Providing additional Saturday rehabilitation can improve functional independence and health related quality of life at discharge and it may reduce patient length of stay, yet the economic implications are not known. The aim of this study was to determine from a health service perspective if the provision of rehabilitation to inpatients on a Saturday in addition to Monday to Friday was cost effective compared to Monday to Friday rehabilitation alone.Cost utility and cost effectiveness analyses were undertaken alongside a multi-center, single-blind randomized controlled trial with a 30-day follow up after discharge. Participants were adults admitted for inpatient rehabilitation in two publicly funded metropolitan rehabilitation facilities. The control group received usual care rehabilitation services from Monday to Friday and the intervention group received usual care plus an additional rehabilitation service on Saturday. Incremental cost utility ratio was reported as cost per quality adjusted life year (QALY) gained and an incremental cost effectiveness ratio (ICER) was reported as cost for a minimal clinically important difference (MCID) in functional independence.996 patients (mean age 74 (standard deviation 13) years) were randomly assigned to the intervention (n = 496) or the control group (n = 500). Mean difference in cost of AUD$1,673  -271 to 3,618) was a saving in favor of the intervention group. The incremental cost utility ratio found a saving of AUD$41,825  per QALY gained for the intervention group. The ICER found a saving of AUD$16,003  in achieving a MCID in functional independence for the intervention group. If the willingness to pay per QALY gained or for a MCID in functional independence was zero dollars the probability of the intervention being cost effective was 96% and 95%, respectively. A sensitivity analysis removing Saturday penalty rates did not significantly alter the outcome.From a health service perspective, the provision of rehabilitation to inpatients on a Saturday in addition to Monday to Friday, compared to Monday to Friday rehabilitation alone, is likely to be cost saving per QALY gained and for a MCID in functional independence.ACTRN12609000973213Australian and New Zealand Clinical Trials Registry November 2009 There is debate about the potential advantages to the health system and broader community of providing health services on the weekend, such as those provided in intensive care and emergency departments,2. The pRehabilitation is an important part of the health system and involves specialized, coordinated, multidisciplinary care to restore functional independence in patients. There iBefore weekend rehabilitation services can be implemented routinely into practice it is important to find out whether they are cost effective from the perspective of the health service. Studies are needed to investigate if the increased cost of a weekend rehabilitation service is offset by savings within the inpatient episode of care. Economic evaluations of additional weekday inpatient rehabilitation programs have shown a lower cost per patient admission-12, but The aim of this study was to undertake cost utility and cost effectiveness analyses alongside a multi-center, single-blinded randomized controlled trial with concealed allocation. The primary research question was to determine from a health service perspective if the provision of a rehabilitation service to inpatients on a Saturday in addition to Monday to Friday compared to Monday to Friday rehabilitation alone, was cost effective per quality adjusted life year (QALY) gained and for a minimal clinically important difference (MCID) in functional independence.An economic evaluation was completed alongside a single-blinded randomized controlled trial that examined both the clinical effects and costs of providing a rehabilitation service  to inpatients on a Saturday in addition to Monday to Friday compared to Monday to Friday rehabilitation alone, for a mixed adult cohort of patients admitted for inpatient rehabilitation. The evaluation was completed from a health service perspective. The trial was registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12609000973213) prior to patient recruitment. Full details of the protocol). The trParticipants randomized to the intervention group received a rehabilitation service on a Saturday in addition to usual care Monday to Friday rehabilitation compared to the control group who received usual care Monday to Friday rehabilitation alone. Usual care typically consisted of interventions focused on task-specific training and discharge planning for independent living in the community. Patients were scheduled to receive about one hour of physiotherapy and one hour of occupational therapy each weekday as well as full nursing, medical and other allied health services. Over the weekend, usual care included full nursing care and a limited medical service to address high priority medical needs. In addition to usual care, the intervention group received rehabilitation therapy on a Saturday. They were scheduled to receive an hour each of physiotherapy and occupational therapy. Additional physiotherapy and occupational therapy were provided because they are the most frequent forms of therapy intervention during inpatient rehabilitation-17, withFrom July 2010 until June 2011, eligible participants who were 18-years-old or older and admitted to one of two rehabilitation facilities were invited to participate. Patients with any orthopedic, neurological or other disabling condition were included. Patients were excluded if they did not give informed consent, were admitted for slow stream geriatric rehabilitation or if they were participating in another intervention trial.Patients were randomized to the intervention or the control group using a concealed method, with 1:1 allocation. The block allocation sequence was generated electronically and assignments concealed in sequentially numbered, sealed, opaque envelopes. Only after the participant was enrolled in the trial and had completed baseline testing was group assignment made by opening the next envelope in the sequence.Based on one of the primary outcome measures (length of stay) from a pilot study, a samplThe trial took place at two publicly funded metropolitan inpatient rehabilitation facilities with a combined total of 90 rehabilitation beds . Prior to being accepted for inpatient rehabilitation, patients are typically assessed in an acute hospital as being able to participate actively in rehabilitation with the expectation that they will improve sufficiently to return to independent living in the community.Patient length of stay for the rehabilitation admission was measured as the number of overnight stays in the rehabilitation unit, from the day of admission until the day of discharge, inclusive of any transfers to an acute ward to manage medical conditions. Any unplanned readmissions to the health service in the 30 day period immediately post discharge from rehabilitation were also recorded and included in the total length of stay. This period was considered relevant in the economic evaluation as readmissions account for a significant portion of all health care expenses and one third of all hospital readmissions are within one month of discharge.Time units for utilization of allied health services during the rehabilitation admission were recorded on the hospital database. This captured Monday to Friday and Saturday interventions for physiotherapy and occupational therapy. Data source, unit definition and unit cost for each of these resources included in the economic evaluation are specified in Table Cost data were collected from the health service and covered two financial years; 2010/2011 and 2011/2012. Data from the 2011/2012 financial year (6% of sample) were discounted at a rate of 3.5%, consistent with the corresponding national Consumer Price Index, so all Total cost included the cost of the rehabilitation admission, acute care costs during the rehabilitation admission, and the cost of any readmission during the 30 day period post discharge from rehabilitation Table . Cost daThe EuroQol (EQ-5D-3L) questionnaire was convt-test to report statistical significance[Outcome and cost data were analyzed according to the intention-to-treat (ITT) principle with multiple imputation used to account for missing outcome data-29 but wificance. IncremeConfidence intervals around the individual ICERs for the quality of life utility score and the functional independence score were calculated using the bootstrap method , the change in outcome measures from admission to discharge and total cost. IndividThe ICER for the quality of life utility score represents the cost per QALY gained. The ICER for a one point change in the functional independence score was multiplied to report the cost difference for a 22 point change in the functional independence score, representing the cost difference for a MCID in functional independence. The likelihood of achieving a 22 point MCID in functional independence was reported as a relative risk (RR) between groups. The rate of change per day was calculated for quality of life utility and functional independence scores by taking the difference between admission and discharge for each outcome and dividing by each patient’s length of stay. Patient readmissions 30 days post discharge from rehabilitation were reported as a relative risk (RR) between groups.Analyses were completed using IBM SPSS Statistics Version 21 and custA sensitivity analysis was performed by removing the penalty loading for Saturday physiotherapy and occupational therapy wage rates. The reason for this is because the working week and the weekend may differ by country, for example in some settings and cultures Saturday is regarded as a normal working day. ConsideA total of 996 patients were randomized to the control group (n = 500) or the intervention group (n = 496) with the flow of the patients through the trial reported elsewhere[Patients had a mean age of 74 years (standard deviation (SD) 13) and 631 (63%) were women. The groups appeared similar for diagnosis and co-morbidities Table . There wParticipants in the intervention group received on average an additional 53 minutes of rehabilitation therapy (95% CI 31.0 to 74.1) per week compared to the control group  for the intervention group and 28.7 days (SD 32.2) for the control group, with a mean difference of -2.9 days (95% CI -6.5 to 0.7) in favor of the intervention group  for the intervention group and 23.1 days (SD 20.2) for the control group, with a mean difference of -1.9 days (95% CI -4.1 to 0.4) in favor of the intervention group. In the 30 day period post discharge from rehabilitation, readmission average length of stay was 4.6 days (SD 16.2) in the intervention group compared to 5.6 days (SD 18.9) in the control group, with a mean difference of -1.0 day (95% CI -3.2 to 1.2) in favor of the intervention group  was $15,859  for the intervention group and $17,532  for the control group, with a mean cost difference of -$1,673  in favor of the intervention group Table .The initial rehabilitation admission cost was on average $13,320 SD 9,894) for the intervention group and $14,275  for the control group, with a mean cost difference of -$955  in favor of the intervention group. In the 30 day period post discharge from rehabilitation, the average cost of admissions back to the health service was $2,539  for the intervention group and $3,257  for the control group, with a mean cost difference of -$718  in favor of the intervention group   per QALY gained for the intervention group compared to the control group. The incremental cost effectiveness ratio showed a cost saving of $727  for a one point change in the functional independence score for the intervention group compared to the control group. This equates to a cost saving of $16,003  for a MCID in functional independence for the intervention group compared to the control group. To put these outcomes in context, patients in the intervention group were 17% more likely to achieve a MCID in functional independence  at discharge compared to those in the control group.The ICER ellipses