Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth. After some time though, tumors become resistant and recur with a poor prognosis. The majority of resistant tumors still expresses a functional androgen receptor (AR), frequently amplified or mutated.To test the hypothesis that AR is not only expressed, but is still a key therapeutic target in advanced carcinomas, we injected siRNA targeting AR into mice bearing exponentially growing castration-resistant tumors. Quantification of siRNA into tumors and mouse tissues demonstrated their efficient uptake. This uptake silenced AR in the prostate, testes and tumors. AR silencing in tumors strongly inhibited their growth, and importantly, also markedly repressed the VEGF production and angiogenesis.in vivo. The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools.Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development  Moreover, we establish the efficiency, safety and specificity of synthetic siRNA to treat those advanced tumors.AR is a ligand-activated transcription factor that plays a pivotal role in the prostate, regulating a large set of genes involved in cell division, apoptosis, and angiogenesis. Prostate carcinomas are initially androgen-dependent (ADCaP) and hormone ablation efficiently inhibits their growth, although some deleterious side effects result from the inhibition of AR signaling in normal tissues. The major limitation of these treatments is the capacity of tumors to escape and recur in vitro and in vivo the function of AR in prostate carcinomas. To establish the technical conditions and specificity of AR silencing, we first used the human androgen-dependent prostate tumor model LNCaP. Androgens stimulate LNCaP cells' proliferation whereas castration and the androgen antagonist bicalutamide inhibit the development of xenografted LNCaP tumors in mice We used in this study RNA interference to investigate in vivo, we implanted LNCaP cells subcutaneously into male nude mice. Tumors started to develop after 45 days. Once the measurement of tumors for 5 days demonstrated their exponential growth (3 (mean±SEM). Mice were then randomized for treatment and received daily for 2 weeks an intraperitoneal injection (i.p.) of 3 µg (125 µg.kg−1) of unmodified synthetic siRNAs diluted in saline. Half of the mice were treated with the panAR-siRNA, the other half with a control-siRNA (cont-siRNA) matching no known mRNA sequences in mouse and human databanks. The growth of tumors treated with AR-siRNA was very rapidly arrested, and after 10 days, the mean tumor volume represented only 15% of the control . Half of the animals in each group were sacrificed on the fourth day of treatment. At that time, the Prostate Specific Antigen (PSA) mRNA was strongly repressed in the hAR-siRNA and in the bicalutamide groups, while AR mRNA level was repressed only in the hAR-siRNA treated tumors , which is a key regulator of angiogenesis in the prostate, is upregulated by androgens in the normal prostate and in androgen-dependent cells, through indirect transcriptional in vivo studies using the LNCaP model demonstrated the efficiency and specificity of the antitumoral effects produced by AR silencing. We then studied the effects of the two different AR-siRNAs on the in vivo growth of castration-resistant tumors. We first grafted C4-2 cells to nude mice and, after a month, once vascularized tumors were exponentially growing, and reached a mean tumor volume of 129.9±29.1 mm3, mice were randomized to receive cont-, or panAR-, or hAR-siRNA. In contrast with castration or bicalutamide, which do not affect the development of C4-2 tumors Our 3) with AR-siRNA markedly repressed the tumor growth , triggers a strong silencing of the target, but therapeutic applications of this approach are limited by risks linked to the stable integration of the viral genome in the host, by the difficulty to infect in vivo a large number of cells and by toxic effects resulting from the saturation of the miRNA maturation machinery Specific gene silencing, and RNA interference in particular, offers new exciting therapeutic perspectives for a number of pathologies, including cancers. In this perspective, RNAi-directed silencing in vivo is still under investigation, it is of note that ongoing clinical trials of siRNAs are performed with naked molecules Several groups injected under a normal pressure small amounts of naked siRNAs into mice and reported the efficient silencing of genes such as VEGF in vivo the closely related mouse and human AR mRNAs: treatment with hAR-siRNA silenced AR in tumor cells and inhibited the tumor growth while preserving AR expression in mouse prostate and testes. Treating tumors with hAR-siRNA was as efficient as treating with the antagonist bicalutamide, which affects AR signaling in prostate and testes. The inhibition of the growth of C4-2 tumors by panAR- or hAR-siRNAs in intact males was comparable. All together, these data demonstrate that the inhibition of AR signaling outside the tumor does not participate in the antitumoral effect, opening promising perspectives for developing tumor-specific treatments with reduced side effects for patients.The role of AR in androgen-dependent prostate carcinomas has been well established over years, and recently confirmed using an inducible AR-shRNA lentiviral construct in LNCaP tumors in vitro, leading to contradictory conclusions: Gosh et al., showed that the main driver of CRCaP cells' division was p70S6 kinase, and not AR in vivo data are difficult to interpret. From all the above studies, it was therefore not possible to foretell whether AR was a key target in established resistant tumors.Prostate tumor cells can escape androgen-ablation therapies by multiple mechanisms involving the androgen receptor. The functionality of AR in these resistant cells is well established, but this does not foretell if AR silencing is sufficient to inhibit proliferation and trigger apoptosis, as other molecular events accumulating along tumor progression could help cells bypassing AR signaling. Of note, DU145 and PC3 cell lines, which no longer express AR, are representative of a proportion of advanced prostate tumors where AR is no longer required for tumor growth 3 in a 24 g-weighing mouse in We used to address this question one ADCaP and two unrelated CRCaP models and checked during at least 5 days that each tumor was exponentially growing before randomization for siRNA treatment. AR-siRNA induced in all three tumor models a strong inhibitory effect. The tumors that were smaller at the start of the treatment  were performed by a standard calcium phosphate precipitation method or, in LNCaP, 22RV1 and C4-2 cells were grown in RMPI containing 10% of fetal calf serum. Synthetic siRNA were purchased from Dharmacon . Sequences are indicated in 6 cells/mouse) were injected subcutaneously in 50% (v:v) matrigel (BD biosciences) to 6–8 weeks old male nude mice and measured as previously described Studies involving animals, including housing and care, method of euthanasia and experimental protocols were conducted in accordance with the local animal ethical committee in the Institut André Lwoff in Villejuif, France. Tumor cells . Relative gene expression was analyzed using QuantiTect SYBR Green RT-PCR kit . Human Cyclophilin A was used as an internal control. For siRNA detection, 500 ng of total RNA was annealed at 30°C for 30 min with a stem-loop primer and then reverse transcribed . The RT product was then amplified, using the same kit as above. Sequences of primers are indicated in Protein detection by immunoblotting was performed using antibodies raised against AR , Gluthation-S Transferase alpha 4 , actin  or tubulin . Vascular Endothelial Growth Factor (VEGF) was quantified using an ELISA kit specific for human VEGF . Testosterone in blood samples was quantified by RIA . Immunodetection on paraffin embedded tissues or cryostat sections was performed using antibodies raised against AR , KI67  to label proliferative cells or Collagen IV  to detect microvessels. TUNEL labeling was performed as previously described Using a t-test, results were considered statistically different when calculated p values were <0.05 (*) or <0.01 (**).Figure S1Control of the exponential growth of tumors before first treatment. Before randomization of mice for siRNA treatment, the exponential growth of tumors was verified by plotting the neperian logarithm of the tumor volume as a function of time. This analysis was performed for each individual tumor studied. The figure represents the ln(tumor volume), mean±SEM, of tumors studied in (0.53 MB TIF)Click here for additional data file.Figure S2Effect of panARsiRNA treatment on the growth of an AR-negative tumor . JT8 fibrosarcoma cells, which do not express AR, were grafted to male nude mice. Starting from day 8, mice received daily i.p. injections of 3 µg of cont-siRNA (black symbols) or panAR-siRNA (white symbols). The tumor volume  was not different between the two groups. VEGF-siRNA injected with the same protocol were shown previously to induce 70% reduction in VEGF production and a marked growth inhibition of these tumors .(0.61 MB EPS)Click here for additional data file.Figure S3Naked siRNAs do not induce in mice a typeI-Interferon response. Interferon-a was quantified by ELISA (R&D) in serum collected from mice bearing LNCaP tumors and treated daily for 15–21 days with 3 µg of either cont-siRNA, hAR-siRNA or panAR-siRNA (experiments shown in (0.61 MB EPS)Click here for additional data file.Figure S4Effects of bicalutamide and siRNA on LNCaP and C4-2 cells in vitro. A: LNcaP (grey bars) or C4-2 (black bars) cells were incubated for 3 days in medium containing 10% of charcoal-filtered serum supplemented with R1881 (0.5 nM) or bicalutamide (10-5M) as indicated. The cell number was compared to that of untreated cells. B: A 4xARE-luciferase reporter gene was co-transfected into LNCaP (grey bars) or C4-2 cells (black bars) along with cont- or hAR-siRNA. Cells were then incubated in medium containing 10% of charcoal-filtered serum supplemented with R1881 (0.5 nM) or bicalutamide (10-5M) as indicated. The luciferase activity was quantified 48 h after transfection.(0.72 MB EPS)Click here for additional data file.Figure S5Inhibition of the 22RV1 tumor growth in female mice by hAR-siRNA treatment. 6 weeks-old female swiss nu/nu mice were grafted with one million 22RV1 cells. Once tumors grew exponentially, mice were randomized (6 mice per group) and received daily i.p. injections of 3 µg of cont-siRNA or hAR-siRNA diluted in PBS.(0.50 MB EPS)Click here for additional data file.Table S1(0.03 MB DOC)Click here for additional data file.Table S2(0.03 MB DOC)Click here for additional data file.


