Ipomoea batatas), which is hindering genetic research in this species. It is necessary to develop more molecular markers for potential use in sweetpotato genetic research. With the newly developed next generation sequencing technology, large amount of transcribed sequences of sweetpotato have been generated and are available for identifying SSR markers by data mining.Currently there exists a limited availability of genetic marker resources in sweetpotato  being the most abundant followed by di- (41.2%), tetra- (9.2%), penta- (3.7%) and hexa-nucleotide (3.1%) repeat types. The top five motifs included AG/CT (26.9%), AAG/CTT (13.5%), AT/TA (10.6%), CCG/CGG (5.8%) and AAT/ATT (4.5%). After removing possible duplicate of published EST-SSRs of sweetpotato, a total of non-repeat 7,958 SSR motifs were identified. Based on these SSR-containing sequences, 1,060 pairs of high-quality SSR primers were designed and used for validation of the amplification and assessment of the polymorphism between two parents of one mapping population (E Shu 3 Hao and Guang 2k-30) and eight accessions of cultivated sweetpotatoes. The results showed that 816 primer pairs could yield reproducible and strong amplification products, of which 195 (23.9%) and 342 (41.9%) primer pairs exhibited polymorphism between E Shu 3 Hao and Guang 2k-30 and among the 8 cultivated sweetpotatoes, respectively.Ipomoea species.This study gives an insight into the frequency, type and distribution of sweetpotato EST-SSRs and demonstrates successful development of EST-SSR markers in cultivated sweetpotato. These EST-SSR markers could enrich the current resource of molecular markers for the sweetpotato community and would be useful for qualitative and quantitative trait mapping, marker-assisted selection, evolution and genetic diversity studies in cultivated sweetpotato and related  Ipomoea batatas) is a hexaploid (2n = 6x = 90) dicot and belongs to the family of Convolvulaceae. Due to its high yielding potential and adaptability under a wide range of environmental conditions, sweetpotato is one of the world's important food crops, especially in developing countries. According to the Food and Agriculture Organization (FAO) statistics, world production of sweetpotato in 2008 was more than 110 million tons, and almost 80% came from China, with a production of around 85 million tons from about 3.7 million hectares [beta carotene content of orange-fleshed sweetpotato plays a crucial role to prevent vitamin A deficiency-related blindness and maternal mortality in many developing countries.Sweetpotato  showed significant similarity to known proteins and matched 30,924 unique protein accessions. As shown in Table http://pgrc.ipk-gatersleben.de/misa/, a total of 8,294 SSRs were identified from 7,163 unique ESTs, with an average of one SSR per 7.1 kb. Of these, 949 ESTs contained more than one SSR, and 539 were compound SSRs that have more than one repeat type. In order to identify the putative function of genes containing the SSR loci, the 7,163 EST sequences were also searched against UniProt database with E-value cutoff less than 10-5. Among of them, 4,911 had BLAST hits to known proteins in this UniProt database.A total of 181,615 ESTs with an average length of 548 bp were used to evaluate the presence of SSR motifs. In order to eliminate redundant sequences and improve the sequence quality, the TIGR Gene Indices Clustering Tools (TGICL)  was usedThe compilation of all SSRs revealed that the proportion of SSR unit sizes was not evenly distributed. Among the 8,294 SSRs, the tri-and di-nucleotide repeat motifs were the most abundant types , followed by tetra- , penta-  and hexa-nucleotide  repeat motifs  were polymorphic between the two mapping parents. A total of 644 alleles at polymorphic loci were detected and the average number of alleles per SSR marker was 3.30 with a range of 2-10, based on the dominant scoring of the SSR bands characterized by the presence or absence of a particular band  primers were polymorphic, with a total of 1,004 alleles detected  [Epimudium sagittatum (3.4%) [Arabidopsis thaliana, 3.4 kb in rice, 8.1 kb in maize, 7.4 kb in soybean, 11.1 kb in tomato, 20.0 kb in cotton and 14.0 kb in poplar [The frequency of SSRs in SSR containing ESTs can accurately reflect the density of SSRs in the transcribed region of the genome. Using Sanger and next generation sequencing, a large number of EST sequences for sweetpotato have been generated. These sequences offered us an opportunity to discover novel genes, also provided a resource to develop markers. However, there is abundant redundancy in these EST sequences due to the non-normalized cDNA libraries and submission by different researchers. In this study, in order to reduce the redundancy and avoid overestimation of the EST-SSR frequency, SSR searching was performed following redundancy elimination. A total of 87,492 potential unique EST sequences (about 58.7 Mb) were used for SSR searching and 7,163 ESTs (8.2%) contained SSR motifs, generating 8,294 unique SSRs. The result of SSR abundance was in agreement with the report by Hu et al (9.1%) . These ta (3.0%) , Epimudim (3.4%) . In thisn poplar . HoweverArachis hypogaea L.) [Epimudium sagittatum [In earlier reports, tri-nucleotide repeats were generally the most common motif found in both monocots  and dicogaea L.) , Epimudigittatum , and mangittatum . The pregittatum  and soybgittatum  also suggittatum ,36,37. Tgittatum ,36,38 angittatum ,39. But gittatum , which aet al. [et al. [et al. [In this study, in order to remove possible duplicate of published EST-SSRs of sweetpotato, the primers from the published EST-SSR markers were compared against the 7,163 unique SSR-containing sequences. A total of 336 pairs of SSR primers were found matching to SSR-containing sequences of this investigation, and the matched sequences were excluded from primer designing later. Among the 336 SSR primers, seven pairs of primers designed by Wang et al.  and seve [et al.  and one  [et al. ) matchedIpomoea species.Based on these non-repeat SSR-containing sequences, a total of 1,060 primer pairs were designed and used for validation of the EST-SSR markers in sweetpotato. Of these, 897 primer pairs (84.6%) yielded amplicons in the two parents of our mapping population. This result was similar to EST-SSR amplification rate in sweetpotato ,28 and mAs is commonly known, polymorphic SSR markers are important for research involving genetic diversity, relatedness, evolution, linkage mapping, comparative genomics, and gene-based association studies. In the present investigation, SSR primer polymorphism was first examined in the two parents of our mapping population. Among the tested primers, 195 were polymorphic between the two mapping parents. These markers would be useful for construction of an SSR-based linkage map. Furthermore, among of these working primer pairs, 342 (41.9%) showed polymorphism in the eight cultivated sweetpotatoes. This value was lower than earlier studies, in which 62.5% and 67.2% SSRs revealed to be polymorphism in different test set ,28. A smIn this study, in addition to the characterization of EST-derived SSR markers in cultivated sweetpotato, we designed and validated 1,060 SSR markers in two parents of our mapping population. Among the effective primers, 41.9% of them showed polymorphism in eight sweetpotato cultivars. These developed SSR markers will provide a valuable resource for genetic diversity, evolution, linkage mapping, comparative genomics, gene-based association studies, and marker-assisted selection in sweetpotato genetic study. Since these markers were developed based on conserved expressed sequences, they may be valuable for functional analysis of candidate genes. To the best of our knowledge, this is the first attempt to exploit EST dababase and develop large numbers of SSR markers in sweetpotato.In the present study, two parents of a mapping population, E Shu 3 Hao and Guang 2k-30, and 8 accessions of cultivated sweetpotatoes were used Table . The leaet al [et al and 58,681 generated in house were used in this study. These ESTs were assembled using the TGICL program [http://pgrc.ipk-gatersleben.de/misa/) was used to mine microsatellites. In this work, the search was conducted for sequences that showed at least six repetitions for di-, five repeat units for tri-, and four repetitions for tetra-, penta- and hexa-nucleotides, excluding polyA and polyT repeat. Frequency of SSR refers to kilobase pairs of EST sequences containing one SSR.A total of 181,615 EST sequences including 66,418  from sweetpotato gene index established by Schafleitner et al , 56,516  program . A Perl In order to remove possible duplicate of published EST-SSRs, comparison was performed using the primers from the published 370 EST-SSR markers  againstSequences that showed the longest repetitions and flanking regions that quantified primer design were selected for PCR primer design using primer premier 6.0 . Primers were designed based on the following core criteria: (1) primer length ranging from 18 bp to 24 bp; (2) melting temperature (Tm) between 52°C and 63°C with 60°C as optimum; (3) PCR product size ranging from 100 to 350 bp; (4) GC% content between 40% and 60% with amplification rate larger than 80%. The parameters were modified when unsuitable primer pairs were retrieved by the program. When two distinct microsatellite sequences were present in one EST sequence at distant sites, primer pairs were designed respectively. When two loci were in close proximity in one sequence, the primer pairs were designed outside of these micorsatellites.2), 200 μM of each of the four dNTPs, 0.2 μM of each of the forward and reverse primers, and one unit of Taq DNA polymerase with the following cycling profile: 1 cycle of 5 min at 94°C, an annealing temperature of 55-65°C for 35 cycles  and an additional cycle of 10 min at 72°C. Each of the primer pairs was screened twice to confirm the repeatability of the observed bands in each genotype. PCR products were separated on 6% polyacrylamide denaturing gels. The gels were silver stained for SSR bands detection.PCR analysis was performed in a total volume of 20 μl reaction mixture that contained 40-50 ng template DNA, 1× PCR buffer , the percentage of polymorphic loci  and polymorphism information content (PIC). The polymorphism was determined according to the presence or absence of the SSR locus. The value of PIC was calculated using the formula ZYW conceived, organized and planned the research, and drafted the manuscript. LJ designed PCR primers and participated in DNA extraction and SSR experiment. ZXL participated in primers designing and SSR experiment. LFH participated in primer designing. XLC participated in polyacrylamide denaturing gel running. BPF participated in design and coordination. YJL participated in manuscript preparation and revision. JYC and XJZ provided the plant material for SSR analysis. All authors read and approved the final manuscript.Table S1-Primer sequences for EST-SSR markers in sweetpotato.Click here for file