for confidence intervals  are presented in Figures Figures Removing the penalty loading for Saturday therapy staff, the health service cost during the initial rehabilitation admission and subsequent admissions in the 30 day period post discharge from rehabilitation was on average $15,824  for the intervention group and $17,532  for the control group, with a mean difference of -$1,708  in favor of the intervention group. This did not alter the results of the primary analysis.Our results show a significant improvement in both outcome measures  as well as a difference in costs in favor of the provision of a rehabilitation service to inpatients on a Saturday in addition to Monday to Friday compared to Monday to Friday usual care rehabilitation alone. The confidence interval ellipses for the ICERs show that all the 50%, 75%, and most of the 95% confidence intervals fall within the bottom right hand quadrant suggesting that the intervention is likely to be dominant over the comparator of usual care. Therefore, from a health service perspective, the provision of a rehabilitation service to inpatients on a Saturday in addition to Monday to Friday compared to Monday to Friday rehabilitation alone, is likely to be cost saving per QALY gained and for a MCID gained in functional independence.The National Institute for Health and Clinical Excellence (NICE) in the UK reports This economic evaluation may have important implications for health services that offer inpatient rehabilitation, with potential to reduce costs per admission, improve patient outcomes and improve patient access. When considering the health service perspective, it is reported that in the inpatient rehabilitation setting the patient length of stay is the largest contributor to health care costs,39. ThisDespite these benefits, implementation of this model of care also needs to consider workforce redesign. Traditionally, allied health clinicians work Monday to Friday, so the clinicians providing rehabilitation services may be reluctant to change their work practices by working on the weekend. This maThe strengths of this economic evaluation are that it was completed alongside a blinded fully powered randomized controlled trial, it used an appropriate alternative intervention as 70% of Australian rehabilitation inpatient health services do not offer a weekend physiotherapy service, and it From a health service perspective, the provision of a rehabilitation service to inpatients on a Saturday in addition to Monday to Friday compared to Monday to Friday rehabilitation alone, is likely to be cost saving per quality adjusted life year gained and per minimal clinically important difference gained in functional independence.ANCOVA: analysis of covariance; AUD$: Australian dollars; CEAC: cost effectiveness acceptability curve; CHEERS: Consolidated Health Economic Evaluation Reporting Standards; CI: confidence interval; EQ-5D-3L: EuroQol; ICER: incremental cost effectiveness ratio; ITT: intention to treat; MCID: minimal clinically important difference; NICE: National Institute for Health and Clinical Excellence; QALY: quality adjusted life year; RR: relative risk; SD: standard deviation; TTO: time trade-off.The authors declare that they have no competing interests.NKB, JJW, NS and NFT conceived the clinical trial and economic evaluation design, analyzed the data, interpreted the results and drafted the manuscript. NFT and NKB are the guarantors. All authors had access to all of the data in the economic evaluation and can take responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/12/89/prepubConsolidated Health Economic Evaluation Reporting Standards (CHEERS) Checklist. Items to include when reporting economic evaluations of health interventions.Click here for file


Currently there is no reliable medical treatment for aortic regurgitation (AR).Thirty-nine Sprague–Dawley rats underwent creation of AR or sham operation. Treated rats were assigned to early or late institution of sildenafil therapy (100 mg/kg/day) for a total of 10 weeks. Treatment–effects were measured by serial echocardiography, invasive hemodynamic measurements, and tissue analysis.Rats assigned to early treatment developed less remodeling than untreated rats. Thus, left ventricular (LV) dilation was blunted by sildenafil with end–systolic diameter being significantly smaller . Also, LV wall thickness was significantly decreased in treated rats compared to controls . Fractional shortening was improved by treatment (p < 0.05). Myocardial fibrosis was borderline decreased by treatment (p = 0.09). Akt was increased in treated compared to controls (p < 0.05).Sildenafil slightly inhibits LV remodeling and improves fractional shortening in rats with AR when treatment is initiated early. Aortic regurgitation (AR) is a common valvular disease that affects men more than women, and whose incidence increases with age Maurer . Severe Three rats died during the study–period; one from the AR (early) group, one from the AR + SIL (late) group and one from the AR (late) group. Only data from surviving rats were included in the final analysis.AR resulted in classic eccentric hypertrophy and led to rightward shift of PV–loops indicating LV remodeling, Fig. Echocardiography showed AR + SIL (early) rats to develop less remodeling than AR (early). LV dimensions increased in all rats with AR, starting immediately after AR–induction. In AR (early) LV dilation continued throughout the study-period while treatment with sildenafil blunted this response, Fig. As seen in Fig. In contrast, invasive hemodynamic measurements showed no differences in measurements of LV performance or filling between treated and untreated groups, Table In the early treated group, histological analysis showed a borderline significant decrease in subendocardial collagen fibrosis compared to AR (early) (p = 0.09), Fig. We did not find any difference in phosphorylated levels of Akt S473 and Akt S308 between controls and sham–operated rats, while we found an increase in phosphorylated Akt S473 in AR + SIL compared to AR (p = 0.002). Total calcineurin and phosphorylated ratio of CamKII, ERK, JNK, and p38 were unchanged between groups, Fig. max and dp/dtmin in AR + SIL (late) compared to AR (late), Table Echocardiographic measurements of LV remodeling showed weaker effect of sildenafil treatment in the late treated group compared to the early treated. We did not find any significant differences in overall analysis of LV remodeling. However we saw a possible trend towards less wall thickness and LV mass (not shown) in AR + SIL (late) compared to AR (late) . Echocardiography showed ventricular function to be unchanged by treatment Fig. , as FS, In this study we demonstrate the effects of PDE5-inhibition in an experimental model of AR. We found PDE5-inhibition to blunt LV remodeling and LV dysfunction, when treatment was administered early. Although only borderline significant, the LV changes occurred along with a small reduction in subendocardial collagen fibrosis. Protein analysis showed increased levels of Akt in early treated rats while five known key pro–hypertrophic proteins were unaltered by treatment. In rats treated late with sildenafil, similar salutary effects were not noted. Our results indicate mild positive effects of PDE5-inhibition in experimental AR, however with a specific treatment-window wherein treatment is effective.In humans the clinical course of AR is slow and more benign than other cardiac overload conditions , early initiated sildenafil treatment has shown to inhibit development of LV hypertrophy and collagen fibrosis  injection. Treatment with a PDE5A-inhibitor was initiated after 2 weeks of AR. Administration was performed by mixing sildenafil  in transgenic dough–diet  that was fed to rats daily for a total of 10 weeks. All animal experiments were approved by the Johns Hopkins University Institutional Animal Care and Use Committee.AR was induced by echocardiography-guided closed-chest operation, described in detail elsewhere  were divided into three groups: Rats with AR treated with sildenafil beginning at 2 weeks after AR (AR + SIL (early), n = 8), rats with severe AR receiving no treatment (AR (early), n = 8), and sham-operated rats receiving no treatment . Sham-operated rats had all procedures performed except perforation of aortic valves. Rats were sacrificed 12 weeks after baseline examination.Fifteen male Sprague–Dawley rats (age 9–10 weeks) were subjected to AR and divided into two groups: Rats with AR treated with sildenafil beginning at 12 weeks after AR , and rats with severe AR receiving no treatment . Rats were sacrificed 22 weeks after baseline examination.Early study: 0 (baseline), 2, 6, 9 and 12 weeks after AR. Late study: 0, 2, 6, 9, 10, 12, 16, 19, and 22 weeks after AR.Echocardiography was performed on a Vivid 7 machine  with a 14 MHZ linear vascular probe. Examinations were performed at following time points: −1. Tracking was performed by using at least three consecutive cardiac cycles in EchoPAC’s strain modality analysis software . The global trace representing averaged circumferential deformation was used. For strain-rate measurements the global traces were used.Diameters, wall thickness (WT), and shortening fraction (FS) were assessed by M–mode. Measurements were averaged from four beats, two parasternal long–axis (PLAX) and two short–axis (SAX) planes. FS was calculated as [LVEDD − LVESD]/[LVEDD]. As a measure of WT, septum and LV posterior walls were averaged. Speckle tracking was performed by acquiring 2D cine loops from at least six cardiac cycles, in mid-ventricular SAX plane at frame rates >70 sInvasive LV hemodynamic measurements were performed at baseline and before sacrifice. In anesthetized rats a micro–tip conductance pressure–volume catheter  was inserted into the heart by right–sided carotid arteriotomy. Pressure–volume loops (PV-loops) were obtained under resting conditions.Formalin (10 %) fixed and paraffin embedded myocardium from LV was sectioned into 5 µm thick slices and stained with H&E and Masson’s trichrome. For analysis, four transmural areas from each circumferential LV-section (one from each rat) were extracted using Scanscope software . For fibrosis quantification FRIDA  was used. This is a custom open-source image analysis software package for color image datasets. Epicardium, pericardium and larger vessels were excluded from this analysis.Tissue samples from snap–frozen hearts were prepared for protein analysis as described elsewhere .AR was documented by comparing sham-operated and untreated (AR) rats. The effect of treatment was documented by comparing medically treated (AR + SIL) with untreated (AR) rats. The difference between multiple groups was analyzed with ANOVA and Tukey’s test for multiple comparisons. Serial echocardiography was compared by repeated measures. All p values are two-tailed and a significance level of 0.05 was used. Statistics are given as mean ± standard error unless stated otherwise. All analysis were performed using SASExperimental AR is different to what is seen clinically, mainly due to the acute nature of the model. However this is a limitation in most experimental valve models and was accounted for by postponing measurements until after the acute phase, which allowed adaptation to the new loading conditions (Olsen et al. Ten weeks of medical treatment is similar to what has been previously used (Takimoto et al. Our sample size is modest but adequate to demonstrate significant differences in the early treatment group. A larger sample size may have revealed additional significant effects.As in clinical settings, speckle tracking imaging faces limitations with frame rates, which is amplified with fast heart rates. However, in small animals the shortened diastolic filling time is followed by a systolic phase that is as long or even longer, which allows >7 frames to reconstruct the systolic part of the strain curve. This number is similar to what is used in human MR tagging studies.In a rat model of chronic severe AR, we found sildenafil to mildly inhibit left ventricle remodeling and systolic dysfunction when treatment was started early in the course of disease.