Pathogenicity islands (PAIs), distinct genomic segments of pathogens encoding virulence factors, represent a subgroup of genomic islands (GIs) that have been acquired by horizontal gene transfer event. Up to now, computational approaches for identifying PAIs have been focused on the detection of genomic regions which only differ from the rest of the genome in their base composition and codon usage. These approaches often lead to the identification of genomic islands, rather than PAIs.We present a computational method for detecting potential PAIs in complete prokaryotic genomes by combining sequence similarities and abnormalities in genomic composition. We first collected 207 GenBank accessions containing either part or all of the reported PAI loci. In sequenced genomes, strips of PAI-homologs were defined based on the proximity of the homologs of genes in the same PAI accession. An algorithm reminiscent of sequence-assembly procedure was then devised to merge overlapping or adjacent genomic strips into a large genomic region. Among the defined genomic regions, PAI-like regions were identified by the presence of homolog(s) of virulence genes. Also, GIs were postulated by calculating G+C content anomalies and codon usage bias. Of 148 prokaryotic genomes examined, 23 pathogenic and 6 non-pathogenic bacteria contained 77 candidate PAIs that partly or entirely overlap GIs.Supporting the validity of our method, included in the list of candidate PAIs were thirty four PAIs previously identified from genome sequencing papers. Furthermore, in some instances, our method was able to detect entire PAIs for those only partial sequences are available. Our method was proven to be an efficient method for demarcating the potential PAIs in our study. Also, the function(s) and origin(s) of a candidate PAI can be inferred by investigating the PAI queries comprising it. Identification and analysis of potential PAIs in prokaryotic genomes will broaden our knowledge on the structure and properties of PAIs and the evolution of bacterial pathogenesis. PAIs are distinct genetic elements of pathogens encoding various virulence factors such as protein secretion systems, host invasion factors, iron uptake systems, and toxins ,2. PAIs in silico methods have been developed to detect HGT 1 and [G+C]3) of every ORF. The compositional bias at the first and third positions were reported to be positively correlated to expressivity and genomic G+C content, respectively [T deviates over 1.5 σ or if deviations of [G+C]1 and [G+C]3 are of the same sign and at least one of them is over 1.5 σ. Mahalanobis distance (dM) was used to evaluate deviation of the codon usage of a gene and mean of the genome [M is a statistic in unit of standard deviation from the mean of 61 codon frequencies and can be calculated as follows:To detect GIs in a chromosome, we first identified horizontally transferred genes (H) based on the algorithm developed by Garcia-Vallve et al. . To alleectively ,27. Extre genome . dM is adM = (X - Xmean)T S-1(X - Xmean)mean correspond to vectors having relative frequencies of the 61 codons for a gene and the mean values for a genome, respectively. S-1 is the inverse of variance-covariance matrix (S) of all the 61 codon frequencies. The higher this value is the more deviation in codon usage [Ms can be converted to p-values using the χ2 distribution function. We considered a gene as extraneous in codon usage if its p-value was less than 0.05. It should be noted that genes longer than 300 bp were used for calculating the mean and standard deviation (σ) of G+C contents and dMs. This is from the observation that genes having shorter than 300 bp have much higher chance of anomalies in G+C content and codon usage.Where X and Xon usage . If Xs aE. coli O157 Sakai, cag PAI of Helicobacter pylori 26695, VPI-2 of Vibrio cholerae, and a PAI of Enterococcus faecalis, were averaged 0.35. Neighbouring regions were merged into larger regions which were referred to as GIs in this study. Some genomic regions had highly biased G+C content compared to the whole G+C content of the chromosome, while their codon usage were not biased. For example, 46.4 kb genomic region ranging from 2,647,129 bp in Yersinia pestis KIM, which contains yersiniabactin genomic island [We ran a genome scan of a 10-gene window and identified regions containing four or more H. This threshold frequency of 0.4 was inferred from the observation that the frequencies of H in known PAIs such as LEE of c island  has consThe detection scheme for the regions of cPAIs is outlined in Figure SHY designed the study, developed the software for implementing the devised algorithm, and wrote the manuscript. CH and HK contributed to the writing the software, and YHK collected and reviewed the data, and TKO assessed the biological significance of the results. JFK supervised the project and contributed to the development of methodology and writing the manuscript. All authors read and approved the final manuscript.The complete list of organisms whose genomes were searched for candidate PAIs in this studyClick here for fileComplete list of the PAI loci used as the query for BLASTP searchesClick here for fileDetailed information of candidate PAIs in prokaryotic chromosomesClick here for fileDetailed information of PAI-like regions not overlapping GIs in prokaryotic chromosomesClick here for file


V), and dimethylarsinic acid (DMAV)—arsenite was the most potent. Similar effects were also observed in aortic rings without endothelium, suggesting that vascular smooth muscle plays a key role in enhancing vasoconstriction induced by arsenite. This hypercontraction by arsenite was well correlated with the extent of myosin light chain (MLC) phosphorylation stimulated by phenylephrine. Direct Ca2+ measurement using fura-2 dye in aortic strips revealed that arsenite enhanced vasoconstriction induced by high K+ without concomitant increase in intracellular Ca2+ elevation, suggesting that, rather than direct Ca2+ elevation, Ca2+ sensitization may be a major contributor to the enhanced vasoconstriction by arsenite. Consistent with these in vitro results, 2-hr pretreatment of 1.0 mg/kg intravenous arsenite augmented phenylephrine-induced blood pressure increase in conscious rats. All these results suggest that arsenite increases agonist-induced vasoconstriction mediated by MLC phosphorylation in smooth muscles and that calcium sensitization is one of the key mechanisms for the hypercontraction induced by arsenite in blood vessels.Chronic exposure to arsenic is well known as the cause of cardiovascular diseases such as hypertension. To investigate the effect of arsenic on blood vessels, we examined whether arsenic affected the contraction of aortic rings in an isolated organ bath system. Treatment with arsenite, a trivalent inorganic species, increased vasoconstriction induced by phenylephrine or serotonin in a concentration-dependent manner. Among the arsenic species tested—arsenite, pentavalent inorganic species (arsenate), monomethylarsonic acid (MMA Arsenic is a ubiquitous element distributed in the environment, and millions of people are chronically exposed to arsenic worldwide . Natural2+ concentration , allowing Ca2+–CaM complex formation, which binds to and activates myosin light chain kinase (MLCK)  . The act relaxes .2+-dependent MLCK, it has been recently suggested that smooth muscle contraction is modulated by Ca2+ sensitization , phenylephrine, and serotonin creatinine sulfate. We obtained monomethylarsonic acid (MMAV) from Chem Service  and anti-MLC and anti-phospho-MLC antibody from Santa Cruz Biotechnology . Fura-2/AM was supplied by Molecular Probe , and all other reagents used were of the highest purity available.The following chemicals were purchased from Sigma : sodium arsenite , sodium arsenate , dimethylarsinic acid  weighing 300–400 g throughout all experiments. Before the experiments, animals were acclimated for 1 week in the laboratory animal facility maintained at constant temperature and humidity with a 12-hr light/dark cycle. Food and water were provided 2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25.0 mM NaHCO3, and 11.1 mM glucose, pH 7.4). The organ bath was continuously gassed with 95% O2/5% CO2 and maintained at 37°C. The rings were stretched gradually to an optimal resting tension of 2 g and equilibrated for 30 min. The change in tension was measured isometrically with Grass FT03 force transducers  and recorded using the AcqKnowledge III computer program .Rats were sacrificed by decapitation and then exsanguinated. We carefully isolated the thoracic aorta and cut it into ring segments. Aortic rings without endothelium were prepared by gently rubbing the intimal surface of the aortic rings with a wooden stick. We then mounted the rings in four-channel organ baths filled with Krebs-Ringer (KR) solution  in minimum essential media (MEM) with 100 U/mL penicillin and 100 μg/mL streptomycin in a 95% air/5% CO−8 M phenylephrine was added to the organ bath for 2 min, and then ice-cold acetone with 10% trichloroacetic acid and 10 mM dithiothreitol was immediately added to stop the reaction. The aortic rings