We provide evidence that increasing glucose concentrations downregulate androgen receptor (AR) mRNA and protein levels through NF-κB activation in LNCaP cells. Moreover, there was a synergic effect of glucose and TNFα in downregulating the AR in LNCaP cells. By contrast, insulin had no effect on AR regulation. In vivo experiments showed that streptozotocin-induced diabetes (STZ-DM) produces tumor growth retardation and a significant reduction in AR expression in PAC120 prostate cancer mice. In conclusion, our results suggest that hyperglycemia and TNF-α play an important role in protecting against prostate cancer by reducing androgen receptor levels via NF-κB.Type 2 diabetes has been associated with decreased risk of prostate cancer in observational studies, and this inverse association has been recently confirmed in several large cohort studies. However the mechanisms involved in this protective effect remain to be elucidated. The aim of the present study was to explore whether different features of type 2 diabetes  protect against the development of prostate cancer. For this purpose LNCaP cells were used for  Type 2 diabetes (T2D) and prostate cancer (PCa) are two major, growing health problems that affect millions of men worldwide. PCa is an androgen-dependent malignancy which constitutes the most common solid organ cancer in men in the US, Canada and Australia, and the second most common cancer in men globally T2D has been recognized as a key factor contributing to the development of solid organ malignancies including liver, pancreas, colorectal, breast, endometrial, uterine, and bladder The precise mechanisms involved in the protective effect of T2D on PCa development remain to be elucidated. It has been reported that individuals with increased genetic susceptibility to T2D have a decreased risk of PCa in vitro system. Moreover, since hyperglycemia is able to activate NF-κB in endothelial cells, pericytes and vascular smooth muscle cells in vivo the effects of hyperglycemia on AR regulation and tumor growth.Since PCa is androgen-dependent, the first aim of the present study was to examine whether insulin or glucose levels had any effect on the androgen receptor (AR) in PCa LNCaP cells  2 at 37°C. All in vitro experiments were done at least in two independent experiments by triplicate.The human prostate cancer cell lines LNCaP were obtained from the American Type Culture Collection  and were plated in 75 cm2 flasks and cultured in RPMI 1640  containing 10% FBS, 1% Penicillin/Streptomycin, 1% Hepes and different insulin concentrations  or glucose concentration  for 4 days. LNCaP cells were also treated with increasing amounts of D-glucose  in the presence or absence of TNFα (50 ng/ml) or QNZ . Cells were maintained in a humidified incubator with 5% COin vivo studies a PAC120 PCa mouse model was used. De Pinieux et alFor ad libitum. Prostate tumors were sliced into 5×5 mm pieces and grafted subcutaneously as previously described Five-week-old athymic swiss mice  were housed in the single pathogen free zone of the Animal Facility Laboratories of the Institut de Recerca de Vall d’Hebron and they were provided with food and water 2, and immediately sacrificed by cervical dislocation. Tissue were collected and frozen at −80° or in formalin.Mice were divided into four groups: a) mice injected with 6 mg of streptozotocin (STZ)  intraperitoneally (IP), one week before the tumor was grafted; b) mice treated with STZ IP after tumor implantation; c) mice treated with the vehicle  IP and d) non-treated mice. Blood samples were taken twice a week by saphenous vein for measurements of their glycemia as a control of the maintenance of diabetic status. At the end of the experiments, treated and control animals were sedated by COTumor samples were fixed with formalin and paraffin-embedded; then five µM-thick tumor sample sections were hydrated with xylene (3×10 min) and ethanol at decreasing concentrations  and stained with hematoxilin (30 seconds)  and eosin (20 seconds) (Sigma-Aldrich). Finally, samples were dehydrated with graded ethanol solutions (as described above) and cleared in xylene before being mounted with DPX .2O2 system. Appropriate negative controls were performed incubating sections without the primary antibody.After the de-waxed and hydration processes, the slides were pre-treated using a microwave antigen retrieval method . Endogenous peroxidase was quenched before overnight incubation at 4°C with anti androgen receptor  used at 1∶50 and with monoclonal mouse anti-human cytokeratin, . After several washes, sections were incubated 30 minutes at room temperature with secondary antibodies conjugated with HRP (Dako Diagnostics SA), and reactions were developed for 1 min with diaminobenzidine and the H5′-TGAAAGCCATGCTACTCTTCAG-3′ and reverse primer 5′-GCTCA CCATGTGTGACTTGAT-3′), human 18S (forward primer 5′- TAACGAACG AGACTCTGGCAT-3′ and reverse primer 5′-CGGACATCTAAGGGCATCACAG-3′). Results were analyzed using the 7000 SDS program and quantified by the comparative CT method (2−ΔΔCT method).Total RNA was extracted from LNCaP D-glucose and L-glucose treated cells using RNeasy Mini kit,  Reverse transcription (RT) was performed at 42°C, for 50 min using 2 µg of total RNA and 200 U of Superscript II together with an oligo-dT primer and reagents provided by Invitrogen. An aliquot of the RT product was amplified in a 25- µl reaction using SYBRGreen  with appropriate oligonucleotide primer pairs corresponding to human androgen receptor . Protein extracts were used for Western blotting with antibodies against AR rabbit polyclonal antibody AR N-20 (Santa Cruz Biotechnology Inc), human phospho-NF-κB  and PPIA rabbit polyclonal antibody  at 4°C. After several washes, immunoreactive bands were visualized by horseradish peroxidase-conjugated secondary antibody (Dako Diagnostics SA) followed by peroxidase-dependent chemiluminescent reagent  by exposure to x-ray film. Densitometric analysis was performed using ImageJ program.The results are expressed as mean ± SD. Data were subjected to the ANOVA test and significance  was attained.Since PCa is androgen dependent, we first wanted to examine if insulin had any effect on AR levels. We treated LNCaP cells with different insulin concentrations  for four days. The results showed that insulin treatments did not change AR mRNA levels when compared with untreated LNCaP cells . MoreoveWe next wanted to study the effects of hyperglycemia on AR levels using LNCaP cells. LNCaP cells were cultured with increasing concentrations of glucose  over the course of four days. The media was changed everyday to maintain constant glucose concentrations. AR mRNA levels were reduced significantly in LNCaP cells cultured in 10 or 30 mM glucose when compared to LNCaP cells cultured in 5 mM glucose . Moreovein vitro system. For this purpose we repeated the glucose treatments in the presence or absence of TNF-α (50 ng/ml). The results showed again that AR mRNA and protein levels were reduced significantly when cells were cultured in 10 or 30 mM glucose when compared with LNCaP cells cultured in 5 mM glucose. Notably, a potent synergic effect leading to a significant further reduction of both AR mRNA and protein levels was obtained when TNFα was added to different D-glucose concentrations in LNCaP cells , an NF-κB inhibitor. The results showed that QNZ co-treatment was able to block the hyperglycemia-induced downregulation of AR mRNA and protein levels .in vivo. In order to do this, we used a PAC120 PCa mouse model with STZ-DM. For this purpose intraperitoneal injection of STZ was administered to swiss nude mice before (n = 7) and after (n = 7) subcutaneously PAC120 tumor implantation. Non-treated (n = 5) and citrate treated (n = 5) mice were used as controls. Non-treated and citrate treated mice showed a similar tumor growth  stained sections from both groups of diabetic mice  did not in vitro experiments showed that hyperglycemia reduced AR levels we decided to explore the possibility that STZ-DM was affecting AR levels in vivo. We performed immunohistochemistry against the androgen receptor, in the tumors of control and diabetic mice. Control animals showed positive and strong nuclear staining for androgen receptor in all epithelial cells  have not been previously examined.PCa has one of the strongest relationships between age for any human cancer, and genetic factors are estimated to account for 42% of the risk de novo steroidogenesis promoted by insulin in PCa Insulin is a growth factor for prostatic epithelium and it also stimulates the growth of a rat PCa cell line in vitro A proposed hypothesis for how hypoinsulinemia may decrease prostate carcinogenesis is by limiting the bioavailability of insulin-like growth factor I (IGF-I) Since PCa is androgen-dependent it is possible that the lower levels of testosterone reported in long-term diabetes  may reach levels below a threshold to a lower risk of PCa in vivo model was detected. Overall, our results suggest that two essential components of T2D: hyperglycemia and the proinflammatory cytokine TNFα participates in reducing the risk of PCa in T2DM. It should be noted that an increased level of TNFα is a constituent of the low-grade inflammatory process characteristic of T2DM  and might be an essential factor accounting for the lower risk of PCa in type 2 in comparison with type 1 diabetic patients.The mechanisms underlying hyperglycemia on AR regulation have not been previously reported. In the present study we found that hyperglycemia downregulates AR through NF-κB activation, and that this inhibitory effect is further increased by TNFα. In addition, the downregulation of AR induced by diabetes was associated with a tumor growth reduction or even no tumor growth in the PAC120 PCa mouse model treated with STZ. Furthermore, a significant reduction in AR expression in the endogenous prostates of this TNF-α is produced primarily by immune cells but it is also produced by numerous other cell types, including epithelial cells of human prostate cancer Two further considerations should be made regarding the potential preventive role of TNFα in PCa development in T2D. First, TNFα downregulates SHBG In conclusion, our results suggest that hyperglycemia and TNF-α play an important role in protecting type 2 diabetic patients against PCa by reducing AR levels via NF-κB activation. Further studies addressed to examining whether other mechanisms of NF-κB activation existing in diabetes such as advanced glycated end products (AGEs) or oxidative stress are involved in the lower risk of PCa observed in T2D are warranted.


There are some evidences in Brazil and others countries pointing out insufficient resources and actions to promote prevention and rehabilitation for substance abuse related disorders. Also the lack of professional qualification contributes to this scenario, generating knowledge deficiencies and inadequate practices. The study evaluates the Regional Reference Centre on Drugs of Juiz de Fora, Brazil (CRR-JF). The CRR-JF aims to train professionals of the local health sector (NHS) and social care system increasing their knowledge of screening and brief intervention (SBI) practices on the use of drugs and strengthening health care activities. Five courses are provided: Motivational Counseling and Brief Intervention, Case Management and Social Reintegration of Drug Users, Improvement of Medical Practices about Drugs, and Comprehensive Drug Care for Hospital’s Professionals. Questionnaires were applied measuring participant satisfaction and changes in the practices. The added workload of the courses was 360 hours. A total of 180 professionals attended the courses . According to results, 83.2% of participants were satisfied or very satisfied about the courses attended; 61.3% evaluated the content of the courses as good, 28% as great, and 60% were very satisfied or satisfied with the applicability of the course content. Regarding SBI, the participants showed a significant increase in their level of trust and ability to approach users of psychoactive substances and counsel them to reduce or stop their consumption. Besides providing knowledge acquisition, participation in the CRR-JF enabled greater discussion of the practices carried out, reflecting on the work of professionals in the development of more effective interventions in prevention, promotion, and psychosocial rehabilitation of users of psychoactive substances. Acknowledgments: National Secretary of Policy on Drugs (SENAD), Coordination for the Improvement of Higher Education Personnel (CAPES).