Manon J. N. L. Benders is also affiliated with: Centre for the Developing Brain, Division of Imaging Sciences and Biomedical Engineering, King's College London and King's Health Partners, St. Thomas' Hospital, London, United Kingdom.An affiliation for the 2


Heredity and environmental exposures may contribute to a predisposition to allergic rhinitis (AR). Autoimmunity may also involve into this pathologic process. FCRL3 (Fc receptor-like 3 gene), a novel immunoregulatory gene, has recently been reported to play a role in autoimmune diseases.This study was performed to evaluate the potential association of FCRL3 polymorphisms with AR in a Chinese Han population.Five single-nucleotide polymorphisms of FCRL3, rs945635, rs3761959, rs7522061, rs10489678 and rs7528684 were genotyped in 540 AR patients and 600 healthy controls using a PCR-restriction fragment length polymorphism assay. Allele, genotype and haplotype frequencies were compared between patients and controls using the χ2 test. The online software platform SHEsis was used to analyze their haplotypes.This study identified three strong risk SNPs rs7528684, rs10489678, rs7522061 and one weak risk SNP rs945635 of FCRL3 in Chinese Han AR patients. For rs7528684, a significantly increased prevalence of the AA genotype and A allele in AR patients was recorded. The frequency of the GG genotype and G allele of rs10489678 was markedly higher in AR patients than those in controls. For rs7522061, a higher frequency of the TT genotype, and a lower frequency of the CT genotype were found in AR patients. Concerning rs945635, a lower frequency of the CC genotype, and a higher frequency of G allele were observed in AR patients. According to the analysis of the three strong positive SNPs, the haplotype of AGT increased significantly in AR cases .This study found a significant association between the SNPs in FCRL3 gene and AR in Chinese Han patients. The results suggest these gene polymorphisms might be the autoimmunity risk for AR. Allergic rhinitis (AR) is defined as an IgE-mediated nasal inflammation, which is induced by allergens and regulated by T lymphocytes . AR has It has been reported that numerous loci and candidate genes show associations with AR , 9. In oFc receptor-like genes (FCRLs), also known as FCRHs (Fc receptor homology), locate at the 1q21–23 region . FCRLs hRecently, polymorphisms of FCRL3 have been reported to be associated with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) , Behcet’Five hundred and forty AR patients referred to us from January 2012 to July 2013 were included in this study. All the patients were of Chinese Han ethnic origin, and all were from the Chongqing city and its neighboring townships in the southwest region of China. All the patients were from and had been treated at the outpatient department of Otorhinolaryngology of the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. The diagnosis of AR was based on the patients’ medical history, symptoms and the presence of a positive skin prick test  in response to a panel of common allergens defined by the ARIA 2008 guidelines . The SPTThe local ethics authorities  provided permission and helped to obtain informed consent from all participants. Written informed consent was obtained from all participants.We studied 5 SNPs rs945635, rs3761959, rs7522061, rs10489678 and rs7528684 in the FCRL3 region on 1q21–23, which were demonstrated earlier by other groups to be associated with certain immune-related diseases. SNP rs7528684 lies in the promoter region, while SNPs rs945635 and rs7522061 are located in the exon2 and exon4 region, SNPs rs3761959 and rs10489678 are located in the intron region in FCRL3 gene, respectively. Genomic DNA was extracted from EDTA-anticoagulated peripheral blood leukocytes using the Wizard Genomic DNA Purification Kit method. Briefly, 300 µl of blood was mixed with cell lysis solution. Leucocytes were spun down and lysed with Nuclei Lysis Solution. The pellet was separated out by Protein Precipitation Solution. Precipitated proteins were removed by centrifugation. Then, two-filar DNA was separated out by methyl alcohol. The DNA on the EP tube was dissolved in 100 μL DNA Rehydration Solution.These five SNPs were genotyped by restriction fragment length polymorphism analysis (PCR-RFLP). The amplification was performed using initial denaturation at 95°C for 5 min, 95°C for 30 s, 56–60°C for 30 s, 72°C for 30 s, and 72°C for 5 min followed by 37 cycles. The PCR products were incubated with restriction enzymes  for at lP values of less than 0.05 were considered statistically significant. To evaluate the quality of the genotyping data, the Hardy–Weinberg equilibrium (HWE) for SNP genotype frequencies was tested using a chi-square teat (χ2 test). Allelic and genotypic frequencies between patients with AR and the control patients were compared by χ2 test. The online software platform SHEsis  was used to analyze the haplotype and probabilities. The association between genotypes/alleles and the AR risk was estimated by calculating odds ratios (OR) and 95% confidence intervals (CI).All statistical analyses were performed using SPSS version 18.0 software . The demographics of the cases and controls enrolled in this study are shown in P>0.01). The genotype and allele frequencies of these five SNPs tested FCRL3 polymorphisms are shown in Our results showed that FCRL3 SNPs rs945635, rs3761959, rs7522061, rs10489678 and rs7528684 were in Hardy–Weinberg equilibrium in the cases and controls  = 1.61×10-9, OR [95% CI] 2.13 [1.67–2.72]; Pc = 1.06×10-8, OR [95% CI] 1.65 [1.39–1.95], respectively). The frequencies of the AG and GG genotype and the G allele were significantly lower in the AR patients than in the controls .The results showed that there were significant differences between the AR patients and control patients concerning the frequencies of rs7522061, rs10489678 and rs7528684. A significantly increased prevalence of the rs7528684 AA genotype and A allele was found in the AR patients compared to the controls  and the G allele , and a lower frequency of the AG genotype  and the A allele  were found in AR patients compared with the controls.For rs10489678, a higher frequency of the GG genotype  and T allele , and a lower frequency of the CT genotype  and C allele  were found in AR patients.Concerning rs7522061, a higher frequency of the TT genotype  and C allele , and a higher frequency of G allele  were observed in AR patients.For rs945635, a lower frequency of the CC genotype . We also found that the distribution rate of the CGTGG and GGCGG haplotypes were notably decreased in the AR patients compared to the control patients . Furthermore, as shown in P = 8.29×10-14, OR [95% CI] 1.978 [1.652~2.368]). On the contrary, GGC, GGT decreased markedly in AR group .Haplotype analysis was performed using the Haploview V3.32 program and the online software platform SHEsis. The 18 possible haplotype frequencies are shown in In short, the CGTGA, GATGA and GGTGA haplotypes were associated with a significantly increased risk of AR, while CGTGG and GGCGG were associated with a notably decreased risk of AR. Moreover, when the haplotypes of the three positive SNPs were analyzed, the haplotype of AGT increased significantly in AR cases, while GGC, GGT were associated with a markedly decreased risk of AR.FCRL3 in human encodes a type I transmembrane protein harboring both cytoplasmic ITAM and ITIM elements that can repress B-cell receptor activation. Despite this inhibitory property, mounting associations for FCRL3 with autoimmune and lympho-proliferative disorders imply a role for it in promoting B-cell pathogenesis. High FCRL3 expression on B-cells and augmented autoantibody production were observed in individuals with the disease-susceptible genotype. Associations were also found between the SNP and susceptibility to autoimmune thyroid disease and systemic lupus erythematosus. Thus, FCRL3 may play a pivotal role in autoimmunity. As an important genetic autoimmunity factor, it functions as the mediator between the innate and adaptive immune system. AR, as an atopic desease, is also regulated by the innate and adaptive immune system, especially the imbalance of Th1/Th2 Cells. However, no public literature has revealed the association between polymorphisms of FCRL3 and AR until now. This study was performed to evaluate the potential association of FCRL3 polymorphisms with AR in a Chinese Han population.In this study, we investigated the FCRL3 gene polymorphisms in a Chinese population with AR. The results demonstrated a significant association between SNPs rs7528684, rs10489678, rs7522061 in the FCRL3 gene and AR in a Chinese Han population. For rs7528684, which lies in the promoter region of FCRL3, the AA genotypes and the A allele significantly increased the risk of AR, but the AG, GG genotypes and the G allele decreased the risk of AR. Concerning rs10489678, located in the intron region of FCRL3, the GG genotype and the G allele were risk factors for AR, but the AG genotype and the A allele provided protection. For rs7522061, located in the extron4 region of FCRL3, the TT genotype and the T allele significantly increased the risk of AR, but the CT genotype and the C allele decreased the risk of AR. There is also a weak protective association between SNP rs945635 (in the exon2 region of FCRL3) and AR cases at the CC genotype and the G allele. However, the expression of SNP rs3761659 (in the intron region of FCRL3) shows no statistical difference between these two groups. Thus it can be seen that these SNPs in the promoter, extron and intron region of FCRL3 might contribute its functional effect on AR. According to the haplotypes analysis of the FCRL3 gene, we found that the CGTGA, GATGA and GGTGA haplotypes were associated with a significantly increased risk of AR, while CGTGG and GGCGG were associated with a notably decreased risk of AR. Concerning the three positive SNPs of FCRL3, the haplotype of AGT increased significantly in AR cases. To the best of our knowledge, this is the first study to evaluate an association between FCRL3 SNPs and AR in a Chinese Han population.Pc = 2.27×10-12 and 7.11×10-13, respectively) to Graves’ disease [For SNPs rs945635 and rs3761959, there are a few inconsistent conclusions to be a risk factor of various autoimmune diseases. And these results are inconsistent between distinct ethnicities. A study  suggests disease  in Chine disease  showed tFor SNPs rs7528684, rs10489678, rs7522061 in FCRL3, this study found that there were significant association with AR in a Chinese Han population. The FCRL3 rs7528684 polymorphism has been shown to be a risk factor of various autoimmune diseases, including SLE, these results are inconsistent between distinct ethnicities. The updated meta-analysis  showed tAs discussed above, The SNPs in FCRL3 gene are strongly associated with ADs by way of mediating the pathogenesis of B cells, Th2/Th1 Cells and Treg Cells, while these cells involve similarly into AR. As shown in In short, our findings that the SNPs rs7528684, rs10489678, rs7522061 in FCRL3 gene, which revealed in the strong association with Chinese Han AR patients, have been testified by a few researches focused on their association with numerous ADs. Moreover, the haplotype analysis reveals that the haplotype of AGT in the FCRL3 gene increased significantly in AR cases. Thus, we postulated that the SNPs, rs7528684, rs10489678 and rs7522061 might be chosen as the tag SNPs to evaluate the high risk of developing AR in Chinese Han population.In the present study, we made many efforts to decrease the influence of confounding factors on the results. We selected the AR patients and controls using strict guidelines and confirmed the genotyping results by direct sequencing. We observed a novel association between rs945635, rs3761959, rs7522061, rs10489678 and rs7528684 of FCRL3 and AR in a Chinese Han population. However, it is worth mentioning that there are some limitations to our study. First, we did not detect the protein level of FCRL3 in the peripheral blood and perform a functional analysis study, so we could not draw a certain conclusion about the influence of these polymorphisms on the cytokine levels. Second, as it is well known that environmental factors are critical for the development of AR, further studies will be needed to clarify the genetic influence of FCRL3 in AR pathogenesis, including gene-gene and gene-environment interactions.In summary, we have demonstrated an association between FCRL3 gene polymorphisms and susceptibility to AR. Our results suggest that there may be an autoimmunity player evolved into the pathogenic process of AR. The FCRL3 are susceptibility genes for the autoimmunity development of AR in the Chinese Han population. However, more intensive studies and advanced methods, such as genome-wide association study, gene-gene, gene-environment, are required to further clarify the molecular mechanisms and the complex interaction between environmental factors and FCRL3 gene polymorphisms in AR. Generally, we hope that our study can provide a deeper insight into AR pathogenesis.