were washed with the acetone solution three times, lyophilized overnight, and stored at −70°C until protein extraction. After the dried tissues were cut into small pieces, we extracted proteins in a 50 μL sample buffer containing 8 M urea, 2% sodium dodecyl sulfate, 5% β-mercaptoethanol, 0.01% bromophenol blue, and 62.5 mM Tris-HCl by vortexing for 3 hr at room temperature. Protein extracts were electrophoresed in a 15% polyacrylamide mini-slab gel  and then transferred onto nitrocellulose membranes in Tris/glycine buffer . We detected the MLC level and the extent of phosphorylation with immunoblotting using anti-MLC antibody and anti-phospho-MLC antibody (Santa Cruz Biotechnology), respectively. Immunoreactive bands were visualized by horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) and an enhanced chemiluminescence kit . For densitometric analysis of MLC-P/MLC, we determined densities of the corresponding bands using TINA software .We measured the extent of MLC phosphorylation using polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis using an anti-phospho-MLC antibody, as previously described . After t2+ levels, we cut the thoracic aorta without endothelium into spiral strips approximately 8 mm in length and 1 mm in width under a dissecting microscope. After the aortic strips were treated with arsenite for 14 hr, they were exposed to 10 μM acetoxymethyl ester of fura-2 (fura-2/AM) and 0.1% cremophor EL in a Krebs-Henseleit solution  for 4 hr at room temperature.To measure intracellular Ca2+ level based on the method described by 2/5% CO2. Passive tension of 2 g was applied and allowed to equilibrate before measurement. We elicited muscle contractions by changing the media with the KH solution containing 12.5, 25, and 90 mM KCl. Muscle strips were illuminated alternatively at 48 Hz in excitation wavelengths of 340 and 380 nm. We measured the intensity of 500 nm fluorescence emitted by 340 nm excitation (F340) and that emitted by 380 nm (F380) successively. We calculated the ratio of F340 to F380 [R(F340/F380)] as an indicator of intracellular Ca2+.We measured the intracellular free CaRats were anesthetized with phenobarbital . We placed a catheter of polyethylene PE-50 tubing  filled with heparinized saline (100 U/mL) in the carotid artery to measure blood pressure, and we placed a catheter of polyethylene PE-10 fused to PE-50 tubing in the jugular vein to administer drugs. Catheters were tunneled subcutaneously and exteriorized at the back of the neck. Wounds were sutured and cleaned with alcohol. Experiments were performed after a 1-day recovery period. On the day of the experiment, the arterial catheter was connected to a pressure transducer (BIOPAC Systems Inc.) and blood pressure was measured using the AcqKnowledge III computer program. We allowed blood pressure to stabilize for a minimum of 30 min before beginning treatment. To determine the effects of arsenite on blood pressure increase induced by phenylephrine, we administered arsenite solution (1.0 mg/kg) by an intravenous bolus injection into the jugular vein. In the controls, equivalent amounts of saline were injected. After 2 hr, we infused the rats with 2.5 μg/kg/min phenylephrine for 3 min via the jugular vein and monitored the change of blood pressure in response to phenylephrine simultaneously. Infusions were performed with a Harvard syringe pump  at a rate of 0.1 mL/min.p-value of < 0.05 to determine significance.We calculated the means and standard errors for all treatment groups. The data were subjected to one- or two-way analysis of variance (ANOVA) followed by Duncan’s multiple range test to determine which means were significantly different from the control. Statistical analysis was performed using SPSS software . In all cases, we used a V and DMAV, on phenylephrine-induced constriction  for 14 hr and then added phenylephrine and serotonin cumulatively to obtain concentration-contraction curves. Arsenite alone did not cause any changes in basal vascular tone (data not shown). Arsenite treatment, however, enhanced the contraction induced by both phenylephrine and serotonin in a concentration-dependent manner . We invetriction . ArsenatTo investigate whether the enhanced contraction induced by arsenite was an endothelium-dependent effect, we performed experiments using aortic rings without endothelium. Treatment with arsenite to aortic rings without endothelium still resulted in a concentration-dependent increment of agonist-induced contraction in blood vessels , suggest2+ elevation in vascular smooth muscles, we investigated the effects of arsenic on intracellular Ca2+ levels when vasoconstriction was initiated by high K+ concentration. We used a high concentration of K+ to induce contraction in blood vessels, since K+ directly induces rapid influx of extracellular Ca2+ through voltage-gated Ca2+ channels in plasma membrane without involving other signal transduction pathway and thus could serve as a simple alternative tool to investigate the current premise. As shown in + in aortic rings without endothelium, which is consistent with the results shown in 2+ in the presence of high K+. Arsenite, however, did not induce intracellular Ca2+ elevation, but rather decreased Ca2+ concentration significantly in the presence of 90 mM K+ (2+ levels, suggesting that arsenite might increase the contractility of blood vessels via Ca2+ sensitization.To examine whether the increased MLC phosphorylation was mediated by intracellular Ca90 mM K+ . These r2+ levels simultaneously using fura-2–loaded aortic strips. After the muscle strips were treated with 25 μM arsenite, contraction was induced successively by 12.5, 25, and 90 mM K+. 2+ elevation (2+ elevation versus contraction relationship (2+ sensitivity of contractile machinery in smooth muscle.To investigate this assumption, we measured smooth muscle contraction and the change of intracellular Calevation . Plottintionship . These rin vivo, we monitored changes in blood pressure after intravenous infusion of phenylephrine into conscious rats (p = 0.029; in vivo and this confirms the previous in vitro results (.To verify the effects of arsenite on blood vessels ous rats . An intr results .+ in a concentration-dependent manner and that the Ca2+ sensitization in smooth muscle largely contributes to arsenite-induced hypercontractility. These in vitro results were consistent with in vivo results in which arsenite potentiated the hypertensive effect of phenylephrine. Recently, the effect of arsenic on platelets has been suggested as a key mechanism in the development of cardiovascular diseases (In the present study we demonstrate the ability of arsenic to enhance contraction of isolated aortic rings from rats. We have shown that arsenite enhances the vascular contraction induced by phenylephrine, serotonin, and high Kdiseases . Blood vdiseases . Our datSuppression of nitric oxide production in endothelium results in the loss of vasodilator activity, causing vasoconstriction , and we V and DMAV showed no significant effect. Arsenate is generally known to exert its toxic effects by replacing phosphate in various biochemical reactions because it has a similar structure and properties to phosphate (As shown in hosphate . Howeverhosphate . Therefo1-adrenoceptor and the 5-HT2 serotonin receptor, respectively, on aortic smooth muscle cells and thus result in increasing intracellular Ca2+ (+ bypasses receptor-signaling pathways and leads directly to intracellular Ca2+ elevation by opening Ca2+ channels following membrane depolarization (2+ increases by phenylephrine, serotonin, and high K+ result in phosphorylation of MLC leading to vascular smooth muscle contraction. It is widely accepted that the degree of MLC phosphorylation is the essential factor that determines the extent to which smooth muscle contracts (Phenylephrine and serotonin act on the αlar Ca2+ . In contrization . These iontracts . As showontracts , consist2+ elevation, the level of responsiveness of contractile machinery to intracellular Ca2+ plays a key role in regulating the contractility of smooth muscle cells (2+ increase in aortic strips revealed that arsenite potentiated the magnitude of contraction without concomitant increase in Ca2+ elevation (2+ sensitization to the hypercontraction induced by arsenite. Previous studies suggested that the mechanism for Ca2+ sensitization was mainly due to modulation of MLC phosphatase activity (2+ concentration, which resulted in hypercontraction. However, it is currently uncertain whether arsenite could inhibit MLC phosphatase directly.In addition to intracellular Cale cells . Simultalevation , suggestactivity . That isin vitro and demonstrated that Ca2+ sensitization in smooth muscle is responsible for MLC phosphorylation-dependent hypercontraction arsenite (in vivo study, arsenite treatment potentiated the hypertensive effect of phenylephrine in rats. These results confirm our in vitro observations and suggest that increased vasocontraction may be a contributing factor in the development of cardiovascular diseases in populations exposed to arsenic.In this study, we showed that arsenite causes enhanced vasoconstriction arsenite . In our