Although a high frequency of androgen receptor (AR) expression in human breast cancers has been described, exploiting this knowledge for therapy has been challenging. This is in part because androgens can either inhibit or stimulate cell proliferation in pre-clinical models of breast cancer. In addition, many breast cancers co-express other steroid hormone receptors that can affect AR signaling, further obfuscating the effects of androgens on breast cancer cells.To create better-defined models of AR signaling in human breast epithelial cells, we took estrogen receptor (ER)-α-negative and progesterone receptor (PR)-negative human breast epithelial cell lines, both cancerous and non-cancerous, and engineered them to express AR, thus allowing the unambiguous study of AR signaling. We cloned a full-length cDNA of human AR, and expressed this transgene in MCF-10A non-tumorigenic human breast epithelial cells and MDA-MB-231 human breast-cancer cells. We characterized the responses to AR ligand binding using various assays, and used isogenic MCF-10A p21 knock-out cell lines expressing AR to demonstrate the requirement for p21 in mediating the proliferative responses to AR signaling in human breast epithelial cells.We found that hyperactivation of the mitogen-activated protein kinase (MAPK) pathway from both AR and epidermal growth factor receptor (EGFR) signaling resulted in a growth-inhibitory response, whereas MAPK signaling from either AR or EGFR activation resulted in cellular proliferation. Additionally, p21 gene knock-out studies confirmed that AR signaling/activation of the MAPK pathway is dependent on p21.These studies present a new model for the analysis of AR signaling in human breast epithelial cells lacking ERα/PR expression, providing an experimental system without the potential confounding effects of ERα/PR crosstalk. Using this system, we provide a mechanistic explanation for previous observations ascribing a dual role for AR signaling in human breast cancer cells. As previous reports have shown that approximately 40% of breast cancers can lack p21 expression, our data also identify potential new caveats for exploiting AR as a target for breast cancer therapy. Breast cancer is a disease in which the pathogenesis can be attributed to hormone exposure, the most notable being estrogens. Successful targeted therapies against estrogen receptor (ER)α have been developed, and this remains an active area of research. Many of these therapies directly target ERα or the ERα signaling pathway, and have been shown to be highly efficacious in treating ERα-positive breast cancers . Howeverin vitro and in vivo studies using anti-androgen therapies have shown promising results [Androgens are another class of sex hormones, and epidemiologic studies have supported their role in breast biology and carcinogenesis -4. In fa results ,18,19. A results ,8,20. Th results . In addi results . Moreove results . However results . Thus, dTP53, a homozygous PTEN missense mutation, HER2 amplification, and an oncogenic mutation in PIK3CA  database; http://www.sanger.ac.uk/genetics/CGP/cosmic) [As mentioned, laboratory studies assessing the role of AR in breast cancer have been limited and conflicting. In part, this is due to the fact that most AR-positive breast cancer cell lines also express ERα and PR ,8,25-28./cosmic) ,33.To circumvent this issue, we expressed AR in a genetically well-defined, non-tumorigenic, human breast epithelial cell line, MCF-10A. This cell line is spontaneously immortalized as a result of homozygous loss of the chromosomal region 9p, but is genetically stable . FurtherIn this paper, we report and characterize a similar model to study AR signaling in human breast epithelial cell lines. We found that in MCF-10A cells expressing AR, co-stimulation of EGFR signaling with AR ligand binding led to a growth-inhibitory effect due to hyperactivation of the mitogen-activated protein kinase (MAPK) pathway. However, MAPK activation with either AR ligand binding or EGFR activation resulted in cellular proliferation. Moreover, using a genetics-based approach, we found that the effects of AR signaling in MCF-10A cells were mediated through the cyclin-dependent kinase (CDK) inhibitor p21. These data further elucidate the mechanisms that affect AR signaling, and therefore may aid in the development of drugs targeting AR for breast cancer therapy.This was a pre-clinical study not involving human subjects, and therefore did not require ethical review by an institutional review board.2. MCF-7, MDA-MB-231, and MDA-MB-453 cells were grown in DMEM  supplemented with 5% FBS , 100 U/ml penicillin and 100 μg/ml streptomycin (both Invitrogen Corp.). Clones of MDA-MB-231 stably overexpressing AR cDNA were isolated and propagated in DMEM:F12 (1:1) without phenol red (Invitrogen Corp.), supplemented with 5% charcoal dextran-treated FBS (Hyclone Laboratories Inc.), 100 U/ml penicillin, 100 μg/ml streptomycin, and 500 μg/ml G418 . The non-transformed human breast epithelial cell line MCF-10A [AR cDNA was cloned into a modified version of the pIRESneo3 vector , a bicistronic vector with an internal ribosomal entry site (IRES) and the gene encoding neomycin resistance . Cells were selected on medium containing 0.4 μg/ml puromycin (Sigma), and propagated in the same medium as used for the ARIBE cells.Generation of the MCF-10A p21eviously . Because2 tissue-culture flasks at 105 cells/flask. The medium was changed 24 hours later and appropriate drug or vehicle added. All drugs and vehicle controls constituted 0.1% of the final volume in the flasks. The synthetic androgen R1881  was diluted in ethanol (EtOH) and used at a concentration of 1 nmol/l. The androgen antagonist bicalutamide  was diluted in methanol and used at a concentration of 10 nmol/l. Control cells were treated with EtOH only. After 3 days, or when the cells in the control flasks were at 90% to 95% confluency, all flasks were stained with crystal violet (Sigma) diluted in formalin to a concentration of 2 mg/ml.For crystal violet staining, ARIBE and control cells were seeded in 25 cm4 cells/well for experiments without EGF and at 5 × 103 cells/well for experiments using 20 ng/ml EGF. Medium was changed 24 hours later (day 0), and the drug or vehicle was added to the appropriate wells. All drugs and vehicle controls constituted 0.1% of the final volume in the wells. Medium was changed every other day until cells were counted. Cells were counted on days 0, 4, and 8 for experiments without EGF, and days 0, 2, and 4 for experiments using 20 ng/ml EGF.All other growth assays were performed in 12-well tissue-culture plates. Cell counting was performed on a viability analyzer , and all counts were performed in triplicate and repeated at least three times. For ARIBE cell assays, exponentially growing ARIBE and control cells were washed with Hank's balanced salt solution  three times, and seeded in DMEM:F12 medium without phenol red, using 2% charcoal dextran-treated serum, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, and 0.1 μg/ml cholera toxin, either without EGF or with 20 ng/ml EGF, as indicated. Cells were seeded at a density of 1.5 × 104 cells/well. Cells were counted on day 4. The MAPK kinase (MEK) inhibitor U0126  [MDA-MD-231 cell assays were performed similarly. Cells were seeded in DMEM:F12 (1:1) without phenol red, supplemented with 2% charcoal dextran-treated serum at a density of 10WI, USA)  was diluWhole-cell protein extracts prepared in Laemmli sample buffer were resolved by SDS-PAGE using 4% to 12% polyacrylamide gels , transferred to PVDF membranes , and probed with primary and horseradish peroxidase-conjugated secondary antibodies. Primary antibodies were rabbit polyclonal anti-p44/p42 MAPK (ERK) , mouse monoclonal anti-phospho-p44/p42 MAPK (ERK) (Thr-202/Tyr-204) , mouse monoclonal anti-AR , mouse monoclonal anti-p21/WAF , rabbit polyclonal anti-glucocorticoid receptor  and mouse monoclonal anti-GAPDH (6C5) . Proteins were visualized with chemiluminescence . All blots were quantified using ImageJ software  and wereCells were seeded in six-well plates, and drug or vehicle was added the following day. Cells were then harvested and analyzed by fluorescence-activated cell sorting (FACS) analysis at 6 hours or 36 hours after drug addition. Cells were fixed in PBS with 3% formaldehyde and 0.4% NP-40, containing 2 μg/ml dye . DNA content was measured with a flow cytometer , and percentages of G1/G0, S, and G2/M phase cells were determined using Modfit LT software .Renilla luciferase plasmid to serve as control for transfection efficiency.All luciferase experiments were performed in duplicate and repeated at least twice. Cells were seeded in 96-well plates  at 15% confluency in the medium described above for the cell-proliferation assays with 20 ng/ml EGF. At 24 hours after seeding, cells were transfected and concurrently treated with either 1 nmol/l R1881 or vehicle control. The transfection mixture per well contained 12.5 μL reduced serum medium , 0.5 μL transfection reagent , and 100 ng total plasmid. The reporter plasmids consisted of either three copies of a wild-type consensus binding site for AR, or three copies of a mutated binding site of AR conjugated to a firefly luciferase reporter. The consensus binding sites used in the construction of the reporters were gtacattGtGttct for AR, and gtaAattGtAttTt for the mutated AR consensus binding sites. Additionally, each well was co-transfected with a Renilla luciferase activity was measured on the luminometer for 2 seconds per well. All firefly luciferase measurements were normalized to Renilla luciferase measurements. AR activity was expressed as the ratio of the luciferase activity in cells transfected with consensus binding site plasmids divided by the luciferase activity in cells transfected with mutated binding site plasmids.At 48 hours after drug treatment and transfection, luciferase activity was assayed using a commercial luciferase assay system  following the manufacturer's protocol. Briefly, cells were lysed with 150 μL of a 1:1 mixture of luciferase  and medium (Opti-MEM), and incubated at room temperature for 15 minutes. Firefly luciferase activity was measured by a luminometer  for 2 seconds per well, then quenched with 75 μL of the provided reagent . Fifteen minutes after quenching firefly luciferase activity, TBP) levels in the same samples in each experiment. FKBP5, NSDHL and IGFR-1 gene expression was reported as a ratio of the expression in cells treated with drug divided by the expression in cells treated with vehicle. Quantitative real-time reverse transcription PCR (qPCR) was performed  according to the manufacturer's instructions, with on-column DNAse I digestion, then complementary DNA was synthesized . Using cDNA as template and SYBR Green (Invitrogen Corp.) to detect DNA products, expression of insulin-like growth factor (IGFR)-1 was assayed using a two-color real-time detection system . Reactions were performed in triplicate, and repeated at least twice. Transcript levels were normalized to TATA binding protein (in situ hybridization (FISH) analysis, tissue pre-treatment was first performed using paraffin wax-embedded tissue sections 4 to 5 μm thick that had been mounted on charged microscope slides, which were dewaxed, rehydrated through a decreasing graded ethanol series, and treated using a commercial tissue protease kit  according to the manufacturer's directions. After halting protease activity with the provided stop solution, slides were washed, then dehydrated through an increasing graded ethanol series and taken to hybridization. Fluorescently labeled hybridization probes for the X centromere  and the AR locus at Xq12  were diluted in hybridization buffer , 10% Dextran Sulfate), mounted