The core challenge is that infants have a limited behavioral repertoire through which brain function can be expressed. Neuroimaging with fMRI has great potential as a way of characterizing typical development, and detecting abnormal development early. But, a number of methodological challenges must first be tackled to improve the robustness and sensitivity of neonatal fMRI. A critical one of these, addressed here, is that the hemodynamic response function (HRF) in pre-term and term neonates differs from that in adults, which has a number of implications for fMRI. We created a realistic model of noise in fMRI data, using resting-state fMRI data from infants and adults, and then conducted simulations to assess the effect of HRF of the power of different stimulation protocols and analysis assumptions (HRF modeling). We found that neonatal fMRI is most powerful if block-durations are kept at the lower range of those typically used in adults (full on/off cycle duration 25-30s). Furthermore, we show that it is important to use the age-appropriate HRF during analysis, as mismatches can lead to reduced power or even inverted signal. Where the appropriate HRF is not known , a flexible basis set performs well, and allows accurate  Perinatal brain injury can be caused by adverse events such as hypoxia, hemorrhage and stroke , and braA fundamental challenge in assessing function is the limited behavioral repertoire of young infants. A promising solution is to use neuroimaging to assess brain function, as this doesn’t require a behavioral response. fMRI has been used in neonates to measure the brain activity evoked by visual ,5, audithttp://www.mccauslandcenter.sc.edu/CRNL/tools/fmrisim). However, no such optimization has been done for infants, and this forms the second goal of our paper.We consider two consequences of the altered HRF. First, conventional fMRI analyses proceed by generating a model of the expected signal, and comparing this to the fMRI time-course. If an incorrect model of the HRF is used, power to detect brain activity may be reduced. A second important consequence is more subtle: the altered HRF will affect which protocols of stimulation will yield the best sensitivity. As an illustration, The power of various stimulation protocols and analysis methods was assessed using simulation: standard statistical measures (t- and F-statistics) were computed to indicate how well a synthesized BOLD fMRI signal could be detected in well-characterized fMRI noise, given a particular model of the hemodynamic response. A realistic model of fMRI noise was obtained from analysis of adult and neonate resting-state fMRI data. Simulated signal was obtained by convolving a range of stimulation paradigms with three HRFs previously measured empirically in adults and infants . Analyses then proceeded assuming either the correct or mismatching HRFs.The power in an fMRI analysis (and thus the accuracy of any simulation) depends not just on the strength of the signal but on the nature of the noise. For a model of noise in the brain, we used fMRI data acquired during resting in adults (N = 26) and newborns (N = 5). The adult data were acquired using a 3T Siemens Tim Trio MRI scanner and the infant data using a 3T Philips Acheiva MRI scanner.http://www.nitrc.org/frs?group_id=296), obtained in 13 males and 13 females, comprising 195 gradient-echo EPI scans with TR = 2.3 s, 34 slices, a matrix size of 70x70, voxel size 3x3x4mm. An MPRAGE T1-weighted anatomical image was acquired in each adult, matrix size 176x256x256, 1-mm isotropic voxels.The adult data was the Marguilies subset of the fc1000 public domain resting-state dataset  were scanned at the Hammersmith Hospital. They were all born prematurely and had no overt brain injury . One infautomatic analysis software [Both infant and adult data were processed using the parallel-processing software ) to pipeThe time-series statistics were characterised in grey matter, as this is of most interest in activation studies. For the adults we normalized and segmented anatomical images and created a mask comprising voxels with a greater than 50% probability of grey matter; while in the infants, due to the difficulty of normalization and segmentation, we manually drew a grey-matter mask on a portion of the right lateral frontal lobe of the un-normalized anatomical images. As can be seen in the results, when motion was of a similar magnitude, similar noise statistics were obtained in infants and adults.where p = power, f = frequency, x, A and W are constantsIt is well established that fMRI noise is strongly auto-correlated , and has strong power at low frequencies . It is ox has been taken to be 0.67 [The exponent  be 0.67  and 1 [1 be 0.67 ,25,27. Wlog (power) against log (frequency). The frequency domain synthesis comprised magnitudes taken from this model of the power spectrum and random phases, which was then transformed to the time domain using a Fourier Transform. The resulting noise had similar amplitude histograms, and appeared visually similar, to the original noise. The third method formed a balance between the time-domain and frequency domain methods above, and was based on the discrete wavelet transform (DWT) analysis of fMRI noise by [To ensure that the noise was well characterized, and to allow for simple extension and replication, we wished to synthesize noise with the same statistics as the measured resting-state noise. Surprisingly, we did not find an existing tool for this, and further, there was no consensus on the best approach. Thus, in pilot work using the adult data, we tested three approaches to synthesizing noise with fMRI-like statistics. The first was to measure the lag-1 autocorrelation of the fMRI time-series, and then to synthesize noise with this autocorrelation. This time domain model was found to be wanting, as it underestimated the magnitude of very low frequency fluctuations in the power spectrum, which have an important impact on the power of slow block designs. The second method was to measure the power spectrum of the fMRI noise, and to synthesize noise with this power spectrum. The measured adult noise was fit well by a linear model of noise by . We perfArichi et al (2012) measured the HRF in pre-term and term infants, and adults, for a 1-second somatosensory stimulation. The resulting curves show a broad range of morphology–delayed in pre-term infants relative to adults, but still broadly monophasic; and biphasic in term infants . For ourBlock designs, in which for example a stimulation block is alternated with a rest block, are simple and robust . We analIn fMRI studies, analyses typically begin with the creation of a model of the signal, by convolving the expected neural activity with the HRF. In the literature, many authors have used mismatched responses, for example by assuming the HRF of infants is the same as adults. To assess the effect of a mismatch, we analyzed the data simulated with each of the three HRFs using three different models (each of the possible HRFs). The fit of each model to the simulated data was estimated using the general linear model using expectation-maximization in SPM 8. Each model contained two columns: the convolved boxcar, and the session mean. The power of the model to detect the simulated activation was assessed using an SPM-T statistic for the first of these two columns.http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FLOBS). First, the three HRFs were fitted to a set of half-cosine functions, allowing each to be parameterized by a set of delays and amplitudes. Second, in this parameter space we interpolated between the 32- and 38-week HRFs, and the 38 week and adult HRFs. Third, we reconstructed a set of HRFs, and conducted Principal Component Analysis. The top three components accounted for >99% of the variance, and fit well the original Arichi or interpolated HRF functions. They thus form an effective flexible basis set. A model using this basis set contained four columns: the boxcar convolved with each of the three basis functions, and the session mean. The power of the model to detect the simulated activation was assessed using an SPM-F statistic for the F-contrast constructed as the identify matrix. To facilitate comparison to the T statistic, this was then transformed by: adjusting for the difference in the degrees of freedom in the two models, and taking the square root. The resulting value thus corresponds to the T value from the two-column model that has the same significance as the F statistic in the four-column model.In practice, it may often be unclear which HRF is appropriate for a participant. Infants develop at different rates, especially when they have faced some challenge such as being born with very low birth weight, or requiring an extended period of mechanical ventilation. It is not yet known under what circumstances “HRF age” mismatches from “chronological age”, or the extent of this mismatch. We thus developed a flexible basis set, using a procedure from FSL’s “FLOBS” . Cubic interpolation with an accuracy of 0.1s was used to find the peak of these curves, and hence the optimal paradigm period for each combination of actual and predicted HRF.To assess the quality of the HRFs as estimated from the flexible basis set, we reconstructed the HRF by weighting the three basis functions with their estimated beta values, and calculated the correlation over the duration of the true HRF (32.5 s). These correlation values were then Fisher transformed to make them Gaussian, the mean and standard deviation calculated, and the upper and lower bounds were then back transformed for display.The power spectrums of the noise derived from resting state data for adults and neonates are shown in The noise average fit across adults was Not surprisingly, greatest power was obtained across all paradigm periods and for all true HRFs when the correct model HRF was used. Modeling with a mismatched HRF had a strong effect on power, in two cases (32 and 38 weeks GA mismatched with each other in either direction) almost entirely eliminating sensitivity to the signal. At 32 weeks GA, power shows a negative trend for rapid protocol alternations (around 16s) when the analysis was conducted using the adult HRF. The relationship of this to reports in the literature of “negative activations” in neonatal fMRI studies is addressed in the Discussion.The goal of the flexible basis set is to capture brain activity when the HRF is at some unknown point in the continuum from 32 to 38 weeks GA, or 38 weeks to adult. To capture this range, the HRFs were parameterized and then the parameters interpolated. The resulting set of interpolated HRFs is shown in This flexible HRF basis set recovered signal for all three true HRFs, but gave less power relative to the case where the true HRF is known  design.For the flexible HRF model, three columns were used. This is displayed as the square root of an adjusted F statistic, which was designed to be comparable to the T statistics, so that similar values correspond to the same Choosing the correct HRF for modeling was found to be critical for neonatal fMRI. A mismatching HRF can eliminate all power in a block design: in some cases, a mismatched HRF even artifactually yielded a negative signal, but only for short on/off paradigm durations of around 16s. There are a number of reports in the literature of negative fMRI signal in newborns or very young infants ,6,30–32.http://www.mccauslandcenter.sc.edu/CRNL/tools/fmrisim). However, our modeling shows that for neonates, longer cycles often used in adults yield much less power when the HRF is biphasic at 38 weeks  measurements of the HRF to a somatosensory stimulus. It will be useful to characterize: the robustness of these results; degree of variability across individuals in the HRF as a function of age ; the effect of brain injury on the development of the HRF; and whether the HRF develops at the same rate in all brain regions.The flexible basis set was effective in recovering signal from the diverse range of HRFs tested. The PCA analysis showed it captured almost all of the variance from Arichi’s three HRFs, and the temporally-interpolated HRF set, using a linear model with just three components. This simple model will be more robust than a more general  basis sets that has a greater number of degrees of freedom. However, they will fail if an infant has an HRF distinct from Arichi’s set.In sum, using simulations with an accurate noise model we found that stimulation paradigms for newborn fMRI must be carefully chosen, as the power is optimal for a narrower range of paradigms than for adults. For block designs, we recommend full on/off cycle durations of 20-30s. Furthermore, we have shown that it is important to analyze newborn data with the correct HRF, or to use a flexible basis set where the correct HRF is unknown.


