan blue-positive cells; samples were blinded prior to counting. Three independent experiments were performed in triplicate with at least two hundred cells counted per condition.PC12 cells were lysed in Triton-DOC lysis buffer  and diluted 1:1 in non-reducing Laemmli sample buffer. Samples were boiled for 5 minutes and electrophoresed through a 1% agarose gel containing 0.1% SDS in 375 mM Tris-HCL, pH 8.8, for approximately 18 hrs at 4 °C. Lysates were then transferred to a 0.45 μm PVDF membrane at 200 mA for 3 hrs using a semi-dry transfer apparatus . A 4 kg weight was placed on top of the transfer apparatus to accommodate thinning of the agarose gel during transfer. Western analysis was performed using anti-AR  or 3B5H10  antibodies.AR expression was induced with doxycycline in PC12 cell lines expressing AR112Q or NES-AR108Q for 24 hrs. Cells were then washed with PBS to remove media containing doxycycline and treated with media containing 10 nM DHT for 2 hrs. Subsequently, cells were treated with media containing 10 nM DHT and 10μg/mL cycloheximide for 10 hrs. Cells were harvested at the given time points after the final media replacement and analyzed by Western blot, as described above.56.The NES-AR plasmid was constructed by excision of pCMV NES-AR   with SnaBI/EagI and ligation into a preexisting pTRE AR112Q plasmid by sequential digests with NheI  fragment) and EagI. The NES utilized in this study (ATGTTAGCCTTGAAATTAGCAGGATTAGACATC) was originally identified in cAMP-dependent protein kinase inhibitor alphaAR-Dendra2 plasmids were constructed by mutation of the AR STOP codon to an AgeI restriction enzyme site by PCR, excision of the AR C-terminus from this PCR product by BstBI/AgeI digestion, and ligation into a pGWI-Dendra2 plasmid . C-terminally tagged AR-Dendra2 was then excised with BstBI/XbaI and ligated into preexisting pTRE AR10Q and pTRE AR112Q plasmids.Mutation of AR Serine 650 to alanine or aspartate was performed by site-directed mutagenesis  of the pTRE AR10Q and pTRE AR112Q plasmids. The AR coding region was then sequenced to verify the absence of secondary mutations.Stable transfection of Tet-On PC12 cells with these AR constructs and a plasmid conferring hygromycin resistance (pTK-hygromycin) in a 1:4 molar ratio was then performed using lipofectamine 2000 according to the manufacturer’s instructions. Stable transformants were selected with 200 μg/mL hygromycin. Single colonies were isolated and screened for AR protein expression by induction with 1 μg/mL doxycycline and western blotting using an anti-AR antibody . Genomic DNA was extracted from positive clones, and the CAG repeat length was verified by PCR and sequencing analysis. The concentration of doxycycline required to induce equivalent levels of AR protein between cell lines was determined and used for all experimentation.PC12 cells were pelleted in PBS and lysed in NP-40 buffer  supplemented with protease and phosphatase inhibitors. Lysates were incubated on ice for 30 min and cleared by centrifugation. Protein concentration was determined using a DC protein assay (Bio-Rad) and samples were diluted to 1 mg/mL for the IP reaction with 10μg reserved for the input control. 0.5 mg of lysates was incubated with 1μg of AR antibody (BD Biosciences) or mouse IgG, crosslinked to Dynabeads M-270 Epoxy (Invitrogen) overnight at 4 °C with rotation. Immunoprecipitated protein was washed 3–4 times with lysis buffer and eluted with Laemmle buffer for western analysis.PC12 cells were plated on poly-D-lysine coated glass coverslips and treated with 500 ng/mL doxycycline and 10 nM DHT for 24 hrs. Duolink® PLA Fluorescence protocol was performed per manufacturer’s instructions  with primary antibodies against total AR  and AR pS650 . Images were acquired with the same exposure time.95. Briefly, spinal cords were dissected from embryonic day 13.5 PrP-AR112Q transgenic mice, dissociated with trypsin, and plated on poly-D-lysine coated coverslips in 24-well plates. Cultures were grown in glia-conditioned media containing minimal essential medium, 3% charcoal-stripped horse serum, 35 mM NaHCO3, 0.5% dextrose, 1% N3, and 10 nM 2.5 S nerve growth factor for 3 weeks. Cultures were then treated with either ethanol or 10μM DHT for 7 days. Motor neurons were identified by the presence of neurofilament-heavy chain (SMI32 immunoreactivity) and morphology.Dissociated spinal cord cultures were established as previously described2. Cells of approximately the same starting intensity were selected for photobleaching in a designated region of interest (ROI) within the cell nucleus, excluding the nucleolus. Photobleaching was performed using the 490 nm later at 10% power for 4 iterations. Fluorescent recovery within the bleached zone was measured every 0.5 sec for 30 sec, using the 490 nm later at 0.7% power. Absolute fluorescent recovery was determined by the percent of fluorescent recovery relative to the starting (pre-bleach) fluorescent intensity of the ROI. Maximum recovery represents the greatest percent of fluorescent recovery within an ROI independent of time.PC12 cells stably expressing AR10Q-Dendra2 or AR109Q-Dendra2 were plated on poly-D lysine-coated glass bottom dishes (MatTek Corp). AR expression was induced with 500 ng/mL doxycycline and cells were treated with 10 nM DHT for 24 hrs. Prior to imaging, media was replaced with Opti-MEM I minus phenol red (Gibco), supplemented with 500 ng/mL doxycycline and 10 nM DHT. Live cell imaging was performed on a Nikon A1R microscope with cells maintained at 37 °C and 5% COFor pairwise comparisons, statistical significance was determined by two-tailed Student’s t-test unless otherwise noted in figure legends. For multiple comparisons, statistical significance was determined by one-way analysis of variance (ANOVA) with post-hoc Tukey test. For comparison of distributions, statistical significance was determined by the Kolmogorov-Smirnov test. The significance level (α) was set at 0.05 for all experiments. Data were compiled and analyzed using Microsoft Excel or Prism 7 (GraphPad).Supplementary Document