on slides, covered with coverslips, and denatured at 95°C for 5 minutes. Hybridization was conducted overnight at 37°C. Slides were then washed for 4 minutes at room temperature in post-hybridization wash buffer , followed by a second wash with SSC at 75°C for 3 minutes, and then a third wash with water at room temperature for 4 minutes. Slides were then counterstained with DAPI  for 5 minutes at room temperature. Coverslips were mounted using anti-fade mounting medium (Prolong Gold[ Invitrogen Corp./Molecular Probes), and the slides were sent for analysis. Slides were imaged with an epifluorescence microscope  equipped with an illuminator  and a 10 ×/1.4 NA oil immersion lens . Fluorescence excitation/emission filters were as follows: SpectrumOrange excitation, 546 nm/10 nm BP; emission, 578 nm LP (Carl Zeiss Inc.); DAPI excitation, 330 nm; emission, 400 nm via an XF02 fluorescence set ; SpectrumGreen excitation, 475 nm; emission, 535 nm via a combination of 475RDF40 and 535RDF45 filters . Grayscale images were captured for presentation using NIS-Elements software (Nikon) and an attached digital camera , pseudo-colored, and merged.For fluorescent A previously described (@) breast cancer tissue microarray was used for FISH analysis. Two blocks were employed consisting of 30 and 35  samples of primary invasive ductal carcinomas. Tissue microarrays were prepared for FISH analysis as described above.For PSA ELISAs, growth assays were performed as above and supernatants harvested at the end of Day 4. Supernatants were then subjected to ELISAs using the Quantikine human Kallekrein3/PSA Immunoassay kit  as per the manufacturer's protocol.P < 0.05 was considered significant.All statistical analyses were performed using GraphPad InStat software . PIK3CA mutations in the kinase domain [As mentioned above, many previous studies have identified AR expression in human breast cancers. However, levels of AR expression , have been infrequently reported due to difficulty with quantification by immunohistochemistry staining. However, recent studies suggest that overexpression of AR in breast cancer does occur, and is associated with overexpression of ERα and in breast cancers with e domain ,39. In ae domain . Althouge domain , we perfe domain  and was To study AR signaling in ERα-negative non-tumorigenic human breast epithelial cells, we transfected MCF-10A cells with an AR cDNA using a bicistronic vector with an IRES and the gene encoding neomycin resistance. Multiple clones were isolated and designated as ARIBE cells with two representative clones, ARIBE-1 and ARIBE-2, used for all subsequent experiments. As a control, MCF-10A cells were transfected with an 'empty' vector and underwent the same antibiotic selection and single-cell dilution process. Western blot analysis identified high levels of expression of AR in ARIBE-1 and ARIBE-2, which was higher than the expression in MDA-MB-453 cells, but comparable with levels in the AR-positive prostate cancer cell line LNCaP  or a mutated ARE that has been shown to have reduced binding affinity for AR. If AR is active, it will drive luciferase expression when transfected with the wild-type plasmid but not with the mutant plasmid. In all experiments, a 0 nmol/l . The rel0 nmol/l . Similar [IGFR-1 ,45, p21 R-1 [p21 , FKBP5 [1 [FKBP5  and NSDH5 [NSDHL . qPCR waP < 0.001) growth inhibition   Figure . Without) Figure . TherefoTo determine whether the growth inhibition induced by R1881 was the result of cell death or cell cycle arrest, we performed FACS analyses on ARIBE cells cultured in the presence of EGF with and without R1881. There was no significant difference between vehicle-treated and drug-treated cells at 6 hours, but at 36 hours, the cells treated with R1881 showed an increase in the G1/G0 cell cycle fraction compared with cells treated with vehicle  . We examCollectively, these data suggest that ARIBE cells exposed to R1881 display physiologic AR signaling, based upon cellular growth patterns that are antagonized by bicalutamide, activation of key signal transduction pathways, and the ability to upregulate gene expression via known AREs.KRAS mutation and a BRAF mutation, both of which could further activate the MAPK pathway. However, it has been shown that this cell line is relatively genetically stable compared with other breast cancer cell lines [To ensure that the results seen with ARIBE cells were due to signaling through AR and were not a unique response of MCF-10A cells or artifacts from random transgene insertion, we created a second AR-expressing cell line. We used the MDA-MB-231 cell line because it is also ERα/PR/HER2-negative, and has a defined number of mutations in key oncogenes (Sanger COSMIC database). This cell line overexpresses EGFR, which leads to autophosphorylation of EGFR and constitutive activation of the MAPK pathway ,55. MDA-A control cell line was also created by transfecting MDA-MB-231 cells with the empty vector and selecting antibiotic-resistant clones. When stably expressing AR, these cells showed similar responses to R1881 as seen in ARIBE cells; that is, growth inhibition occurred in a dose-dependent manner but with a higher IC50 compared with ARIBE cells, and this effect was blocked by co-culture with bicalutamide (data not shown). A potential caveat to these studies is that R1881 has been shown to bind to the glucocorticoid receptor (GR) , and theRAS and RAF mutations, given that MEK is distal to these proteins in the MAPK pathway. R1881 had no effect on empty vector control cells but caused marked growth inhibition in two AR-expressing clones . Collectively, these results and the ARIBE cell line data show that AR signaling with concurrent MAPK activation via the EGFR pathway can lead to cell cycle arrest. Additionally, these observations led us to hypothesize that the cells are undergoing a phenomenon similar to oncogene-induced senescence, whereby the hyperstimulation of growth-promoting pathways and/or DNA damage may induce cellular death/arrest or induce senescence [Because of the aforementioned genetic alterations in the MAPK pathway in MDA-MB-231 cells, no exogenous growth factors are needed for propagation. Therefore, to simulate EGF removal, we used pharmacological inhibitors of the MAPK pathway and then assayed the response to R1881. We used the MEK inhibitor U0126  because s Figure . Additionescence .p21 gene expression increased in ARIBE cells in response to R1881 , and somatic cell gene knock-out, as described previously [p21 gene knock-down, the ability of R1881 to cause cell cycle arrest under full EGF conditions (20 ng/ml) was dramatically reduced compared with control cells (P < 0.05). Because of the transient nature of siRNA and the longevity of the cell-proliferation assays in conditions with no EGF, effects of p21 knock-down on increased cell proliferation mediated by AR signaling could not be assessed.Although the striking increase in p21 protein levels upon R1881 exposure and the known role of p21 in mediating cell cycle arrest suggested that p21 might be a key mediator of AR-induced arrest, we sought to definitively confirm this using two complementary techniques: knock-down of eviously . After tp21 gene knock-down under conditions of increased cell proliferation , and the fact that gene knock-down can produce non-specific toxicity and significant biologic differences compared with gene knock-out [-/- cells were stably transfected with the same AR cDNA used to create the ARIBE cell line. Clones with antibiotic resistance underwent a single-cell dilution process and multiple clones were isolated. Expression of AR was assayed by western blotting. Two representative clones (p21-/- AR-1 and p21-/- AR-4) had levels of AR expression comparable with those of the p21 wild-type ARIBE cells  gives confidence that targeting AR for breast cancer therapy could be of tremendous importance in treating this disease, and indeed clinical trials are currently underway to test this hypothesis = . HistoriWe characterized these cell lines using a variety of methods, and found that physiologic AR signaling is present in these cells and can induce increased transcription of genes via AREs and increased MAPK signaling. Importantly, our studies provide a number of mechanistic insights. First, R1881 bound to AR leads to increased MAPK signaling regardless of the growth phenotype. Second, AR signaling is dependent on the CDK inhibitor p21, as gene knock-down and knock-out largely abrogated all AR-mediated proliferation in these cell lines. Third, hyperactivation of the MAPK pathway by both EGFR and AR signaling leads to cell cycle arrest, whereas stimulation by either EGFR or AR alone results in cellular proliferation. Cellular arrest by EGFR and AR signaling may be similar to the phenomenon of oncogene-induced senescence, whereby activation of growth-promoting pathways beyond a critical threshold induces cell cycle arrest followed by senescence. Importantly, because our unique model is capable of displaying both growth phenotypes within the same cell line, it allows for the further study of genetic effectors that specifically mediate a growth stimulatory versus inhibitory response to AR signaling in human breast cells.p21 gene expression regardless of the growth phenotype. This may have important clinical considerations, as we have previously reported that loss of p21 expression is seen in up to 40% of human breast cancers [PIK3CA mutations [PIK3CA and AR expression would have a favorable therapeutic response to AR ligand binding. By contrast, molecular apocrine tumors may benefit from dual MEK and AR inhibition as previously reported [The fact that p21 is necessary for AR signaling leading to MAPK activation is consistent with previous reports that the p21 promoter contains an ARE . Further cancers . It mighutations , our resreported , and simIn this study, we constructed new models for AR signaling in human breast epithelial cells. We found that activation of the MAPK pathway by either EGFR or AR signaling leads to cellular proliferation, whereas simultaneous input by both EGFR and AR leads to further increases in MAPK activation and cellular arrest. These findings help elucidate past observations of disparate growth responses to AR ligand binding in various human breast cancer cell lines. Additionally, methods to quantify activation of the MAPK pathway on human tissues may allow for the development of predictive markers of AR signaling that leads to a growth proliferative versus inhibitory phenotype. Moreover, we found that p21 is essential for mediating AR signaling in human breast epithelial cells, regardless of the growth response to AR ligand. Thus, AR expression in conjunction with the presence or absence of p21 may also be useful for predicting sensitivity versus resistance to AR-directed therapies. Significantly, our system provides an ideal model for further study. The use of ARIBE cell lines will help reveal genes and pathways that are crucial for mediating these growth effects, and potentially identify additional predictors of response to AR ligands, thereby accelerating the development of drugs targeting AR for breast cancer therapy.AR: androgen receptor; ARIBE: Androgen Receptor In Breast Epithelium; DAPI: 4'-6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; ERα: estrogen receptor-α; ERK: extracellular signal-regulated kinase; FCS: fetal calf serum; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; MAPK: mitogen-activated protein kinase; PR: progesterone receptor; PVDF: polyvinylidine fluoride; qPCR: quantitative PCR; RNAi: RNA interference; SSC: saline sodium citrate; siRNA: small interfering RNA; TBP: TATA binding protein.B.H.P. has received previous research funding from GlaxoSmithKline (GSK) although none of the studies reported here were supported by GSK. B.H.P. is a consultant for GSK and is on the scientific advisory board for Horizon Discovery, LTD and is entitled to payments for these services. These arrangements are managed according to the Johns Hopkins University conflict of interest policy. All of the other authors declare that they have no competing interests.JP Garay, BK, AMA, MJH, DPC, DJ, AW, PJM, AM, AMD, PA, and BHP designed the experiments; JP Garay, JP Gustin, HK, YK, MM, GMW, CASB, AT, AM, GL and AS performed the experiments; JP Garay, BK, AMA, DPC, MJH, DJ, CASB, PA, AMA and BHP analyzed the data; and JP Garay and BHP wrote the paper. All authors read and approved the final manuscript.Supplementary Figures 1 to 8, Tables 1 and 2. Supplementary figures (eight) and tables (two).Click here for file