Background: Hypospadias is one of the most common congenital abnormalities in the male which is characterized by altered development of urethra, foreskin and ventral surface of the penis. Androgen receptor gene plays a critical role in the development of the male genital system by mediating the androgens effects.Objective: In present study, we looked for new variations in androgen receptor promoter and screened its exon 1 for five single nucleotide polymorphisms (SNP) in healthy and hypospadias Iranian men.Materials and Methods: In our study, at first DNA was extracted from patients (n=100) and controls (n=100) blood samples. Desired fragments of promoter and exon 1 were amplified using polymerase chain reaction. The promoter region was sequenced for the new variation and exone 1 screened for five SNPs  using restriction fragment length polymorphism technique.Results: The results showed a new single nucleotide variation (C→T) at -480 of two patients’ promoter region (2%). None of the mentioned SNPs were detected in patients and controls groups (0%). Conclusion: This finding indicates that new single nucleotide polymorphism in androgen receptor promoter may have role in etiology of hypospadias and development of this anomaly.This article extracted from Ph.D. thesis. (Nasim Borhani) Hypospadias is the most common congenital malformation of the male external genitalia in which the urethral meatus opens on the ventral side of the penis . It affeAR gene is located on chromosome Xq11-12 and consists of eight exons, in which exon 1 encodes the transactivation domain that activates transcription of several different AR-responsive downstream genes , and in addition, AR gene promoter region was scanned for new variations in hypospadias disease in Iranian population.Impaired signaling through the androgen receptor (AR) leads to failure of urethral fold formation and fusion and consequently leads to hypospadias . The AR am genes , 7. The am genes , 9. ThesPatients and controlsThe patients in this analytical association study refered to the Mofid’s children Hospital  between March 2012 to August 2012. The patients should have criteria including diagnosis by urologist and having no sign of other genetic or congenital disease or genital malformations; (the properties distribution of patients in our study are listed in =100) and control group (n=100) were taken using venoject tube containing EDTA (0.5 M). The controls were selected from healthy males in Shahid Beheshti School of Medicine. The control samples should not have any sign of genetic disease, genital malformations or any addiction. Peripheral blood samples of hypospadic patients (nDNA extraction and polymerase chain reaction (PCR)AR transactivation domain were amplified using the primer pair: 5’TCCAGAGCGTGCGCGAA GTG3’ (sense) and 5’CCGACTGCGGCTGT GAAGGT3’ . Genomic DNA was extracted from peripheral blood of patients and controls using the whole blood extraction kit . Nucleotides -756 to -366 of the AR 5’ promoter region (based on Ensemble.org genome browser) were amplified using the primer pair: 5’TCTCCAAA GCCACTAGGCAG3’ (sense) and 5’ACCGAA GAGGAAAGGGCAGCTC3’ (antisense). Nucleotides +604 to +888 of the Restriction fragment length polymorphism (RFLP) analysisFor transactivation domain genotyping, PCR, RFLP and 8% polyacrylamide gel electrophoresis (PAGE) were used. The RFLP reaction condition was detailed and presented in Promoter region sequencingFor AR promoter region sequencing, PCR products were purified using DNA extraction kit  and then scanned for variations using direct sequencing .Ethical considerationsThe informed consent was obtained from all adult participants (control groups) and from the parents or legal guardians of minors (patients). This study had been reviewed and approved by a certified Ethical Committee in Shahid Beheshti University of Medical Sciences.RFLP resultsThe 285 bp PCR product containing five SNP sites was digested using suitable enzymes; SsiI, PstI, CaiI, SfaNI, and BseG1. The two SNPs rs62636528, and rs62636529 produce gain of sites compared to wild type allele, while rs78686797, rs139767835, and rs145326748 supposed to have loss of sites in digestion with the mentioned enzymes . The SsiSequencing resultsAR gene promoter region was scanned for new variations in subjected samples and a new single nucleotide polymorphism (SNP) was detected in two patient samples (2%)  at position -48oC→T (the normal sequence and two sequences curves with a new nucleotide variation are shown in AR gene is a member of the nuclear receptor superfamily that is located on Xq11-12. This receptor plays a critical role in male sexual differentiation by mediating the biological effects of androgens (AR amount or structure may disrupt this patterning (AR for the first time in hypospadic patients and we found a new single nucleotide polymorphism in two patients at -480 using direct sequencing. There have been many reports on AR promoter sequencing. ndrogens . Early det al scanned AR gene promoter and untranslated region in 44 clinical prostate cancer specimens and 36 normal controls; but they did not find any different sequence variations (et al screened promoter region of androgen receptor gene in 240 men with idiopathic infertility and 30 healthy men and this study did not show presence of any deletions or mutations in infertile or healthy men and they suggested that AR gene promoter mutation are not common in men with idiopathic infertility (AR gene promoter region of 100 healthy men suggested that DNA sequence alterations are rare in the human androgen receptor gene promoter (In 2006, Waltering riations . Likewisertility . Furtherpromoter . et al in which mutation screening of genomic DNA of men with hypospadias did not show any point mutation in AR gene exons (et al reported that the majority of patients with hypospadias did not carry androgen receptor gene mutations. In addition Sasagawa et al suggested that alteration of the AR gene is rare in males with isolated hypospadias, cryptorchidism, micropenis, or idiopathic male infertility (et al reported that none of the studied hypospadic patients in their research had AR gene mutations (AR gene mutation is rare in hypospadic patient (Additionally, we did not find any of mentioned single nucleotide polymorphisms in control and patient groups using PCR and RFLP techniques. This finding may declare that these five polymorphisms are not be involved in hypospadias etiology. These data are consistent with the previous report by Muroya ne exons . Also Hiertility . Furtherutations . Likewis patient , 17. et al it was found that a SNP in the AR gene region was associated with susceptibility to severe hypospadias. They concluded that AR rs5919436 polymorphism may act as a new gene marker for increased susceptibility to severe hypospadias in Caucasians (AR gene with the hypospadias; and suggested that expanded CAG repeat length has a role in modifying the risk and development of hypospadias (In addition, In a recent study by Adamovic ucasians . BesidesAR gene expression in the genital system due to decreasing the transcription machinery affinity for binding toward regulatory elements.In addition, in another study on Chilean population, the CAG and GGN polymorphisms in the AR gene has been investigated and showed that in isolated hypospadic boys, there are longer CAG alleles in their AR gene that might be associated with the development of hypospadias . Taken t


Astragalus membranaceus (Fisch.) Bge (AR), one of the most important medicinal plants in Asia, was found to exhibit various bioactivities. Due to limited genomic and transcriptomic data, the biosynthetic pathway of the major bioactive compound in AR, is currently unclear. In this study, 454 GS FLX technology was employed to produce a substantial expressed sequence tag (EST) dataset from the AR. In all, 742721 high-quality reads from the AR were produced using Roche GS FLX Titanium. A total of 9893 unique sequences were obtained and annotated by a similarity search against the public databases, and involved in the secondary metabolic pathway, which would facilitate deciphering the molecular mechanism of secondary metabolism in AR. The assembled sequences were annotated with gene names and Gene Ontology (GO) terms. GO revealed the unique sequences that could be assigned to 34 vocabularies. In the KEGG mapping, unique sequences were established as associated with 46 biochemical pathways. These results provided the largest EST collections in AR and will contribute to biosynthetic and biochemical studies that lead to drug improvement. With respect to the genes related to metabolism and biosynthesis pathway were also found. Our work demonstrated the utility of 454 GS FLX as a method for the rapid and cost-effective identification of AR transcriptome, and this EST dataset will be a powerful resource for further studies such as taxonomy, molecular breeding, and secondary metabolism in AR. Identification of candidate genes involved in the biosynthetic pathway will significantly contribute to the understanding of the biosynthetic and medicinal chemistry of the plant Astragalus membranaceus (AR) is one of the most important medicinal plants in Asia, and has received much of the world attention in recent years, as listed in the 2010 edition of Pharmacopoeia of the People’s Republic of China Samples of AR  were collected from the Medicinal Botanical Garden of Heilongjiang University of Chinese Medicine. After cleaning, AR leaves were immediately frozen in liquid nitrogen and stored at −80°C until use. AR was dried in 105°C for 0.5 h and 80°C drying to constant weight, vacuum packed until DNA extraction. Voucher specimens for every sampled population were deposited at the College of Pharmacy of Heilongjiang University of Chinese Medicine.The total RNA was extracted as previously described Sequencing was based on the 454 GS FLX platform and Titanium reagents. Preparation of the 454 library was performed according to the supplier’s instructions. Approximately 5 µg of amplified cDNA was nebulized and selected for length, which ranged from 300 to 800 bp. The DNA fragments were then denatured to generate single-stranded DNA, which was then amplified by emulsion PCR for sequencing. The sequencing of the libraries was performed on a 454 GS FLX.www.geneontology. org), NCBI’s nr (http://www.ncbi.nlm.nih.gov/), KEGG (www.genome.jp/kegg) and Orthologous Groups of proteins (www.ncbi.nlm.nih.gov/COG) protein databases using BLASTX to identify the proteins that had the highest sequence similarity with the given unigenes along and to retrieve their functional annotations. GO is a standardized gene functional classification system that offers a dynamic-updated controlled vocabulary and a strictly defined concept to comprehensively describe properties of genes and their products in any organism. It has three ontologies: molecular function, cellular component and biological process. The Blast2GO program was used to assign GO annotations to the unigenes. The KEGG database contains metabolic pathways that represent molecular interactions and reaction networks. We used the KEGG annotation to assign pathway annotations to the unigenes that were assigned to special biochemical pathways according to the KEGG standards using BLASTX. The COG is a database in which orthologous gene