Successful instruction of a complicated subject as Physical Rehabilitation demands organization. To understand principles and processes of such a field demands a hierarchy of steps to achieve the intended outcome.This paper is intended to be an introduction to a proposed pyramid scheme of general physical rehabilitation principles. The purpose of the pyramid scheme is to allow for a greater understanding for the student and patient. As the respected Food Guide Pyramid accomplishes, the student will further appreciate and apply supported physical rehabilitation principles and the patient will understand that there is a progressive method to their functional healing process. Musculo-skeletal dysfunction requiring physical rehabilitation can be quite diverse in cause, severity, chronicity, complicating factors, location of injury, and the anatomy involved. Because of the multifactorial involvement of musculo-skeletal dysfunction, it can be a challenge knowing when and where to start the physical rehabilitation process – not to mention teaching this process to students in the fields of chiropractic, medicine, physical/physio and occupational therapies.After a thorough physical assessment of an acute or chronic musculo-skeletal dysfunction (or injury), the clinician may determine a need for progressing the patient into a physical rehabilitation program. The first issue is to avoid inducing any more harm to the patient than what has already occurred. This element of safety involves the implementation of correct diagnosis, timely rehabilitation intervention, correct rehabilitation program design, and correct progression within the program .The pyramid introduced in this paper will assist the clinician by adding further safety and guidance to physical rehabilitation implementation. This paper will introduce a "general" pyramid for guidance that can be adapted to most of the regions of the body. Future publications from this author will include modified pyramid guides for physical assessment and rehabilitation application for specific regions of the human body.As a musculo-skeletal clinician and lecturer in the areas of sports injury care and physical rehabilitation, the author finds this pyramid to be beneficial in instructing patients on their process of rehabilitation and is extremely helpful when teaching physical rehabilitation principles to students.st edition publications, this pyramid will evolve as others in the field provide input. The author looks forward to this evolution process and participating in further discussions on this topic.Like all 1Pyramids are used by professions across the health care scheme. Some of the first health care professions to use the pyramid as a guide were the nutritionists or dieticians. The Food Guide Pyramid expresses to the lay-person and practitioner the emphasis on specific foods (such as grains) and the need to limit other food types (such as simple sugars). These food guide pyramids are now replicated across the world to meet the diverse cultures; Mediterranean, Asian, Latin, Puerto Rican, Vegetarian, Soul Food , JapanesThe Fitness Professionals also boast of a well-designed pyramid expressing their concern for limiting sedentary lifestyles and focusing on plenty of lifestyle physical activities .A complicated Sports Rehabilitation Pyramid was published quite some time ago, Fig. This sports rehabilitation pyramid/structure Figure  is more O'Connor, et al. described another, five step management pyramid in the field of sports medicine and rehabilitation that included 1.) control of inflammation, 2.) promote healing, 3.) increase fitness, 4.) control abuse, and 5.) return to activity.Although, not in a pyramid format, Hyde and Gengenbach nicely noted four phases of rehabilitation and appropriately encouraged progressing the rehabilitation process from phase I to phase IV . This auThe various pyramids and concepts differ for certain reasons that exceed the interest of this article, but the important point is that the pyramid framework of educating the lay-person and practitioner is easy to understand and appears to be internationally accepted.nd and 3rd steps of O'Connor's article. It is expected that the treating physician and/or therapist will have determined that a patient is beyond the acute phases of inflammation control and that, based on objective data, they are a good candidate for non-surgical care, including physical rehabilitation.The proposed pyramid that this paper will formally introduce defines tiers of specific physical rehabilitation progression, consistent with the categories of the 2Similar to other pyramid schemes, the bottom tier should be considered the first and most important and implemented before moving to the next tier, figure . Each paThroughout the physical rehabilitation process, it should be understood that the patient must be educated appropriately by the clinician. It is this author's experience that an educated patient is an inspired and compliant patient. If the patient understands that there are desired goals to meet before progressing to the next tier, the patient may be more focused on their in-clinic and home assignments in order to reach their goals and progress. Additionally, the clinician needs to monitor how the patient naturally moves and performs their exercises; looking for muscle substitution , asymmetry in movement (such as demonstrating greater hip extension with one hip v. the other during gait), and/or sub-optimal regional function (such as diminished core stability during a squatting manoeuvre). If these aberrant movements are occurring, the patient should not progress to the next tier and the patient should be educated about these findings so they may be able to apply the conscious changes during the exercises and also during activities of daily living.This tier has the greatest importance. One might have appropriate motor patterns or inappropriate motor patterns. Motor patterns require a great degree of muscle coordination that may either be under conscious or unconscious control. When a clinician wishes to train a motor pattern, it will require numerous conscious attempts before this motor pattern becomes an unconscious pattern, or engram  If the p®. The Bodyblade® is a reactive, oscillating device that utilizes inertia to generate up to 270 muscle contractions per minute [It is well appreciated that early intervention of cardiovascular conditioning enhances tissue healing via tissue oxygenation and nutrition; decreases potential for muscle atrophy and physical stress on the newly formed collagen fibres ; and hasr minute . The patr minute . Also ror minute . When rer minute , and theThere are numerous philosophies for classifying muscles of a joint: phasic v tonic, and stabilizers v. mobilizers are two examples. Stabilizers are defined as smaller muscles that perform joint stability functions such as joint surface centration . These mAs discussed previously, the mobilizer muscles may tend to be larger, further from the joint, and may play a role in larger and more powerful joint function . They teth tier and assure that the ADL movements are performed with evidence of good engrams, strength, endurance, etc. For athletes, this is an appropriate time to implement special motor skills such as speed and agility [During this tier of the Physical Rehabilitation Pyramid the ultimate goal is to prepare the patient for a safe return to their Activities of Daily Living (ADL). This of course could include laying bricks, child-care, house-work, or playing footy. During this tier's rehabilitation, the clinician will focus on progressing from the 4 agility . For the agility  that thiThroughout this process, continually re-assessing, using outcome measurements, is important in order to confirm that the patient is benefiting from care. Lastly, before discharge from this physical rehabilitation program, there is a need for an exit physical examination to confirm tissue integrity, psychological readiness, and appropriate education for decreasing the risk of re-injury .The scope of this paper is to consider the active physical rehabilitation process and the safe and effective progression of these processes. However, acute injuries may require passive care initially for pain management, inflammation control, etc. Such implementation would include PRICE  . AdditioPhysical rehabilitation is a complicated process of musculo-skeletal healing and recovery which should be patient-sensitive and condition-specific (i.e. not every shoulder condition (e.g. rotator cuff injury) can go straight into three sets of ten repetitions of tubing exercises for internal and external rotation). Like a toddler learning to walk for the first time: if they are not strong enough to weight bear or have the necessary static proprioception, they will not be able to stand. If they do not have the motor control and dynamic proprioception to shift weight from one leg to the other, they will not walk. Simply because a toddler can stand does not mean she can safely walk. Each level of physical progression requires added neuromuscular and cognitive ability and conditioning.Just as the Food Guide Pyramid has progressed over the years, it had to start from something, and this proposed pyramid, just in its infancy, will also progress. If accepted by the rehabilitation community, such progress will include addition of pictures demonstrating actions at each tier (similar to the Food Guide Pyramids), rigorous clinical testing to demonstrate it's effectiveness, and specific pyramids for different regions of the body. This author encourages feedback and discussion on rationale for changes and improvements in this process for enhanced safety and efficacy of patient management and clinician education.The author(s) declare that they have no competing interests.