We dissected the functional domains of p44 for protein interaction with transcription factors, transcriptional activation, as well as the functional domains in p44 related to its growth inhibition in prostate cancer. Using a yeast two-hybrid screen, we identified a novel transcription complex AR-p44-Smad1, confirmed for physical interaction by co-immunoprecipitaion and functional interaction with luciferase assays in human prostate cancer cells. Yeast two-hybrid assay revealed that the N-terminal region of p44, instead of the traditional WD40 domain at the C-terminus, mediates the interaction among p44, N-terminus of AR and full length Smad1. Although both N and C terminal domains of p44 are necessary for maximum AR transcriptional activation, the N terminal fragment of p44 alone maintains the basic effect on AR transcriptional activation. Cell proliferation assays with N- and C- terminal deletion mutations indicated that the central portion of p44 is required for nuclear p44 mediated prostate cancer growth inhibition. Prostate cancer (PCa) is the most common malignancy in men in the United States. In 2011, prostate cancer alone leads to 29% of all newly diagnosed cancers and is the second leading cause of death as a result of cancer An increasing number of AR cofactors have been identified in recent studies Several signal transduction pathways, including transforming growth factor-β superfamily (TGF-β) and bone morphogenetic proteins (BMP) pathways, converge with AR signaling and alterations in these pathways have been implicated in PCa development and growth In this study, we report that Smad1 physically interacts with p44 and the interaction is mediated by AR, subsequently regulating AR transcription activity in the presence of both BMP and androgen. We further delineated three functional domains responsible for AR interaction, AR transcriptional activation and growth inhibition in p44.Antibodies against AR, Smad1 and β-actin, as well as secondary antibodies, were purchased from Cell Signaling Technology, Inc.  or Santa Cruz Biotechnology, Inc. . Affinity purified p44 antibody was described previously LNCaP cells  and its derivate C4-2B were maintained in RPMI 1640  with 10% heat-inactivated bovine serum (FBS). The androgen-independent LNCaP-AI cells, a derivative of LNCaP, were maintained in RPMI 1640 medium containing 10% charcoal-stripped, heat-inactivated FBS (CSFBS)  and 5 µg/ml of insulin, as described previously LNCaP cells stably expressing NLS-p44 were cultured in 100 mm dish in either androgen  or androgen free media until 80%–90% confluence. Cells were then transfected with Smad1 plasmid using lipofectamine 2000 (Invitrogen) following manufacturer user manual. 48 hours after transfection, cells were treated with BMP-2 (GenScript) at a final concentration of 100 ng/mL and harvested in lysis buffer  supplemented with protease inhibitor cocktail (Sigma). Whole cell lysates were collected by centrifugation at 10,000 rpm for 10 minutes at 4°C and total protein concentration measured using a BCA quantitation assay (Pierce). Lysates were precleared by adding 20 µl resuspended Protein G-Agarose beads (Kirkegaard & Perry Laboratories) 4°C for 30 minutes with rotation. 500 µg total proteins was immunoprecipitated with either 1 µg or 2 µg anti-Smad1 (Millipore) or anti-p44 antisera overnight at 4°C. Antibody was captured with 20 µl of slurry of 50% protein A/G beads equilibrated in lysis buffer by mixing for 4 hours at 4°C, and beads were washed three times with lysis buffer. Bound protein was eluted in 2x loading buffer and analyzed by SDS-PAGE electrophoresis and immunoblotting as described below.5-TCCTCCTCTTCCTCAATCTCG-3, ActinR: 5-AAGGCAACTTTCGGAACGG-3) and p21FR , respectively.Chromatin IP (ChIP) was performed as described previously After electrophoresis on SDS-PAGE, protein was transferred to a nitrocellulose membrane for Western blot analysis. Immunoblots were blocked for 30 min in 3% nonfat dry milk in TBST . Blots were incubated with anti-AR, anti-p44 or anti-Smad1 antibodies overnight at 4°C, washed with TBST three times, and incubated for 1.5 h with secondary antibody  (Amersham Biosciences). Antibodies were diluted with 2% BSA in TBST. The protein bands were detected by an enhanced chemiluminesence kit (Amersham Biosciences).The p44 cDNA was deleted every 200 bp from ether N-terminus or C-terminus to generate series of deletions N8, N6, N4, N2, C8, C6, C4 and C2. These deletions were cloned into plasmid pBabe-NLS and fused with nuclear localization signal (NLS) to give plasmids pBabe-NLS-N8, pBabe-NLS-N6, pBabe-NLS-N4, pBabe-NLS-N2, pBabe-NLS-C8, pBabe-NLS-C6, pBabe-NLS-C4 and pBabe-NLS-C2. Those plasmids were used for luciferase assay or construction of stable cell lines. Construction of stable cell lines and luciferase assay were performed as described previously MATa leu2::6xLexAop-LEU2 trp ura ade his) was transformed with pSH18-34 reporter vector  (Origene Technologies), pLexA or pLexA::p44. Resulting yeast strain was then transfected with a yeast two-hybrid library derived from human prostate (Origene). Transformants were selected and target cDNA was puified as described previously Yeast media were prepared as described previously MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) Direct yeast two-hybrid assays were performed to delineate the structural and functional p44 domains. Yeast strain W303-1A . We reasoned that p44 and full length Smad1 may interact indirectly, via a mediator. Since both proteins are known to be involved in androgen receptor (AR)-mediated gene regulation, we decided to test whether the introduction of AR into the two-hybrid system will restore p44-Smad1 interaction. Vector only plus TGFβand FKBP1a were used as a positive control. The resulting system then contained p44-LexA fusion that binds to the LexA operators in the reporter plasmid, Smad1 fused to the B42 transcription activation domain, and AR. Coexpression of Smad1, p44, and AR resulted in a positive two-hybrid signal . We therefore constructed a two-hybrid vector expressing full length Smad1. Full length Smad1, however, did not interact with p44 in two-hybrid tests , right. d signal , suggestTo perform a more careful examination of the interactions among p44, AR and Smad1 in mammalian cells, serials of co-immunoprecipitation were performed using LNCaP cells stably expressing NLS-p44. With anti-p44 antibody, we were able to precipitate and then detect AR in androgen supplemented media but not in androgen-free media , which iTo determine the p44 domain(s) that are involved in the tripartite interaction, we constructed two-hybrid vectors containing individual domains or fragments of AR, Smad1, and p44. The domain structure of p44 is unknown. Therefore we created a series of N- and C-terminal 200 bp deletion mutants . Smad1 dIn order to identify the domain of p44 responsible for AR mediated transcriptional activation, the same serial deletions of p44 were used for transfection in PC3 cells and their effects were examined using dual luciferase assay with a 4XARE luciferase reporter. Consistent with its role as AR coactivator, full length p44 significantly increased levels of AR mediated transcriptional activation . On the AGAACAGCAAGTGCT) Smad1 acts as a corepressor First we tested whether p44 is a cofactor of Smad1/Smad4 complex acting independently of AR with the synthetic Smad1 reporter containing 12X SRE . As showNext, we asked whether Smad1 exerts an effect on p44-dependent stimulation of AR-dependent transcription activation in AR-expressing LNCaP cells. Luciferase assay showed that p44 increased transcription activation approximately 3 fold from the reporter under the control of 4XARE synthetic promoter in the presence of R1881 . Co-tranFinally, to study the interplay of p44 and Smad1/Smad4 on transcriptional activation, we tested various combinations of p44 and Smad1/Smad4 with synthetic reporter (12XSRE and 4XARE) or reporter containing p21 promoter (native single copy of ARE and SRE) in the presence and absence of their respective ligands. As shown in in vitro cell proliferation and in vivo tumor xenografts in an AR-dependent manner Nuclear expression of p44 is decreased in PCa and overexpression of nuclear p44 caused growth arrest both in In this study, we determined the functional domains of p44 responsible for protein-protein interaction with AR and Smad1 transcription factors, transcriptional activation, as well as the functional domains related to growth inhibition in prostate cancer. Using a yeast two-hybrid screen and co-immunoprecipitation, we have identified a novel transcription complex AR-p44-Smad1 in prostate cancer cells. The N-terminus of p44 was found to be not only responsible for its interaction with AR and Smad1, but also for its AR transactivation. The central portion of p44 is revealed to be required for nuclear p44 mediated prostate cancer growth inhibition in cell proliferation assays with series of deletion mutations .p44 was initially identified as a subunit of the methylosome complex and later characterized as a steroid receptor coactivator that enhances androgen receptor -1 as welFive WD40 repeats have been identified in the C-terminus region of p44 and these WD40 repeats are speculated to mediate the protein-protein interactions in p44 multiple protein complexes. Recently, Antonysamy et al. determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The interaction between seven-bladed β-propeller MEP50 and the N-terminal domain of PRMT5 mediates the complexes of PRMT5 and MEP50 In study of the domain(s) responsible for AR transcriptional activation, we found both N-terminus and the central region are critical for p44 to maintain maximum effect on AR transcription activation, while N-terminus (up to N6) alone exhibits minimal effect on AR transcription activation. Deletion N8, containing the 266 amino acids from N-terminus is the minimal length for exerting effect on AR transcriptional activation . InteresNuclear p44 inhibits PCa cell proliferation Worthy to mention, the p44 functions are regulated by its subcellular localization In summary, we have identified AR-p44-Smad1 transcription complexes. This result provides better understanding on the molecular mechanisms in regulation of AR transcriptional activation. A balance is present between Smad1-mediated negative regulation and p44-mediated positive regulation of AR activity. Of interest would be the dominant role of p44 in AR-p44-Smad1 complexes on transcriptional activation. We also identified three different regions related to interaction with AR, AR transcriptional activation, and prostate cancer growth inhibition in p44. Our studies provide new insights into the correlation between structures and functions of p44.Figure S1The interaction among AR, p44, Smad1 was detected by Co-immunoprecipitation in C4-2B cells. AR-p44-Smad1 is in an endogenous complex.(PPT)Click here for additional data file.