products are classified. The unigenes were aligned to the COG database to predict and classify the possible functions of the unigenes.Unigene sequences were searched against Gene Ontology .In order to distinguish redundant sequences from homologous sequences, “unigene” was used in this study to minimize redundancy, each unique sequence was assigned a unigene ID according to the accession number of the best-hit homologue in the Nr database, and finally 9732 unigenes were obtained. A further functional classification of all unigenes was performed using a set of plant-specific GO slims . A totalTo further evaluate the completeness of our transcriptome library and the effectiveness of our annotation process, we used the annotated unigene sequences to search for the genes in the COG classifications. Among the 26 COG categories  that werAR, we mapped the 9893 annotated sequences to the reference canonical pathways in KEGG. A total of 2877 unigene sequences were assigned to 45 KEGG pathways (KEGG analysis provides an alternative functional annotation of genes. We next determined the overall expression levels for each KEGG pathway as described above for GO codes. To identify the biological pathways that were active in pathways . The mospathways , “diterppathways  and “flapathways , “anthocAR belongs to leguminous plant of the Astragalus family, and is one of the most popular traditional Chinese medicines, is widely used as an antidiabetic, immunostimulating, hepatoprotective, cadiotonic, and antiviral drug AR, and will allow for the identification of novel genes involved in the secondary metabolite synthesis pathways. KEGG pathway analysis suggested that transcripts of the glycan biosynthesis, metabolism of terpenoids and polyketides, biosynthesis pathways of other secondary metabolites were highly abundant in AR. Sequence similarity searches against public databases identified the unigenes that could be annotated with gene descriptions, conserved protein domains, and/or gene ontology terms etc., are important for the quality of AR as a medicinal plant. This is the first time that the novel candidate genes of these secondary pathways have been discovered in AR.In this study, we had totally identified 9893 unique sequences which involved in primary and secondary metabolites biosynthesis, earching the annotation information from the KEGG, COG, GO and Nr database. All the genes in these pathways were found in the transcriptome dataset. These unigene sequences and their annotations will provide a valuable resource for investigating specific processes, functions and pathways in AR with increased secondary metabolite yields, different compound compositions and better medicinal characteristics.The quality of herbal medicine has been very difficult to control and to evaluate, primarily because of the complexity and incomplete knowledge of the active compounds. The primary methods that have been used for quality evaluation of herbal medicines are chemical and pharmacological analyses. However, content and fingerprint analysis of one or more of these compounds are not indicative for the medicinal value of the plant, and our genomic approach provides more comprehensive understanding of AR as medicinal plant. Our study generated gene expression data for terpenoids and fatty acid biosynthesis and increased valuable knowledge on other AR compounds. These results provided a meaningful basis for evaluating the bioactive components and their action mechanisms of AR. In this study, we obtained a sufficient amount of transcriptomic data from AR, and carried out an integrated analysis on the gene expression of active compounds. It can be used to produce genetically improved varieties of AR and identify genes involved in the metabolism pathway, the 454 GS FLX technology was used in this study to generate substantial transcriptome sequence data. With this work, we initiated a large-scale investigation of the transcriptome of AR in terms of functional genomics, molecular biology and biochemistry. A large number of novel candidate genes involved in primary and secondary metabolites biosynthesis, were identified in our EST dataset. A total of 9732 sequences have been successfully annotated with GO terms based on the known sequences, and unique sequences are involved in the secondary metabolic pathway, which would facilitate deciphering the molecular mechanism of secondary metabolism in AR. Our study provided an initial description of the expression profiles of AR, and also gives a first insight into the gene expression patterns of AR. More importantly, the putative transcriptome information explored in this study will provide a significant contribution towards understanding the metabolism and biosynthesis and may help us to enhance the quality of AR.The transcriptomes can be informative for the analysis of active medicinal compounds in herbal plants. To explore the transcriptome of


AR) gene, located on the X chromosome, is not known to be involved in sex determination. In the Japanese frog Rana rugosa the AR is located on the sex chromosomes . Phylogenetic analysis shows that the AR on the X chromosome (X-AR) of the Korean R. rugosa is basal and segregates into two clusters: one containing W-AR of Japanese R. rugosa, the other containing Y-AR. AR expression is twice as high in ZZ  compared to ZW  embryos in which the W-AR is barely expressed. Higher AR-expression may be associated with male sex determination in this species. To examine whether the Z-AR is involved in sex determination in R. rugosa, we produced transgenic (Tg) frogs carrying an exogenous Z-AR. Analysis of ZW Tg frogs revealed development of masculinized gonads or ‘ovotestes’. Expression of CYP17 and Dmrt1, genes known to be activated during normal male gonadal development, were up-regulated in the ZW ovotestis. Testosterone, supplied to the rearing water, completed the female-to-male sex-reversal in the AR-Tg ZW frogs. Here we report that Z-AR is involved in male sex-determination in an amphibian species.In mice and humans, the androgen receptor ( R. rugosa (2n = 26) has two sex-determining systems within one species R. rugosa are found in the Korean R. rugosaR. rugosa evolved through two independent inversions on chromosome 7 R. rugosa.Sex is genetically determined in most vertebrates. As in other vertebrate species, heterogametic sex chromosomes in amphibians determine the male (XX/XY) or female (ZZ/ZW) fate AR-knockout mice AR knock-in mice AR does not participate in sex determination in mice. The Sry on the Y chromosome is recognized as the master genetic determinant of male fate in this species Androgens exert a variety of effects in target tissues such as male reproductive organs, brain and skeletal tissues. Androgenic effects are mediated by tissue-specific transcriptional control of target genes via nuclear androgen receptor (AR) R. rugosa the AR gene is located on the sex  chromosomes and reportedly on the inverted region of the Y and W chromosomes AR gene is in the process of evolutional degradation from lack of recombination between the inverted and non-inverted regions of the sex chromosomes . In addition, it has been proposed that degradation of the W-AR began just after or at the origin of the ZW sex-determining system in R. rugosaW-AR expression levels are extremely low in R. rugosa embryos, perhaps owing to promoter sequence variation and cognate transcription factor interaction between the W- and Z-AR promoters W-AR is transgenically expressed as demonstrated in reporter assays using Xenopus laevis kidney-derived A6 cells R. rugosa W-AR is still in an early phase. During sex determination, R. rugosa male gonads synthesize more androgens than females AR expression is up-regulated in the male gonad of R. rugosa tadpoles prior to sex determination AR may be involved in male sex determination in this species. To test this hypothesis, we produced transgenic (Tg) frogs carrying an exogenous Z-AR driven by the promoter region of both Z-AR and EF1α genes. Strikingly, a subset of the Z-AR-Tg female (ZW) frogs formed composite gonads or “ovotestes”. Here we report that the Z-AR is involved in male sex determination in a vertebrate species.In All the animal experiments in this study were performed in respect of the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions  and the Prevention of Cruelty to Animal Act . This included official approval from the Committee of Animal Experimentation of Waseda University (Permit Number: 2013-A005).R. rugosa frogs were used in this study. Unfertilized eggs were artificially ovulated and inseminated Female heterogametic (ZZ/ZW) AR using primers shown in AR gene tree was constructed using the UPGMA method [Genetyx Mac version 14.0.9].To construct a phylogenetic tree of the AR gene, we employed PCR to amplify the promoter region of the Z-AR cDNA (pink bar) and the promoter of Z-AR (blue arrow) into the p(I-SceI)DPCG construct  into the pT2AL200R150G (prrT2ARG). First, we amplified the Z-AR cDNA by PCR using a pair of primers specific for the nucleotide sequence of Z-AR cDNA: forward 5′-AAAAAAGATATCATGGAGGTGCACATTGGACT-3′ and reverse 5′-GAGATGAAGGTGTCAAGTACCTATAGAAAAAA-3′, and the R. rugosa Z-AR (DDBJ Accession No. AB491761) or EF1α (AB822986) promoter region. Then we inserted the Z-AR  or EF1α  promoter region ligated to the 2,312-bp Z-AR cDNA into the p(I-SceI)DPCG vector between the SalI/Agel sites and the pT2AL200R150G vector SalI/XhoI sites, respectively. Each AR expression vector carried either the promoter of the AR gene or of EF1α. V5 (yellow bar) represents the small epitope tag polypeptides (GKPIPNPLLGLDST) of the V5 protein from the paramyxovirus of simian virus 5 (SV5). Restriction enzyme sites for I-SecI and TolII recognition sites are indicated by black arrowheads.To construct the vectors for transgenesis we inserted the 2.3-kbp  pARPAR,  [18], orI-SceI meganuclease- I-SceI meganuclease (NEB) and the I-SceI-cleaved plasmid encoding Z-AR and V5, and the Tol II mRNA Z-AR integration into genomic DNA, we extracted DNA from the tail tip of all Tg frogs just after metamorphosis, using the AllPrep DNA/RNA Micro Kit (QIAGEN). The PCR primers used were: forward, 5′-GCGGTTTTTCCAACTTACCA-3′ and reverse, 5′-CGAGACCGAGGAGAGGGTTA-3′).To produce Tg frogs, we used the Z-AR/V5 expression in Tg ZW gonads. Total RNA was prepared from the gonads of Wt and Tg frogs just after metamorphosis using ISOGEN (NIPPON GENE) and cDNAs were synthesized Z-AR/V5, CYP17A1, and Dmrt1) or 25 cycles (GA3PDH) 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), CYP17A1 , Dmrt1  and GA3PDH  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 Z-AR cDNA (AB372103) encoding polypeptide RVISCKRNNPASSSRRFFQL (AR20) corresponding to residues 695–714, and the other the CYP17A1 cDNA (AB284119) encoding polypeptide DEKEWVNPHLFNPDRFLDENGNRVYS (CYP1726), corresponding to residues 409–434. The DNA fragments were inserted into the BamHI/XhoI sites of the pGEX-4T expression vector . The GST-AR20 and GST-CYP1726 expression cassettes were transfected into E. coli BL21. The transfected cells were sonicated in ice-cold water using an Ultrasonic Processor . We purified GST-AR20 and GST-CYP1726 proteins according to the manufacturer's protocol  and immunized 8-week-old BALB/c female mice three times with 50 µg of protein in complete Freund's adjuvant (Wako) at 