Pristionchus pacificus has originally been developed as a satellite organism for comparison to Caenorhabditis elegans. A 10X coverage of the whole genome of P. pacificus is available, making P. pacificus the first non-Caenorhabditis nematode with a fully sequenced genome. The macroevolutionary comparison between P. pacificus and C. elegans has been complemented by microevolutionary studies of closely related strains and species within the genus Pristionchus. In addition, new understanding of the biology of Pristionchus from field studies, demonstrating a close association with various scarab beetles and the Colorado potato beetle, supports consideration of this nematode in studies of ecosystems. In the course of field studies on four continents more than 1,200 isolates were established from 15,000 beetle specimens representing 18 Pristionchus species. Two remarkable features of the Pristionchus – beetle association are the high species specificity of the interaction and the interception of the beetle's sex communication system for host recognition by the nematodes, as suggested by chemotaxis studies. Evolutionary interpretations of differences in developmental, behavioral and ecological patterns require a phylogenetic framework of the genus Pristionchus.The nematode Pristionchus species based on a set of 27 ribosomal protein genes encompassing a total of 10,971 bp. The phylogenetic tree provides evidence for North American and European clades, which are embedded in a deeper clade that includes Asian species. It also indicates putative invasion events. Of the 18 Pristionchus species, 13 are gonochoristic and five are hermaphroditic. The phylogeny indicates that all hermaphroditic species have arisen independently within the genus Pristionchus.Here, we provide a robust phylogeny of all 18 available Pristionchus for nematode biodiversity assessments. While some species are represented by more than 100 isolates, others were found less than four times. Such patterns were observed on all continents and in all phylogenetic clades indicating that species asymmetry is a widespread phenomenon, which can now be further investigated by molecular tools.Combined ribosomal protein cDNA data can provide the basis for reconstruction of a robust phylogenetic framework for microevolutionary and biogeographic analyses. An additional major implication of our studies is the use of  Pristionchus pacificus is a laboratory model organism to study developmental biology, evolutionary biology, and behavior in comparative context to other nematodes such as Caenorhabditis elegans  and dung beetle (Geotrupes ssp.), respectively ,3,19P. pPristionchus nematodes we could demonstrate that the SSU serves as a fast and reliable barcoding marker for species identification with sufficient resolution within the genus. The importance of a fast identification procedure as shown above, which distinguishes morphologically very similar species, is emphasized by the observation of the frequencies of the different Pristionchus nematodes in the isolates. Whereas in the total sample size of more than 15,000 beetles and soil samples some species were easily detectable since they occurred several hundred times or were present in at least moderate frequencies, seven species were found by serendipity only in single or very few isolates (Table Pristionchus sp. 3 is represented by a single strain isolated from a soil sample from Cold Spring Harbor (New York), Pristionchus sp. 11 was found twice in soil samples from Japan, Pristionchus sp. 14 was isolated once and P. sp. 15 twice from Oriental beetles in Japan. Two isolates of P. sp. 10 were obtained from soil samples from Nepal, two isolates of P. sp. 4 were found on beetles in western Europe, and P. americanus was detected three times on North American beetles .RNA was reverse transcribed into cDNA with the help of the Omniscript reverse transcriptase kit  and the primer RH5620 (s. construction of EST libraries). Complete transcripts were synthesized by RT-PCR in two overlapping fragments per gene. The SL1-specific primer BJ234 (5'-GGTTTAATTACCCAAGTTTGAG-3') was used together with the gene-specific antisense primers to obtain the 5' parts of the transcripts and the combination of RH5620 and the sense primers to obtain the 3' parts. The PCR was performed with the help of the HotStar C. elegans and C. briggsae orthologues. Genes that were either duplicated in Caenorhabditis or appeared to be present in more than one copy in Pristionchus were omitted from the analysis. Reliable coding sequences  were concatenated and subjected to analysis by Modeltest 3.7 to determine the best-fit nucleotide substitution model for maximum likelihood analysis (ML) by likelihood ratio tests (LRT) using the Akaike information criterion [Pristionchus sequences were used for parameter determination. The selected settings corresponded to the GTR+G+I model with 6 substitution types , assumed nucleotide frequencies of A = 0.25540, C = 0.31530, G = 0.25700, T = 0.17230, assumed proportion of invariable sites I = 0.5307, rates at variable sites following the γ distribution with shape parameter α = 0.7072.Sequences were aligned manually using the Seqpup 0.6 f software for Macintosh . The aliriterion ,46. OnlyThe phylogenetic reconstruction was performed by the heuristic search algorithm of the PAUP*4.0b10 software using the parameters of the selected substitution model and the likelihood optimality criterion . The braAIC Akaike information criterionCDS Coding sequenceCPB Colorado potato beetleEST Expressed sequence tagML Maximum likelihoodMP Maximum parsimonyNJ Neighbor-joiningSSU small subunit ribosomal RNA geneWEM generated isogenic female lines, designed and carried out all of the molecular experiments, performed all of the phylogenetic analysis and wrote the manuscript. MH carried out all of the fieldwork, generated isogenic female lines and did the mating experiments. RJS designed and coordinated the experiments and wrote the manuscript. All authors read and approved the final manuscript.Oligonucleotides used in this study. Oligonucleotides used for cDNA synthesis, RT-PCR and sequencing are shown. a, sense; as, antisense; cDNA, cDNA synthesis; seq, sequencing.Click here for file


For organisms living or interacting in groups, the decision-making processes of an individual may be based upon aspects of both its own state and the states of other organisms around it. Much research has sought to determine how group decisions are made, and whether some individuals are more likely to influence these decisions than others. State-dependent modelling techniques are a powerful tool for exploring group decision-making processes, but analyses conducted so far have lacked methods for identifying how dependent an individual's actions are on the rest of the group.Here, we introduce and evaluate two easy-to-calculate statistics that quantify how dependent an individual's actions are upon the state of a co-player in a two-player state-dependent dynamic game. We discuss the merits of these statistics, and situations in which they would be useful.Our statistical measures provide a means of quantifying how independent an individual's actions are. They also allow researchers to quantify the output of state-dependent dynamic games, and quantitatively assess the predictions of these models. The development of state-dependent modelling techniques applicable to evolutionary biology has allowed us to address many questions about the behaviour and life history strategies of organisms. Behavioural or life history decisions induce changes in the state of the organism  ,2. TheseIn evolutionary and behavioural ecology, one area that has been the focus of a great deal of attention recently is how groups of animals come to make combined decisions  ,24. Statet al. [When we look at group decision-making strategies, it is important for us to know how decisions are being made, and who is making them. It is possible to explore this using dynamic game models. For example, Rands et al.  developeet al. , we can et al.  for furtet al. , it was The potential complexity of the results generated by state-dependent dynamic games needn't be a barrier to using them as tools for investigating group decision-making and social behaviour, provided we are able to develop techniques for quantifying and comparing pertinent aspects of the models, such as the degree of autonomy different individuals have in determining their behaviour. In this