P. aeruginosa strain PAO1 during growth in a defined medium (BHI) was examined and compared with some neuraminidase features of K. pneumoniae in this investigation.The properties of neuraminidase produced by P. aeruginosa which was used in a sensitive fluorometric assay by using 2′-(4-methylumbelliferyl) α-D-N acetylneuraminic acid as substrate.The enzyme was isolated from concentrated culture supernatants of P. aeruginosa PAO1 paralleled bacterial growth in defined medium (BHI) and was maximal in the late logarithmic phase of growth but decreased during the stationary phase, probably owing to protease production or thermal instability. Highest production of P. aeruginosa PAO1 neuraminidase was in BHI culture media. The neuraminidase of P. aeruginosa PAO1 possessed an optimum temperature of activity at 56°C and the activity was maximal at pH 5. Heating the enzyme to 56°C for 45 min., in the presence of bovine serum albumin destroyed 33.1% of it's activity and addition of Ca+2, EDTA and NANA also decreased activity markedly.Neuraminidase production in p. aeruginosa PAO1.The results revealed that the highest specific activity is for  Pseudomonas aeruginosa is a motile, gram negative, non-spore forming rod shaped bacterium  moiety exposed on asialylated glycolipids , Brucella broth (BB), Minimal Media (M9), Pepton water (PW) and Tryptose soy broth (TSB) for neuraminidase production and activity. Meanwhile, the effects of different environmental conditions such as pH, temperature, added cation (Ca+2) and components  on the expression and activity of the neuraminidase were investigated.The aim of the present study was to investigate the details of the production and properties of neuraminidase produced by Bacterial strains and culture media.Pseudomonas aeruginosa strain PAO1 was kindly provided by Dr. E.A. Worobec  and Klebsiella pneumonia ATCC 10031 was obtained from the American Type Culture Collection. Bacteria were cultured in brain heart infusion broth  at 37°C with continuous shaking up to the stationary phase of growth, which was monitored by measuring cell turbidity at 600 nm  as substrate   , 12. UpoThe quantitative determination of neuraminidase activity with 2′-(4-methylumbelliferyl)-α-D-N- acetylneuraminic acid (MUN) was detected with an F-2500 fluorescence spectrophotometer Hitachi (fluorimeter) using excitation light at 365 nm and measuring emission light at 450 nm , 13. A 1+2) and compounds such as N- acetylneuraminic acid (NANA) and ethylenediaminetetraacetic acid (EDTA) on neuraminidase activity, as well as the effect of pH, Ca+2 and ethylenediaminetetraacetic acid (EDTA) on extracellular and cell-bound neuraminidase activity was tested in the system described above. Also, the relationship between P. aeruginosa PAO1 growth curve and neuraminidase production, the effect of pH and temperature on the stability of enzyme and influence of growth medium on the production of neuraminidase by P. aeruginosa PAO1 was examined.All assays were performed in duplicate. , 13 The P. aeruginosa PAO1 growth curve and neuraminidase production.Relationship between P. aeruginosa PAO1 was grown in BHI broth, at 37°C. At given intervals (1 hour), a portion of culture supernatant was removed, and bacterial growth was measured at 600 nm by a biophotometer (Eppendorf BioPhotometer plus). A portion (50 µl) of the cultures supernatant was filtered through a membrane filter  and dialyzed against acetate buffer (0.1 M) and the activity of enzyme was determined  to 1:25 of the original volume. The suspension was divided into three parts and treated as follows: (i) Tween 80 (0.1%) added to the cell suspension and the mixture was shaken at 30°C for 30 min, (ii) the cells in phosphate-buffered saline (PBS) suspension were lysed by twice freezing and thawing the suspensions in liquid nitrogen, which were allowed to thaw completely before re-freezing , dialyzed against acetate buffer (0.1 M) and assayed for neuraminidase activity  is obtained after removal of nuclei, cell debris and fat layer by serial centrifugations  and centrifuged for 5 minutes at 600×g at 4°C and repeating the wash step once again. Supernatant was discarded in all of these steps. The packed cell was filled with extraction buffer and vortexed to achieve an even suspension. The cells were broken in a 7 ml Dounce homogenizer using Pestle B . After every 5 strokes with the pestle, the cells were checked under a microscope using Trypan Blue staining to ascertain the degree of breakage. The supernatant from this portion was passed through a Millipore filter  and assayed for neuraminidase activity .Effect of various concentrations of NANA on enzyme production by  Data showed that a 4 mg concentration of N-acetyl neuraminic acid (NANA) has the greatest effect on production of neuraminidase by P. aeruginosa PAO1. Further incremental increases had no effect on enzyme production.Influence of growth medium on production of neuraminidase. The greatest enzyme yield was in BHI containing complicated components whereas less enzyme was produced in minimal media (M9) media. The pattern observed for neuraminidase production closely paralleled the growth curve of the organism  release decreased within 40 to 60 min. The assay is linear with respect to enzyme concentration.P. aeruginosa PAO1 neuraminidase activity.The optimum pH and temperature of  The optimum temperature and pH was determined using 4-methylumbelliferyl-α-DN- acetylneuraminic acid (MUN) as the substrate. A peak of maximal activity occurred at 56°C, but greater than 95% of the enzyme activity was destroyed at 65°C. Optimal activity was achieved at pH 6.4.P. aeruginosa PAO1 neuraminidase.Thermal stability of  Heating the enzyme in the presence and absence of albumin led to approximately 30% and 42.5% reduction of the P. aeruginosa PAO1 neuraminidase activity respectively within 45 min at 56 °C.P. aeruginosa PAO1 neuraminidase at different pH after 1 hour.Stability of  Incubation of the enzyme at various pH showed that the lowest and highest stability of the enzyme occurred at pH 7 and 2 respectively.2+ ions, ethylenediaminetetraacetic acid (EDTA) and N-acetyl neuraminic acid (NANA) on neuraminidase activity of P. aeruginosa PAO1.Effects of Ca  Added cations and other reagents were examined for their effects on the neuraminidase activity. Calcium chloride (Ca2+), ethylenediaminetetraacetic acid (EDTA) and N-acetyl neuraminic acid (NANA) led to a decrease in activity. The results indicated that calcium ions, ethylenediaminetetraacetic acid (EDTA) and N-acetyl neuraminic acid (NANA) have inhibitory effects on the enzyme activity  and Calcium chloride (CaCL2) on extracellular and cell-bound neuraminidase activity.P aeruginosa PAO1 and K. pneumonia ATCC 10031. In P. aeruginosa PAO1, increasing the pH led to an initial drop in activity at pH 6 followed by an increase whereas in K. pneumoniae ATCC 10031, the enzyme activity increased with increasing pH and an appreciable drop in activity was observed at pH 7. As well, there were some observed increases and decreases in enzyme activity when the whole-cell bacteria was incubated in the presence of various CaCl2 and EDTA concentrations for the cell-bound and extracellular neuraminidase activity of sheep kidney and rat liver lysosome  production which has occurred at the stationary phase and causes breakup of the enzyme . Beside pH effect on activity of the enzyme, there may be changes in enzyme structure that affects the enzyme stability.P. aeruginosa PAO1 is susceptible to Ca2+. The Calcium ions have increasing effects on the activity of neuraminidase of Vibrio cholerae and are capable to make activity of the enzyme two-fold at a concentration of 1mM  that competitively prevents activity of the enzyme  was induced to increase of enzyme reaction to membrane and being bound to it. But at the concentration more than 0.03 M, the salt had destructive effects over this reaction and was induced to release the enzyme from the membrane. Divalent cations such as Mgmembrane . These tmembrane , 25.Soluble form of the enzyme at the high concentrations of salt, is due to change in the enzyme conformation and at the tertiary structure of it that affects on catalytic activity of the enzyme as well. The amount of conformational change established by salt is depend on their reaction with the polar bounds at the peptide sequence , 30.2+, the opposing effects of EDTA (more and less than 1.5 mM) could be explained by EDTA acting as a chelating agent and resulting in a loss of positive ions, causing an increase in the ionic repellent forces between the enzyme and membrane and consequently becoming soluble. At concentration of more than 1 mM, the enzyme adhesion is due to the increasing ionic forces of solution (Also similarly to Casolution , 25.P. aeruginosa PAO1 and it is 1.82, 11.94, 8.63 and 40.6 fold of Klebsiella pneumonia ATCC 10031, sheep kidney, rat liver and Hep-2 cell neuraminidase specific activity respectively and the neuraminidase of P. aeruginosa PAO1 is more an extracellular enzyme than cell-bound.Taken together, these results suggest four contrasting organisms. The highest activity is for neuraminidase of


Long-term disability following natural disasters significantly burdens survivors and the impacted society. Nevertheless, medical rehabilitation programming has been historically neglected in disaster relief planning. ‘NHV’ is a rehabilitation services program comprised of non–governmental organizations (NGOs) (N), local health departments (H), and professional rehabilitation volunteers (V) which aims to improve long-term physical functioning in survivors of the 2008 Sichuan earthquake. We aimed to evaluate the effectiveness of the NHV program.510 of 591 enrolled earthquake survivors participated in this longitudinal quasi-experimental study (86.3%). The early intervention group (NHV–E) consisted of 298 survivors who received institutional-based rehabilitation (IBR) followed by community-based rehabilitation (CBR); the late intervention group (NHV–L) was comprised of 101 survivors who began rehabilitation one year later. The control group of 111 earthquake survivors did not receive IBR/CBR. Physical functioning was assessed using the Barthel Index (BI). Data were analyzed with a mixed-effects Tobit regression model. Physical functioning was significantly increased in the NHV–E and NHV–L groups at follow-up but not in the control group after adjustment for gender, age, type of injury, and time to measurement. We found significant effects of both NHV  and sponaneaous recovery . The effect of NHV-E  was marginally greater than that of NHV-L . It could, however, not be determined whether specific IBR or CBR program components were effective since individual component exposures were not evaluated.Our analysis shows that the NHV improved the long-term physical functioning of Sichuan earthquake survivors with disabling injuries. The comprehensive rehabilitation program benefitted the individual and society, rehabilitation services in China, and international rehabilitation disaster relief planning. Similar IBR/CBR programs should therefore be considered for future large-scale rehabilitation disaster relief efforts. Long-term physical disability following natural disasters significantly burdens the impacted society, even more so than immediate medical needs The large volume of traumatic injuries overwhelmed the severely damaged local medical infrastructure of Sichuan province, resulting in mass evacuation of medically stable patients to hospitals across China IBR was administered in county hospital rehabilitation departments where patients participated in individualized physical rehabilitation programs. Rehabilitation interventions included muscle strengthening and range of motion exercises, training in self care and mobility activities, education in bladder, bowel and skin care management, and provision of assistive devices. Traditional Chinese therapies including acupuncture and massage were also provided. Following discharge to the community, CBR health sector services including medical care, rehabilitation, assistive devices, health prevention, and health promotion were provided Currently, NHV operates in two counties which accounted for more than 20% of the earthquake casualties This research has been approved by the ethical review board of the Medical Faculty of the Nanjing Medical University. Informed consent has been obtained from all study participants. All clinical investigations have been conducted according to the principles expressed in the Declaration of Helsinki.We use a longitudinal quasi–experimental design with two points of measurement and two intervention groups and a control group.NHV IBR was implemented initially in a hospital in County A (NHV-E) which provided specialized rehabilitation care in its newly established rehabilitation department as part of comprehensive medical management of victims. CBR services were concurrently implemented in the community for patients' benefit following discharge by an international rehabilitation services NGO. Due to the urgent need to rehabilitate additional victims and given NHV's operational efficiency the program was implemented in neighbouring County B after one year (NHV-L). Due to resource and political constraints NHV was not implemented in neighbouring county C which served as the control group. Earthquake impact and