2-week intervals. Sera were collected and tested 5 days after the last booster injection and used for immunohistochemistry.For production of mouse antibodies, we designed two sets of primers. One pair amplified the R. rugosa adult testes protein (30 µg) and molecular weight markers (BIO-RAD) were subjected to electrophoresis using a 10% polyacrylamide gel and 30 mA for 1 to 2 hrs R. rugosa adult testes.The primary antibodies against Vasa Gonads were surgically excised from Wt and Tg frogs just after metamorphosis, fixed and frozen as previously described Images were scanned and adjusted for brightness and contrast by Adobe Photoshop CS2.R. rugosa is found in the Korean R. rugosaW-AR gene was inherited from the ancestral type X-AR of the Korean R. rugosa, we constructed an AR gene tree using the UPGMA method [Genetyx Mac version 14.0.9], based on the nucleotide sequences of the promoter region of the AR in the 6 local populations. The X-AR in the Korean population appeared to be basal and segregated into two clusters: one contained the X-AR of western Japan, the Z-AR northern Japan including Sado island and the Y-AR of the central population; the other contained the X-AR of eastern and central Japan and the W-AR of the northern population  testis. Wt female (ZW) and male (ZZ) frogs developed normal bilateral ovaries and testes, respectively   (Tg ZW frogs carrying the exogenous  145 nM) . Many ge 145 nM) . Similar 145 nM) .To examine the internal structure of non-Tg and Tg gonads, tissue sections were immunostained for laminin, a marker of the basement membrane AR driven by the Z-AR promoter (pARPAR) into 450 fertilized eggs of R. rugosa. Two hundred and thirty injected eggs (51.1%) developed into tadpoles at St. I (limb buds are visible) and 222 tadpoles (49.3%) grew into frogs just after metamorphosis, of which 111 were characterized  developed an intermediary gonad, a hybrid of testis and ovary, called ‘ovotestis’. These hybrid organs were classified into the three categories mentioned above .We introduced an exogenous cterized . In the AR-Tg ZW frogs formed testes.When T was present in the rearing water, 35 tadpoles metamorphosed into frogs. All 14 of the Wt male (ZZ) frogs developed testes, as expected. All 12 of the Wt ZW frogs and all 9 of the AR driven by the R. rugosa EF1α promoter (prrT2ARG) into 200 fertilized eggs  of Type 3, but not the Tg left gonad  frogs formed ovotestes  frog formed ovotestes co-expressing AR and GFP. Hence, it is reasonable to conclude that Z-AR has become a gene involved in sex determination in R. rugosa possessing the ZZ/ZW sex-determining system. To the best of our knowledge, this is the first report showing that AR-transgenesis induces female-to-male sex-reversal in a species of vertebrate.Dmrt1 and CYP17 expression is enhanced in the XX sex-reversed gonad as well as in the normally differentiating testis of R. rugosaDmrt1 expression is up-regulated during the late stages of male sex differentiation Dmrt1 in XX tilapia fish results in partial to complete sex reversal Dmrt1 expression in the ZW ovotestes are compatible with these previous findings. Furthermore, we found that the Type 3 Tg gonad, the masculinized part (the ovotestis) that expressed the exogenous AR also expressed Dmrt1 and CYP17, while the non-masuculinized part (the ovary) did not express any of these 3 genes   . HoweverAR when T is supplied. In the ZW testis, Dmrt1 and CYP17 mRNA levels increase, as they do in the developing ZZ testis, indicating that T regulates Dmrt1 and CYP17 expression directly or indirectly through the AR. Threshold levels of T and AR in the ZW gonads required for sex determination have yet to be determined; amounts available may be insufficient to trans-activate cognate genes, or to initiate the gene cascade necessary for specification of the male fate. However, a ZW gonad would be expected to mature into testes if the combined levels of T and AR were sufficient for testis formation. In support of this is the finding that more androgens are synthesized in R. rugosa Wt ZZ gonads during sex determination than in Wt ZW gonads Z-AR can induce sex-reversal in ZW R. rugosa, suggesting that androgen and its receptor are involved in non-genotypically programmed male sex determination in this species, and moreover, this report provides evidence for the first time that evolutionary degradation of the W-AR has led to a critical role for the AR (Z-AR) in sex determination in a species of vertebrate.ZW female frogs can develop testes in the absence of Tg Figure S1Histology of the type 3 gonads. All 3 of the Type 3 Tg ZW gonads obtained in this study are shown . Dashed lines indicate the borders of the gonads.(TIF)Click here for additional data file.Figure S2AR expression in Wt ZW (+T) gonads. The PCR analysis was performed to examine Z-AR expression in Tg and Wt ZW gonads treated with (+) or without (−) T as described elsewhere Z-AR and GA3DPH expression are shown in Z-AR and GA3PDH mRNA level in the gonad, respectively.(TIF)Click here for additional data file.Figure S3Immunohistology of Wt ZW (+T) gonads. Frozen sections from the Wt ZW (+T) testis were stained with the antibodies of AR, CYP17, Vasa and laminin (Lam), and counterstained with DAPI and also HE. AR- and CYP-17 positive signals were observed in the Wt ZW testis treated with T.(TIF)Click here for additional data file.


Neuropilin 1 (NRP1), a receptor of vascular endothelial growth factor (VEGF), promotes angiogenesis, tumor growth, tumor invasion and metastasis. However, the function of NRP1 in melanoma progression, as well as the effect of NRP1 expression on the prognosis of patients with melanoma remains unknown. In the present study, NRP1 expression was examined in 460 cases of melanocytic lesions  at different stages, using a tissue microarray. The correlation of NRP1 expression with melanoma progression, and its prognostic value in patients with melanoma was examined. In addition, the correlation between matrix metalloproteinase 2 (MMP2) and NRP1 expression in patients with melanoma was analyzed. The results demonstrated that NRP1 expression was significantly increased in primary (56%) and metastatic melanoma (62%), compared with common nevi (11%) and dysplastic nevi (24%). Notably, increased NRP1 expression was correlated with a poorer overall, and disease specific, 10-year survival . Multivariate Cox regression analyses indicated that NRP1 is an independent prognostic marker for melanoma. Furthermore, a significant positive correlation between NRP1 and MMP2 expression in melanoma biopsies was observed, and their concomitant expression was closely correlated with melanoma patient survival, further supporting the hypothesis that the expression of NRP1 is associated with melanoma invasion and metastasis. In conclusion, increased NRP1 expression is associated with disease progression and reduced survival in patients with melanoma, and is a promising prognostic molecular marker for this disease. Melanoma is the cutaneous malignancy with the highest mortality and its incidence has continued to grow over the last 30 years . AlthougThe neuropilins are multifunctional proteins that are involved in neural and vascular development, immunity and cancer . NeuropiNRP1 is involved in angiogenesis, axon guidance, cell survival, invasion and migration . A numbeMatrix metalloproteinase 2 (MMP2) is a zinc-dependent proteinase that is capable of cleaving extracellular matrix substrates. Degradation of the matrix is a crucial event in the progression, invasion and metastasis of cancer cells. Increased MMP2 expression was shown to predict adverse outcomes in patients with breast cancers . Recent The use of human skin tissues was approved by the Clinical Research Ethics Board of the University of British Columbia  . The preThe collection of melanoma specimens and the construction of the tissue microarray (TMA) have been previously described ,23. Brie2O2 (Sigma-Aldrich) for 30 min and blocking with universal blocking serum for 30 min, slides were incubated with a primary mouse monoclonal anti-NRP1 antibody  at 4°C overnight. Negative controls were produced by omitting the NRP1 antibody during the primary antibody incubation step. The slides were then incubated with a biotinylated streptavidin conjugated horseradish peroxidase anti mouse and anti-rabbit universal secondary antibody  for 30 min each, followed by developing with a diaminobenzidine substrate kit (DAKO Diagnostics) and counterstaining with hematoxylin.Immunohistochemistry was performed as described previously ,23. TMA Positive NRP1 immunostaining was defined as cytoplasmic and membrane staining, and graded according to the intensity and percentage of cells with positive staining. The evaluation of NRP1 staining was done microscopic examination of the tissue sections by two observers (including one pathologist), who were blinded to the status of the samples, using a microscope . NRP1 staining intensity was scored as 0, 1+, 2+ and 3+. The percentage of NRP1-positive cells in the samples was also assigned to one of four categories: 1, 0–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. On the basis of the immunoreactive score, the staining pattern was defined as: Negative, (0); weak, ; or stro2 tests. Survival time was calculated from the date of melanoma diagnosis to the date of death or of the last follow-up. The effect of NRP1 expression on the overall and disease-specific survival was evaluated using Kaplan-Meier analysis and a log-rank test. Univariate and multivariate Cox proportional hazard regression models were performed in order to estimate the hazard ratios (HRs) or adjusted HRs, and their associated 95% confidential intervals (CIs). P<0.05 was considered to indicate a statistically significant difference. SPSS version 16  software was used for all analyses.Differences in the demographic and clinical characteristics, and in NRP1 expression between patient subgroups were evaluated by the Kruskal-Wallis test and χ2 test revealed that the percentage of high NRP1 staining was significantly greater in primary melanoma (56%) and metastatic melanoma (62%), compared with that in common nevi (11%) and dysplastic nevi (24%)  to AJCC II–IV  to enhance the binding of VEGF165 to VEGFRs, and promotes VEGF165-mediated tumor angiogenesis, cell migration and tumorigenicity ,30. In anti-VEGF , which mnti-VEGF .Increasing evidence suggests an important role for MMPs, a large family of secreted peptidases, in tumor invasion and metastasis . MMP2, oNRP1 is known to be an important receptor for VEGF. Therefore, in the present study, greater emphasis was placed on elucidating the correlation between NRP1 and MMP2 expression . The resIn conclusion, the current study demonstrated that NRP1 expression is significantly correlated with the progression of human melanoma. Notably, high NRP1 expression was correlated with a poorer 5-year and 10-year survival in patients with melanoma, and was shown to be an independent prognostic factor. Furthermore, there was a significant positive correlation between NRP1 and MMP2 expression in melanoma biopsies, and their concomitant expression was inversely correlated with the survival of patients with melanoma. These data suggest that NRP1 is involved in melanoma pathogenesis and that it may serve as a prognostic marker for patients with this disease.