paper, we develop two statistics for quantifying the degree of independence that individuals show in a two-player game. Using these statistics give us a new tool for investigating decision-making processes using state-dependent dynamic games.pxy = 0.25 for a pair of players in states x, y, the focal player should rest 25% of the time, and forage 75% of the time . Note tx, and that of its co-player as y. The solution to the game defines a behavioural response  for each player based on the current state value of both itself and its co-player, and so for a focal player in state x with co-player in state y, its action is pxy as defined by the game's solution. We refer to the full set of behavioural responses for all possible combinations of state pairs as the 'policy' of an individual. For simplicity, we assume that there is no dependence upon time in this example, meaning that the players are following the same policy at each decision period, and their actions are only dependent upon the current state of their co-player . Note also that the statistic we derive is independent of the measures of fitness used to derive the policy .x, y, the proportion of the current population in that state pair is dxy, where ∑x,ydxy = 1.We also assume that the expected state distribution of a stable population has been determined, for instance by forward-running a population of player pairs following the calculated policy using a Markov-chain process , or by cx, but does not know the state of its co-player y. Although the focal knows its own state, its policy may define a large range of possible behaviours in response to the possible states of its co-player. Although exact information about the co-player's state doesn't exist, it is possible to calculate the probability that the co-player is in each state y because we have already calculated the expected state-distribution of the population. Therefore, we can calculate gx, a 'best guess' as to the behaviour to conduct based upon weighting the policy-determined behaviours for all possible co-player states by the probabilities that the co-player is in those states . Therefore, this best guess represents the behaviour that maximises the proportion of time that the focal individual in any state x responds in the appropriate manner (as defined by its policy) to a co-player in state y, despite the focal player not being able to assess y.Our first statistic considers a situation where the focal individual knows its own state x, y, the best guess is calculated asBased on the likelihood of the pair being in states gx = ∑y(pxydxy)/∑ydxy.     (1)C quantifies the 'incorrectness' of this best guess. For a state pair x, y, the 'incorrectness' of a best guess is given byThe statistic gx - pxy|.     (2)|x, the size of the worst mistake it can therefore make is the maximum value of gx and 1 - gx. Therefore, recalculating eqn. 2 asFor a focal individual in state x, y relative to the worst mistake possible. For a given value of x, we can sum this over all values of y (adjusted accordingly for the population distribution) to calculate the severity of the mistake made when the focal individual is in state x. In this way, we can calculate an overall incorrectness statistic C, considering all possible state pairs and their distributions, asgives us the size of the mistake made at C defines the degree of independence an individual has in its actions. If C = 0, then even when the co-player's state is unknown, the focal player's 'best guess' as to the action specified by its policy is always correct. This implies that its actions are completely independent of the co-player's state. By contrast, if C = 1, the focal individual's best guess is no better than if it were to make the worst mistake possible, implying that its actions are maximally dependent upon the state of the co-player.3 instead of eqn. 2 becomes apparent with a few examples. Consider a state distribution in a population of pairs in which the focal players are all in the same state, whilst 50% of the co-players are in state 1, and the other 50% are in state 2. If we represent the state-dependent policy set of the focal individual as py = {b1, b2} where bi is the likelihood of performing a set behaviour if the co-player is in state i, then it is straightforward to show that if py = {0,0} or py = {1, 1}, then C = 0, meaning that the actions of the focal player are not dependent upon the state of its co-player. If we consider an adjusted version of the incorrectness statistic, C' = ∑x,y(|gx - pxy|)dxy which uses eqn. 2 instead of eqn. 3, C' is also equal to 0.The necessity of using eqn. 2 is more apparent if we consider py = {0, 1}. This is the policy where the focal can make the greatest potential mistakes if it is unable to assess its co-player's state, where it performs the principal behaviour 50% of the time, and consequently performs the behaviour that is appropriate to its co-player's state 50% of the time. For this policy, we find that C = 1 and C' = 0.5. Now compare this to a policy py = {0, 0.5}. If the focal player is again unable to assess its co-player's state, it should perform the principal behaviour 25% of the time, and will therefore be performing the behaviour that is appropriate to its co-player's state 66.7% of the time. The statistics for this policy are C = 0.667, and C' = 0.75. We see here that C' is therefore not a useful statistic, as it increases despite an decrease in the inaccuracy of the choice made. On the other hand, C accurately reports the decrease in incorrect choice-making.The inherent problem with using eqn. C (which is arbitrarily based upon the size of the maximum mistake possible), we can instead compare the uncertainty of an observer about an individual's likely behaviour, when the observer has and when it does not have knowledge of the co-player's state . The difference between these two values, i.e. the reduction in uncertainty due to knowledge of the co-player's state, provides another measure of the extent to which the focal player's behaviour is influenced by the state of its co-player. This statistic relies upon quantifying the ability of an observer to predict the behaviour of a focal individual in a given state in the cases where the observer does or does not know the exact state of the co-player.The previous case defined a statistic that quantifies how dependent the actions of an individual are upon the state of its co-player by calculating the degree to which its ability to correctly respond to its co-player is reduced when it lack information about the co-player's state. Instead of using x with a co-player in state y, we can calculate an observer's Shannon-Weaver uncertainty regarding the individual's choice of action asThese measures are relatively straightforward to quantify. We base our definitions of uncertainty on the measure of uncertainty (or entropy) defined by Shannon and Weaver  (see 3030 for dipxy is 0 or 1 (and therefore the observer can predict exactly the behaviour that will be performed), and a maximum value of 1 when pxy is 0.5 . Using eqn. 5, the expected uncertainty of the observer when it has perfect knowledge of the co-player's state can be calculated asNote that this measure gives a minimum value of 0 when the xyU(pxy)dxy.     (6)x, the probability with which it performs the principal action  is gx, and so the uncertainty regarding its behaviour isWe can calculate in a similar way the observer's uncertainty about the behaviour of the focal player, when it (the observer) does not know the state of the co-player. If the focal player is in state The expected uncertainty for an observer with no information about the co-player's state is thereforeHaving calculated the mean uncertainties when the observer does and doesn't know the co-player's state, we can use the absolute difference between these values to determine how uncertainty changes with knowledge of the co-player's state, giving the statisticS = 0, that implies that a player's actions are completely independent of the state of its co-player. If S is greater than 0, this means that knowing the co-player's state allows us an observer predict the behaviour of the focal individual with a greater degree of success, which implies that the actions of the focal player are to some extent dependent upon the state of the co-player. S can reach a maximum value of 1, which would imply that the focal player's behaviour is completely dependent upon the state of its co-player .If there is no change in uncertainty, C and S give broadly similar summaries of the degree of independence shown by individuals. Table a-c are completely independent of state, and give C and S values of 0 regardless of the population distribution. Policies d and e are dependent upon the state of the co-player but not the state of the focal individual, and give differing values of C and S based upon the population distribution. Note that for a population in which pairs of individuals are distributed evenly across all state-combinations (distribution I), policy d gives a C value of 1, whilst policy e gives a C value of 0.5 – this corresponds to the fact that the best-guess of the focal individual