victim demographics were comparable in the neighboring counties evaluated.Five hundred and ninety-one earthquake survivors were originally identified and enrolled. Eligibility criteria for this study were: 1) age of 18 years and older; 2) diagnosis of a disabling injury requiring physical rehabilitation caused by the earthquake, and 3) no complicating internal injury. Based on these criteria, two patients were excluded due to complicating internal injuries. Written informed consent was obtained from participants in the intervention groups. Verbal telephonic consent was obtained from patients in the control group due primarily to limited volunteer availability. Identical consent forms were used. Five hundred and ten subjects completed the study of which 298 subjects were assigned to NHV–E, 101 participants to NHV-L, and 111 to the control group .Physical functioning was assessed with the Chinese version of the Barthel Index (BI), a measure of performance of ADLs, comprised of personal hygiene, bathing, eating, toileting, ambulation, dressing, bladder and bowel control, transfers, and mobility Diagnostic data on injury type were retrieved from patient hospital charts. Demographic information including gender and age as well as date of assessment were also recorded. Baseline intervention group data were obtained prior to the onset of IBR; NHV-E data were obtained at 4–10 months post-earthquake and data for the NHV-L group at 16 months. Baseline control group data were collected at 10 months post-earthquake. Follow-up data for the three groups were collected at 27.5 to 34 months . MeasureDue to a ceiling effect of the BI  we compuA ceiling effect implies that variance of BI scores cannot be measured above the scale maximum score due to the scale design. Scores, however, can be mathematically estimated with the Tobit model which employs a latent dependent variable that can assume values above the scale maximum. Estimation of supramaximal values allows correction for the baseline imbalance of physical functioning across study groups as the NHV-L and control groups had higher baseline values see , leavingi) at different time points (level 1) were nested in individuals j  (level 2) who in turn were nested in different intervention groups (level 3) or counties k , random intercepts δj and φk were introduced on the individual and county level, respectively. To model individual responsiveness to recovery over time, a random slope γj for the time from the earthquake to the respective measurement points in days (TimeEQMP) was considered. In the fixed part of the model, BI values Y* (latent) were estimated using patients' age, TimeEQMP, and receipt of NHV rehabilitation services at follow up in the early (NHV_E_fu) or late intervention group (NHV_L_fu) as time-varying covariates. Gender , injury type , and presence in an intervention (NHV_E_base vs. NHV_L_base) or control county (reference) were time-constant covariates. Random effects and residuals ε were assumed to be normally distributed with a mean of zero and variance of σ2.Since observations i  for multiple testing.To differentiate the effect of IBR/CBR from the effect of recovery over time attributable to other factors, counterfactual predictions To determine the possible degree to which study dropouts may have resulted in estimation bias, the model was re-estimated by imputing BI dropout data for three scenarios: a) BI remained constant over time; b) BI decreased by ten points; c) BI remained constant in dropouts from the intervention groups while increasing by ten points in controls (censored observation stayed at 100). Fatalities were excluded.All data were analysed with Stata 12. The three-level Tobit model was implemented with the gllamm command Parameter estimates of the multi-level Tobit model  show basMarginal predictions show significant differences in baseline and follow-up BI scores in NHV-E  and NHV-L , while the difference is no longer significant in the control group  due to the absence of the NHV effect .2 = 22.9, p<0.001). Dropouts were also significantly younger  and had higher baseline BI scores in the intervention groups . Sensitivity analysis showed the greatest relative decrease in NHV rehabilitation effects for imputation scenario B in which dropout BI scores were decreased by ten points  benefitted. Statistically meaningful improvement in physical functioning was thus shown in both early and late intervention groups, demonstrating benefit from rehabilitation delivered nearly 1.5 years after injury.To compare our results with findings from the literature, we performed a systematic review (May 2012) in PubMed, Embase, and CINAHL using the search terms “rehabilitation” AND “earthquake” AND (“outcome”* OR” effectiveness”). Four studies reporting quantitative data on functional outcomes of rehabilitation interventions on adult earthquake subjects were identified; all investigated populations from Sichuan. Two employed a cross-sectional design with a control group Evidence on the effectiveness of medical rehabilitation in earthquake victims is scarce. Of the four relevant studies, two were of questionable methodological quality and none employed a pre-post intervention design using a control group. Our study is the first to employ a longitudinal design with a control group, allowing estimation of a causal effect of rehabilitation programming on physical functioning. The comparably large sample sizes allowed for appropriate adjustment for patient demographics and injury types. This study thereby contributes significantly to the evidence base on medical rehabilitation of earthquake victims.Limitations of our research require that its results be viewed with caution, however. Counterfactual analysis allowed us to differentiate the effect of NHV IBR/CBR from that of recovery over time. However, although effectiveness of the NHV IBR/CBR program was demonstrated, it could not be determined whether specific IBR or CBR program components were effective since individual component exposures were not evaluated. IBR is believed to have made a significant contribution as has been found in other studies Although the BI ceiling effect was corrected using a Tobit model, actual improvement of patients who started IBR with censored scores was nonetheless not measured in this study. Future studies could employ other functional status measures which allow more precise evaluation of functional improvement, potentially based on WHO's International Classification of Functioning, Disability and Health (ICF) Increased dropouts in controls compared to intervention groups may have also biased results; nonetheless, the NHV effect remained significant in our sensitivity analysis. Younger persons with better functional status at baseline dropped from the study, presumably migrating to other counties for employment Our study has significant implications for rehabilitation disaster-related research, for the study population, for the impacted area, and for rehabilitation disaster relief within China and internationally. Regarding disaster-related research, this analysis reinforces the significance of systematic needs and functional assessment data collection in support of long-term outcomes follow up studies. Such studies are required to improve the scientific base of disaster relief planning Sichuan earthquake victims with improved physical functioning are able to perform daily activities more independently, requiring less assistance. Moreover, they can perform household domestic tasks and work outside the home, contributing to the overall recovery of post-disaster society with reduced demand on health and social services. Greater victim community integration also enhances personal empowerment and well-being. The NHV rehabilitation services programme has thereby reduced the potential long-term burden of the earthquake on individuals and the community in this resource-constrained area.The significant development of rehabilitation infrastructure via NHV demonstrates how large-scale natural disasters, particularly earthquakes, present an opportunity to expand rehabilitation services in low-resource regions. When the international rehabilitation services NGO departs in 2013 at the end of its five-year commitment, significantly developed IBR and CBR services will remain in place. Rehabilitation departments of hospitals in County A and B are now treating traumatic victims of motor vehicle accidents as well as other disabling injuries and rehabilitation diagnoses.The rapid, close, and continued coordination between multiple rehabilitation stakeholders, including local authorities, national and international NGOs, and professional societies, serves as an example to guide future disaster responses in China and internationally. Capitalizing on the established operational mechanism and employing experienced program leadership, NHV was implemented in only half the time in County B (two weeks). The use of a professional volunteer recruitment database to hasten response underscores the recognized need within the international humanitarian response community for an international register of foreign medical team provider organizations In conclusion, our study provides evidence that the NHV program was effective in improving physical functioning of injured earthquake survivors of the 2008 Sichuan earthquake. The comprehensive rehabilitation program benefitted the individual and society, rehabilitation services in China, and international rehabilitation disaster relief planning. Similar IBR/CBR programs should therefore be considered for future large-scale rehabilitation disaster relief efforts.


Hereditary recurrent fevers (HRF) are autoinflammatory disorders whose etiology remains unknown in many cases.To identify a new HRF geneTNFRSF11A was screened by Sanger sequencing in other patients. TNFRS11A expression was quantified by fluorescence-activated cell sorter analysis (FACS). NF-k B activation was assessed using a luciferase assay in HEK293 cells transfected with plasmids encoding wild-type and mutated TNFRSF11A.Comparative genomic hybridization  was performed in the proband. TNFRSF11A. This transmembrane receptor binds the TNFSF11 cytokine, activates NF-k B signaling, and regulates fever in rodents, consistent with a possible role in HRF. TNFRSF11A screening in other patients with genetically-unexplained HRF revealed a heterozygous frameshift mutation in a patient and her affected mother. The mutated protein is expressed at similar levels as the normal receptor on leukocytes. Most importantly, this mutation results in a gain of function on NF-k B signaling, since the mutated protein is more responsive to TNFSF11 stimulation than the wild-type receptor. Since TNFRSF11A  was previously known for its key role in osteoclastogenesis, the medical history of our patients was reassessed and revealed minor symptoms also found in patients with TNFRSF11A-associated bone disorders.Array-CGH analysis performed in a patient with multiple congenital anomalies and a recurrent fever syndrome revealed a de novo heterozygous chromosomal rearrangement encompassing a duplication of TNFRSF11A in HRF reveals a key role of this receptor in autoinflammation and opens up new fields of research at the crossroads between bone metabolism and innate immunity.The implication of None declared


Fixation in 10% neutral buffered formalin prior to transfer to 70% ethanol for one week has been shown to adequately preserve immunorecognition of PCNA, cytokeratins AE1/AE3 and EGFr. This study investigated whether 12 hrs fixation in 10% NBF plus transfer to 70% ethanol for 4 weeks would similarly preserve immunorecognition to an extent where antigen retrieval (AR) used to reverse the masking effects of fixation on some antigens would not be necessary. Two cell lines, DU145 and SKOV3 were grown on coverslips and fixed either for 684 hrs in 10% NBF or for 12 hrs in 10% NBF which was then replaced with 70% ethanol for 672 hrs. The second experiment had the same design except an additional set of cells were subjected to heat-induced AR concomitantly. PCNA, cytokeratins AE1/AE3, and EGFr (membrane and cytoplasmic) were used to evaluate the effects of immunorecognition. Fixation in 10% NBF for 12 hrs plus transfer to 70% ethanol for 672 hrs did not preserve immunorecognition of PCNA adequately in either cell lines. Cytokeratins immunoreactivity was preserved by transfer to 70% ethanol. Cytoplasmic EGFr antigens were not adversely affected by 10% NBF fixation in either cell line and transfer to 70% ethanol had limited effects. With AR, there was little recovery of PCNA immunorecognition on cells fixed in only 10% NBF, but almost complete recovery for cells transferred to 70% ethanol. For cytokeratins there was complete recovery of immunorecognition either with only 10% NBF or 12 hrs plus transfer to 70% ethanol. For EGFr, AR resulted in complete loss of immunorecognition following either treatment. This study indicated that 12 hrs of fixation in 10% NBF plus transfer to 70% ethanol for 4 weeks with AR resulted in recovery of immunorecognition for PCNA and cytokeratins, but standard methods of AR caused loss of immunorecognition of EGFr. The reaction of 10% NBF with primary amines (e.g. lysine) and thiols (e.g. cysteine) results in the formation of the hydroxymethyl groups For too long the practice of transferring tissues from 10% NBF to 