Prostate cancer begins as an androgen-responsive disease. However, subsequent accumulation of multiple sequential genetic and epigenetic alterations transforms the disease into an aggressive, castration-resistant prostate cancer (CRPC). The monoallelic Androgen Receptor (AR) is associated with the onset, growth and development of Prostate cancer. The AR is a ligand-dependent transcription factor, and the targeting of androgen- and AR-signaling axis remains the primary therapeutic option for Prostate cancer (PCa) treatment. A durable and functional disruption of AR signaling pathways combining both traditional and novel therapeutics is likely to provide better treatment options for CRPC. Recent work has indicated that expression of AR is modulated at the posttranscriptional level by regulatory miRNAs. Due to a relatively long 3’ untranslated region (UTR) of AR mRNA, the posttranscription expression is likely to be regulated by hundreds of miRNAs in normal as well as in disease state. The main objective of the article is to offer a thought-provoking concept of “andro-miRs” and their potential application in AR gene expression targeting. This new paradigm for targeting constitutively active AR and its tumor specific splicing isoforms using andro-miRs may pave the way for a novel adjunctive therapy and improved treatment of CRPC. Prostate Cancer (PCa) is the most commonly diagnosed malignancy among the male population in North America and continues to be the second leading cause of cancer-related deaths . Recent Acquired resistance to anti-androgen therapy remains a major obstacle in the treatment of advanced stage metastatic cancer. Although the exact molecular mechanisms responsible for the progression of the disease from androgen-dependent state to androgen-refractory state is unclear, CRPC tumors display revived capability of androgen signaling through AR, a ligand dependent transcription factor belonging to nuclear receptor super-family ,4. TestoThe AR protein structure is composed of three major functional domains: N-terminal transactivation domain (TAD), central DNA-binding domain (DBD) and C-terminal ligand-binding domain (LBD) ,8. MolecIn this review, we briefly remind the reader of potential mechanisms mediated through AR; though we mainly intend to focus on the implication of miRNAs mediated regulations of PCa, with AR-centric approach. We discuss the regulation of AR activity through andro-miRs and their potential application as therapeutic agents in treatment of CRPC patients.Discovery of a crucial role for AR in PCa dates back to the 1940s when Huggins and Hodges demonstrated the effectiveness of surgical castration in men with PCa . Since tMajorities of PCa are dependent on AR signaling—even as they progress into castration-resistant state; AR continues to remain the favorite target for cancer therapy. Overexpression of AR at both the mRNA and protein levels has been reported in CRPC tumors ,17. AR ghttp://androgendb.mcgill.ca). Over 800 of these mutations appear to promote androgen -independence [Accumulation of a significant number of AR mutations is another molecular mechanism exploited by PCa cells to acquire androgen-independence and become castration-resistant. AR gene is one of the most abundant mutated of the nuclear steroid receptor family with 1,029 mutations have been reported in the AR gene mutations database (pendence . The incpendence .The first AR gene mutation (T877A) was identified in hormone-dependent LNCaP PCa cells derived from lymph-node metastasis . InteresSeveral AR splice variants have been identified and altered splicing has emerged as another significant molecular mechanism potentially contributing to the recurrence of PCa. AR splice variants AR3, AR4, AR4b, AR5 and AR8 lacks the LBD and have been found to be over expressed in CRPC tumors . The absAR protein is known to interact with a plethora of diverse proteins including other transcription factors as well as AR coregulators with distinct coactivating or corepressing properties. Interaction of AR with coactivator proteins is important for assembly of the transcriptional complexes to modulate transcriptional activities of its target genes. A recent update of AR-database registers over 300 proteins as AR coregulators. As of February 2013, the list of proteins that have been identified as AR-coregulators in the Nuclear Receptor Signaling Atlas (NURSA) contains 283 entries. These coregulators are involved in a multitude of functions. The first AR co-activator to be identified was SRC-1 . The steAlthough the list of proteins functioning as coregulators for AR is vast, AR may regulate the expression of most of the coregulators, since modulation of expression of the cofactors are dependent on an AR feedback mechanism to regulate the coregulator complex formation . AlteratIn the last few years, there have been numerous reports supporting the impact of microRNAs (miRNAs) in the progression of androgen-sensitive PCa to CRPC. Only recently, however a couple of reports have shown that the AR is a direct target of miRNAs ,36. ReguC. elegans and D. melanogaster in the 1990s have shed light on the role of miRNAs in carcinogenesis [miRNAs are short (~20 – 24 nucleotides) noncoding RNAs that regulate gene expression by facilitating cleavage of its target mRNA in plants; and mostly by translational repression in animals. In animals, miRNAs target mRNAs by imperfect complimentary base-pairing to the 3’ untranslated region (3’ UTR) to downregulate target’s protein synthesis in actively translating ribosomes . Currentogenesis . In the ogenesis . Studiesogenesis .Our knowledge of miRNA mediated regulation of PCa still presents an unclear picture owing to the heterogeneity of the disease and the complexity of cellular signaling presumably involved in the conversion of androgen-sensitive to CRPC. Expression profiling of miRNAs in androgen-dependent versus androgen-independent cell lines reveal that miRNA expression is significantly increased in metastatic invasive cell lines such as the PC3, DU145, and MDA PCA 2B indicating the importance of miRNAs in the progression of the disease to androgen-independence . Of the *, miR-330, miR-148a and miR-449. Each of these affects the expression of growth regulatory genes in PCa [Several other miRNAs have been reported in PCa including miR-126s in PCa –61. Upres in PCa . Converss in PCa . Similars in PCa . CollectGreater than 50% of miRNA genes are coded in the introns of protein coding genes. Some studies have shown that there appears to be a coordinated expression of the host genes and the miRNAs that are coded in the introns, because most intronic miRNAs must be transcribed from the host gene’s RNA polymerase II type promoters . NeverthNumerous miRNAs express differentially in PCa cells and large number of these miRNAs have potential to target androgen-centric signaling pathways in addition to targeting of AR expression directly. An experimental undertaking to identify all the miRNAs, which have potential to directly interact with AR 3’ UTR is clearly lacking. A validation of all the miRNAs targets found within the 3’ UTR of AR and fine-tuned influence of these andro-miRs in AR expression is of paramount importance to realistically understand the progression of disease to metastatic stages. It is interesting to note that a modest 3–4 fold increase in expression of AR expression is sufficient for tumors to develop androgen-independence , and dowet al., using a tour de force approach analysis of miRNAs targeting AR expression, revealed 71 potential andro-miRNAs that down-regulated endogenous AR protein levels in multiple AR positive PCa cell culture models [* can directly bind to the 3’ UTR of the AR to downregulate AR expression both at the mRNA and protein levels resulting in decreased cell proliferation and increased apoptosis in LNCap and C4-3B cells [As noted earlier, due to a relatively large 3’ UTR, the AR is likely to be targeted by a plethora of miRNAs. It is logical that all the predicted andro-miRs may not express in prostate cells or in tumors. Nevertheless, it is expected that experimental introduction of miRNAs into PCa cell or tumors is likely to downregulate AR expression. An elegant recent study by Östling e models . Among t3B cells . Our recA comprehensive study to biochemically catalog miRNAs that have the potential to bind to the AR 3’ UTR would help the PCa and AR research community to start thinking towards potential application of miRNA based adjunctive therapy of PCa. However, off-target effect of miRNAs should also be considered cautiously in order to establish a comprehensive picture of miRNA mediated PCa therapy.We propose that miRNAs which have been validated to interact directly with the AR 3’ UTR by base-pairing and downregulate AR mRNA and protein levels, can be conveniently termed as andro-miRs, albeit in PCa context only. A list of target-validated andro-miRs is shown in AR mRNA species of length 8.5 kb and 11 kb with variable 6.6 to 6.9 kb to 3’ UTR lengths have been identified ,57,74, hDecades of research have presented us with complex scenarios for the development of CRPC; however, the role of androgen and AR in this progression is irrefutable. The heterogeneity and inherent complex molecular circuitry of disease continues to rely on androgen and its cognate receptor, leading to drug-resistance and failed-treatments. Because of these intrinsic biological properties, the current practice of targeting the AR protein activity does not appear to be completely effective. Development of curative therapies for the treatment of CRPC is urgently needed; especially when resistance to AR-antagonist Enzalutamide (MDV3100) poses a grim prognosis. Discovery of andro-miRs has opened up a new avenue, which must be further explored to establish the efficacy of miR-based adjunctive therapeutics in CRPC treatment. This approach would require the development of an efficient systemic delivery method for andro-miRs to directly target the expression of AR mRNA isoforms, in addition to conventional therapeutics which are designed to target androgen and the AR protein. Last but not least, the 3’ UTR of the AR remains an uncharted territory needing further exploration to understand the regulatory role of this region: the important cis-acting RNA motifs and structural elements, as well as the alternative polyadenylation events responsible for the activation of multiple isoforms. Understanding the RNA-processing events and their potential role in AR function would comprehensively enhance our knowledge of this very fascinating, yet under studied molecule.