is a 50% chance of performing the principal behaviour with policy d, and a 25% chance with policy e, which is analogous to the numerical example given above. The S values in e are greater than 0.5 however, highlighting how the two statistics can differ.Both f and g are dependent upon the state of the focal player, but not the co-player, and so the focal individual's behaviour should be independent of its co-player: correspondingly, the C and S values are all 0. Policy h gives a typical case in which the focal player's behaviour may be dependent upon the state of both itself and its co-player, and we can see that the C and S values of this policy are intermediate between those of policies e and g. Policies i and j demonstrate that these statistics can also be used to consider policies with continuously distributed likelihoods of performing behaviours – these particular cases are dependent upon the state of the co-player but not the focal player itself, and should be compared to policies d and e. Finally, policies k and l demonstrate the sorts of C and S values expected for a randomly generated discrete and continuous policies.Policies C, we used a simple example model where the focal player was always in a single state, and its co-player could be in one of two possible states. In Figure C and S calculated for this simple example change in response to changes in the focal player's policy . This figure shows that C may be more sensitive than S to detecting small improvements in predicting how dependent an individual's actions are upon its co-player's state . This suggests that C may be a more useful statistic to use when it is likely that there is little dependence of players' actions on those of their co-player. S, on the other hand, gives a much smoother convex shape, which may be more useful at detecting small changes in dependence of action between policies where dependence is going to be high.In describing C and S change in response to differing probabilities of the co-player being in each of its possible states in the simple example described. This figure demonstrates that if we change the state-distribution within the population, the values of C and S will change, and the values of these statistics will be low if the state distribution is highly skewed within the population , meaning that we should take the distribution of states into consideration if we are comparing the statistics gained for a range of different policies with separately calculated stable population distributions. Therefore, it may be sensible to combine the S or C statistic with some other measure of state distribution within the population.The panels of Figure C, this measure involved quantifying the change in the focal player's ability to respond correctly to its co-player, whilst the second statistic S quantified the change in the amount of uncertainty an observer faces in predicting the response of the focal individual to its co-player. As can be seen from these examples, C and S give broadly similar results , and so we leave it to the reader to decide which of these statistics they prefer to implement. We do however suggest that S may be more meaningful biologically:C is defined relative to the size of maximum mistake that an individual can make when it is in any given state, and is thus scaled to give a numerical value ranging between 0 and 1. S also gives a numerical value between 0 and 1, but is based upon concepts from information theory that capture how individuals use (or fail to use) the information available to them within the structure of the state-dependent policy. As described above, C may be more sensitive to differences in policy in situations where there is likely to be little dependence of a player's actions upon those of its co-player, whilst S may be more sensitive when it is very likely that there is dependence.In this paper, we have presented two statistical measures that both give a means of measuring how dependent the actions of a pair of players are on each other within a state-dependent dynamic game, by comparing some measurement of an individual's ability to respond appropriately to its co-player when information does or does not exist about the exact state of that co-player. In the first statistic et al. [C and S in Figure S gives a better illustration of the immediate shift from relative independence  to greater dependence . Therefore, we suggest that S is a more sensitive statistic in this case, where players are likely to show a high degree of dependence of action upon the exact level each others' state.This is demonstrated in the output given in Figure et al. . In the The examples discussed above (such as the foraging game described in ) focus ovice versa. We could, for example, use this to explore how differences in the size and energetic requirements of predators and prey affect their strategies: how do predators feeding on similarly-sized prey items differ from those that eat prey that are usually much smaller or larger than themselves? As an extreme case, we could consider the relationship between a parasite and its host as an extremely asymmetrical game , we couies, see ,32): parWe can see from the examples given that these techniques give useful and relatively straightforward methods of assessing the independence of an individual's action from the output generated by a state-dependent dynamic game. The statistics we describe here give us a means of quantifying the effects of changing the parameter values of dynamic game models, and consequently a means of exploring the effects of both the environment and the life history traits of the players (dependent upon the assumptions made in formulating the model). These summary statistics should be useful for assessing consensus decision making, leadership decisions, antagonistic relationships, and other situations in which there is a potential conflict of interest between individuals that base their decisions upon some aspect of the states of their group's members.As well as giving us a means of quantifying the effects of changing parameters upon these group processes, they should allow us a means of comparing how policies and their associated stable distributions of individuals change in response to sensitivity analysis, which should contribute to our use of these models to identify behavioural rules that can then be explored both experimentally and theoretically ,33,34.The statistics used in this paper are developed and described in the 'results and discussion' section above. For illustrative purposes, these statistics are used to explore the policy and distribution sets outlined in figure a) never perform the principal behaviour; b) always perform the principal behaviour; c) perform the principal behaviour 50% of the time, regardless of state; d) behaviour is dependent upon the co-player's state – 'only perform the principal behaviour if y ≤ 10'; e) behaviour is dependent upon the co-player's state – 'only perform the principal behaviour if y ≤ 5'; f) behaviour is dependent upon personal state – 'only perform the principal behaviour if x ≤ 10'; g) behaviour is dependent upon personal state – 'only perform the principal behaviour if x ≤ 5'; h) behaviour is dependent upon the state of both individuals – 'only perform the principal behaviour if x ≤ 5 or y ≤ 5'; i) the probability of performing the principal behaviour is dependent upon personal state, at 1 - ((x - 1)/19); j) the probability of performing the principal behaviour is dependent upon personal state, at 1 - ((x - 1)/19)2; k) for any given state pair, the likelihood of performing the principal behaviour is either 0 or 1 (chosen randomly with a uniform distribution), with the additional constraint that gx = 0.5 for all x; l) for any given state pair, the likelihood of performing the principal behaviour is between 0 and 1 (chosen randomly with a continuous uniform distribution), with the additional constraint that gx = 0.5 for all x.In the policy sets, a white square represents 'perform principal behaviour with a probability of 1.0' and a black square represents 'perform principal behaviour with a probability of 0.0' . Grey squares represent the corresponding continuum between performing the principal behaviour at 0–100% of the time. Policies illustrated represent: dxy = 1/400; II) population where focal individuals are more likely to have lower state values, where dxy = (20 - x)/4200; III) population where co-players are more likely to have higher state values, where dxy = y/4200; IV) population where focal individuals are more likely to have lower state values, and co-players are more likely to have higher state values, where dxy = (20 - x + y)/8000; V) state distribution similar in form to those found by Rands et al. [For the population distributions, shading represents the distribution of pairs within the population, with darker squares representing higher densities at a pairing. Distributions illustrated represent: I) uniform distribution of pairs across all state combinations, where s et al. ; VI) ranThe statistics were devised by SAR and RAJ. SAR wrote the manuscript. Both authors read and approved the final draft.