70% ethanol to avoid masking effects of formalin on some antigens has been anecdotal. A recent study demonstrated that immunorecognition of PCNA, cytokeratins AE1/AE3, EGF-receptor and Ki67 MIB-1 was preserved by the transfer from 10% NBF to 70% ethanol for up to one week; however, effects of long term fixation or storage of cells in ethanol was not determined The current study examines the effects of long term storage of cells  in 70% ethanol after an initial 12 hrs in 10% NBF and specifically seeks to determine which approach (AR versus transfer to 70% ethanol) better preserves or recovers immunorecognition. This study demonstrated that after 4 weeks of fixation in either condition, neither approach was optimal to preserve or recover immunorecognition for all antigens.The study included two experiments: the first experiment was a 4 week fixation study which compared cells grown on coverslips and fixed for 684 hrs in 10% NBF with 12 hrs fixation in 10% NBF followed by transfer to ethanol for 672 hrs. The second experiment was identical but included AR i.e., 4 weeks of the two conditions of fixation with and without AR. For comparison, each of the two experiments was run with 5 min (0.083 hrs) of 10% NBF fixation representing a condition of minimal fixation. This short period of fixation was necessary to ensure that cells remained attached to the coverslips Experiment 1  was desiThe results from experiment 2 indicated losses of immunostaining for cytoplasmic and membrane EGFr, which was not expected. Because the pH used in experiment 2 might have caused the loss in staining for EGFr, additional cells grown on coverslips were fixed in 10% NBF for 5 min, 180 hrs or 12 hrs followed by transfer to 70% ethanol for 168 hrs to enable simultaneous staining of four AR solutions as well as no AR. The AR solutions tested were: 0.01 M citric acid pH 6.0, 0.01 M EDTA pH 8.0, Tris EDTA pH 9.0 and un-buffered distilled water (GIBCO) (pH∼5.8 before AR and ∼pH 6.8 after AR). In this ‘one week fixation-experiment’ positive control coverslips staining for cytokeratins AE1/AE3 were included for each staining run. The effects of pH 6.0 and 9.0 on immunorecognition of EGFr after one week of fixation were evaluated and loss of staining similar to experiment 2 were obtained (data not shown).2 at 37°C. In the ‘4-week fixation’ experiment, the cells were fixed in 10% NBF at RT for either 5 min, 684 hrs or 12 hrs followed by transfer to 70% ethanol for 672 hrs at RT. Prior to immunostaining, the cells were placed in Tris buffer, pH 7.6, for 10 min, permeabilized by dehydrating through graded concentrations of ethanol, i.e., 70%, 95%, and absolute ethanol and treated with acetone for 15 seconds. The cells were then rehydrated through graded concentrations of ethanol, i.e., absolute, 95%, and 70%, before transferring to a Tris bath. This method of permeabilization was found to result in optimal staining for the antigens under study.The cell culture techniques, immunostaining methods, and evaluation were performed as previously described Following permeabilization, endogenous peroxidase was quenched with 3% hydrogen peroxide and non-specific staining was blocked with 3% goat serum for 1 hr at room temperature. The three primary murine monoclonal antibodies used for immunostaining are described in The primary antibodies were diluted in 1% bovine serum albumin, 1 mM EDTA in PBS, (PBE), pH 7.6. The antibodies were incubated on cells for 1 hr at room temperature. The cells were washed in Tris buffer and incubated with multispecies biotinylated goat anti-mouse/rabbit secondary antibody  for 10 min, washed again, and incubated with HRP-conjugated streptavidin (Signet) for 5 min. For each independent experimental repeat, all immunostaining was performed concomitantly, i.e., 5 min of 10% NBF fixation was performed just prior to immunostaining. Diaminobenzidine (DAB) substrate kit  was used for visualization of the antigen/antibody complex and after immunostaining cells were counterstained with Mayer's hematoxylin  for 1 minute 15 seconds. The coverslips were dehydrated through graded ethanols, cleared in xylene and mounted using Permount (Fisher Scientific).To investigate the effect of AR on 12-hr 10% NBF fixation followed by transfer to 70% ethanol, cells were grown on coverslips in two sets and exposed to either AR or no AR prior to immunostaining so that the recovery of immunorecognition could be evaluated with AR (+AR) and without AR (−AR) for each of the three conditions of fixation . As in experiment 1, all immunostaining for these conditions were performed simultaneously .At the designated end points of fixation, the −AR cells were placed in a Tris bath at room temperature, while the +AR were placed in a Tris bath for 10 min, rinsed in DI water, transferred to a metal coverslip rack, and placed in a Pyrex bowl containing AR solution  at 120°C. The Pyrex bowl was transferred into a pressure cooker and the pressure was maintained at 15 psi for 5 min. Pressure was allowed to drop gradually and cells in the AR buffer cooled before proceeding with immunostaining. Both sets of cells were then permeabilized and immunostained as described above.Blinded evaluation was performed by a board certified diagnostic pathologist (W.E.G.). Each antibody for a specific cell line at each condition and for each of the three replicate experiments was evaluated during the same session to maximize consistency. Two parameters were evaluated: the percentage of cells staining and an immunostaining score based on the sum of the intensity from 0 (no staining) to 4 (strongest staining) multiplied by the proportion of cells staining at each intensity p≤0.01. This higher level of statistical significance (compared to p≤0.05) was chosen to minimize identifying statistically significant but small changes in immunostaining that would not be experimentally important.For statistical analysis, two-tailed t-tests in the Statistics Toolbox of MATLAB (Mathworks) were performed to assess the differences in immunostaining between the means of different samples. Differences were considered statistically significant if Although there were statistically significant differences between the means of the some replicate experiments at some time points, overall the differences between replicate experiments were not experimentally significant; therefore data from all three replicate experiments were combined to determine percentage of cells staining and their immunostaining score. Figures and statistical analyses of immunostaining score are presented because the immunostaining score was found to be more sensitive to changes in immunorecognition than the percentage of cells staining In both DU145 and SKOV3 cells, immunostaining for PCNA was undetectable  after 10p<0.01) in staining after 10% NBF fixation for 684 hrs when compared to 10% NBF fixation for 5 min in both DU145 and SKOV3 cells. In both cell lines immunostaining score after 10% NBF fixation for 12 hrs followed by transfer to 70% ethanol for 672 hrs was significantly higher than 10% NBF fixation for 684 hrs; however, for DU145 cells, staining following 12 hrs of 10% NBF and transfer to 70% ethanol for 672 hrs was less than 50% of immunostaining following 5 min of fixation in 10% NBF (p<0.01)   Panel IIIn DU145 cells, evaluation of cytoplasmic staining for EGFr demonstrated no significant difference when 10% NBF fixation for 684 hrs or 10% NBF fixation for 12 hrs plus 672 hrs in 70% ethanol was compared with 10% NBF fixation for 5 min (p<0.01). In SKOV3 cells, there was no significant difference in immunostaining for membrane EGFr between 10% NBF fixation for 5 min compared with 10% NBF fixation for 684 hrs, however the immunostaining score for membrane EGFr staining of SKOV3 cells fixed in 10% NBF fixation for 12 hrs followed by transfer to 70% ethanol for 672 hrs was statistically higher than for cells fixed for 5 min in 10% NBF (p<0.01). The extent of this higher expression of membrane EGFr would likely be experimentally important.In DU145 cells, the membrane staining for EGFr was statistically different between either 10% NBF fixation for 684 hrs or 10% NBF fixation for 12 hrs followed by transfer in 70% ethanol for 672 hrs when compared to 10% NBF fixation for 5 min (p<0.01) than 5 min of fixation in 10% NBF e.g., DU145 cells .In both DU145 and SKOV3 cells, immunostaining scores of cells staining for PCNA after AR were significantly higher when compared with respective pairs not exposed to AR  except in SKOV3 cells where no statistical difference was observed in immunostaining scores for PCNA when 10% NBF fixation for 5 min −AR was compared with 10% NBF fixation for 5 min +AR  in staining when compared with 10% NBF fixation for 5 min. There were cell line differences observed in evaluation of immunostaining scores for cytokeratins after fixation in 10% NBF for 12 hrs followed by transfer to 70% ethanol for 672 hrs. In DU145 cells, comparing 10% NBF 5 min staining with 10% NBF fixation 12 hrs plus 672 hrs in 70% ethanol was not statistically significant (p = 1.0) (p<0.01). In both cell lines, exposure to AR resulted in an immunostaining scores for cytokeratins AE1/AE3 similar to those obtained with 10% NBF fixation for 5 min without AR.When immunostaining for cytokeratins AE1/AE3 without AR was evaluated, in both DU145 and SKOV3 cells, 10% NBF fixation for 684 hrs resulted in a large and statistically significant decrease (p = 1.0)  Panel IIp<0.01). In SKOV3 cells, 10% NBF fixation for 12 hrs followed by transfer to 70% ethanol for 672 hrs was significantly higher when compared with either fixation of 10% NBF for 5 min or 10% NBF fixation for 684 hrs. This was unexpected given our hypothesis that all values for immunostaining at other conditions of fixation should be less than that for 5 min of 10% NBF fixation.In experiment 2, results of immunostaining score for cytoplasmic EGFr were similar to experiment 1. For DU145 cells, like in experiment 1 there were no statistically significant differences in cytoplasmic expression of EGFr between 10% NBF fixation for 5 min and 10% NBF fixation for 12 hrs followed by transfer to 70% ethanol for 672 hrs; however, when fixation for 5 min in 10% NBF and 10% NBF fixation for 684 hrs were compared, cytoplasmic staining for EGFr was significantly but only slightly decreased, (p = 0.45) or between 10% NBF fixation for 5 min and 10% NBF fixation for 12 hrs plus 672 hrs in 70% ethanol (p = 0.06) (p<0.01), when compared with 10% NBF fixation for 5 min or 10% NBF fixation for 684 hrs. This also was unexpected.In DU145 cells, there was no statistically significant change in immunostaining score for EGFr membrane staining between 10% NBF fixation for 5 min and for 684 hrs  storage in 70% ethanol following 12 hrs of fixation in 10% NBF could similarly prevent the loss on immunorecognition and if antigen retrieval would just be as effective in recovering immunostaining.The extensive loss of immunorecognition of PCNA and cytokeratins AE1/AE3 was similar upon fixation with 10% NBF for 1 or 4 weeks; however, fixation in 10% NBF for 12 hrs followed by transfer to 70% ethanol for 4 weeks (672 hrs) preserved less than 50% of immunorecognition of PCNA in DU145 and SKOV3 cells in contrast to the almost complete preservation immunorecognition of PCNA after 168 hrs of storage in 70% ethanol. The immunorecognition of cytokeratins AE1/AE3 was more variable; however, based on experiments 1 and 2 it is likely that the immunorecognition of cytokeratins is preserved to a large extent upon storage in 70% ethanol for up to 4 weeks after initial fixation for 12 hrs in 10% NBF.As was noted previously, neither cytoplasmic nor membrane expression of EGFr is very sensitive to long term fixation in 10% NBF The extent to which heat-buffer AR unmasks immunorecognition of formalin fixed epitopes is still not known for many antigens In contrast, 10% NBF fixation for 12 hrs followed by transfer to 70% ethanol plus AR resulted in recovery of immunorecognition of PCNA and cytokeratins AE1/AE3; however, unexpectedly, AR resulted in almost complete loss of staining for both cytoplasmic and membrane EGFr regardless of whether the cells were fixed in only 10% NBF or fixed in 10% NBF for 12 hrs followed by transfer to 70% ethanol. Although manufacturer instructions suggest that AR is not required for EGFr immunorecognition (pepsin pretreatment is recommended), this observation emphasizes that heat induced AR is not suitable for some antigens. In this case, two typical methods currently used for AR, pH 6.0 citrate buffer and pH 8.0 EDTA solution actually destroyed the ability to detect EGFr.Although the term “storage in 70% ethanol” is used because it describes the approach, this terminology does not imply that such storage in 70% ethanol is a passive process with respect to molecular effects  as is indicated by our prior study This study was performed using a cell model without tissue processing to paraffin. A prior study using a similar cell model found that fixation followed by tissue processing to paraffin combined have to a greater effect on immunorecognition that just fixation or tissue processing alone This study concludes that transfer of cells to 70% ethanol for 4 weeks following 12 hrs of fixation in 10% NBF does not preserve immunorecognition of PCNA and cytokeratins AE1/AE3 as reported for shorter periods of 180 hrs in 70% ethanol