AR gene with diabetic macroangiopathy has not been investigated. Therefore, the present study was conducted to determine whether genetic variations of AR may determine susceptibility to diabetic macroangiopathy.The aldose reductase (AR) gene, a rate-limiting enzyme of the polyol pathway, has been investigated as a candidate gene in determining susceptibility to diabetic microangiopathy. However, the association of the There were 378 Type 2 diabetic patients enrolled in this study. A single nucleotide polymorphism in the promoter region (C-106T) was genotyped and the AR protein content of erythrocytes measured by ELISA.P = 0.019 and P = 0.012). The erythrocyte AR protein content was increased in patients with the CT and TT genotype compared with those with the CC genotype. After adjustment for age, duration of diabetes, body mass index, systolic blood pressure, HbA1c, and serum creatinine, triglycerides, and total cholesterol in multivariate logistic-regression models, the association between this AR genotype and stroke remained significant.There were no significant differences in genotypic or allelic distribution in patients with or without ischaemic heart diseases, but there was a significant increase in the frequency of the CT + TT genotype and T allele in patients with stroke (AR gene might be a genetic marker of susceptibility to stroke in Type 2 diabetic patients. This observation might contribute to the development of strategies for the prevention of stroke in Type 2 diabetic patients.Our results suggest that the CT or TT genotype of the  AR gene may be one of the factors that determine genetic susceptibility to diabetic microvascular complications [There is familial clustering in the incidence of diabetes-related chronic complications , which sications ,4. Recenications –12.in vivo[in vitro[AR gene could also be a predisposing factor to diabetic macroangiopathy. However, the association of the AR gene with macroangiopathy in diabetic subjects has never been reported.Other reports have indicated a role of polyol pathway hyperactivity in the development of diabetic macroangiopathy in vivo,14 and i[in vitro,15,16. TAR gene determines susceptibility to diabetic macroangiopathy, such as ischaemic heart disease and cerebrovascular disease, in Japanese Type 2 diabetic patients and non-diabetic subjects.To clarify these issues, the present study investigated whether the C-106T polymorphism of the We screened 417 consecutive patients attending Nagoya University Hospital. Patients with Type 1 diabetes (14), malignant diseases (13) and other types of diabetes  were excluded. During screening, two patients declined to participate. A total of 378 Type 2 diabetic outpatients  were enrolled. A total of 334 non-diabetic subjects  who underwent a medical check-up in our hospital from April 2001 to December 2003 served as the control group. They had fasting blood glucose levels < 6.1 mmol/l and had no family history of diabetes. Five had an abnormal electrocardiogram and 20 a history of atherosclerotic diseases.Assessment and definition of diabetic macroangiopathy was based on the following criteria. Cardiovascular disease was defined by a history of ischaemic cardiovascular diseases . Stroke (ischaemic cerebrovascular disease) was diagnosed by means of neurological signs and symptoms, together with computed tomography or magnetic resonance imaging. According to the Acute Stroke Treatment (TOAST) classification , only laAR gene was determined by the polymerase chain reaction restriction fragment length polymorphism method using the primers and conditions described by Kao et al. [Bfa-I at 37°C. Before enzymatic digestion with Bfa-I, the product was purified with QIAquick PCR Purification Kit (Qiagen). The AR protein content in erythrocytes was measured by ELISA using anti-human AR monoclonal antibody .DNA samples were prepared from whole blood using a QIAamp DNA Blood Mini Kit . The C-106T polymorphism of the o et al. . The presd, unless otherwise indicated. Statistical significance of the differences between the groups was determined by chi-square (χ2) tests, unpaired Student's t-tests or anova where appropriate. The Hardy–Weinberg test was performed using a standard observed-expected χ2 test. Multiple logistic-regression analyses were used to assess the association of the CT + TT genotype with diabetic complications. Odds ratios (ORs) and their confidence intervals (CIs) were used to estimate relative risk. A two-sided P-value less than 0.05 was considered statistically significant.Statistical analyses were performed using SPSS for Windows version 12.0 . Data are presented as means ± The clinical characteristics of diabetic patients and non-diabetic subjects are presented in AR gene were 259 (68.5%) for CC, 105 (27.8%) for CT, and 14 (3.7%) for TT in the diabetic group, and 213 (63.8%) for CC, 113 (33.8%) for CT, and 8 (2.4%) for TT in the non-diabetic group. The frequencies of the C allele were 82.6% in the diabetic group and 80.7% in the non-diabetic group. The distribution of genotypes was in Hardy–Weinberg equilibrium. There were no significant differences between the two groups .The genotype distributions of the C-106T polymorphism of 1c and fasting blood glucose levels, SBP, DBP, serum levels of total cholesterol, triglycerides, HDL cholesterol, low-density lipoprotein (LDL) cholesterol and creatinine.Because the number of diabetic patients with the TT genotype was only 3.7%, subjects with the CT and TT genotypes were combined for further analyses. The genotypes were not associated with gender, duration of diabetes, age, diabetes treatment, BMI, HbAP = 0.002 and 0.001 vs. CC; We then compared the AR protein content in erythrocytes among the genotypes. The AR content was significantly increased in a genotype-dependent manner , SBP (P < 0.001), serum creatinine levels (P = 0.008) and serum levels of triglycerides (P = 0.048) were higher in patients with a history of stroke.Age (P = 0.019). Furthermore, in model 2, after additional adjustment for HbA1c, BMI, serum total-cholesterol levels and duration of diabetes, this relationship remained .AR gene as a candidate gene for susceptibility to diabetic macroangiopathy, and found that the CT or TT genotype is associated with increased risk of stroke in Type 2 diabetic patients.We have investigated the C-106T polymorphism in the promoter region of the Diabetes mellitus is one of the major risk factors of coronary heart disease and cerebrovascular disease. The high prevalence of these macrovascular diseases in diabetic patients can be explained by hyperglycaemia per se, as well as by the increased frequency of conventional risk factors such as hypertension, dyslipidaemia, and obesity.Accelerated proliferation of vascular smooth muscle cells (SMCs) is one of the characteristic features of atherosclerosis , and it et al. [AR gene in patients with nephropathy. Furthermore, the CT or TT genotypes and the T allele of this polymorphism have been identified as genetic markers of susceptibility to diabetic microangiopathy in several reports [AR gene. However, a functional reporter gene assay reported by Yang et al. [Hodgkinson et al.  reported reports –12. Therg et al.  showed tAR promoter region are thought to be a risk factor for stroke. Some independent effects of genetic variants  on the risk of cerebral ischaemia have been reported previously [AR gene exerts a stronger effect on the risk of stroke.We showed that the CT and TT genotypes and the T allele of the C-106T polymorphism in the eviously . Compareet al. reported, for the first time, the relationship between the C-106T polymorphism of the AR gene and erythrocyte AR content in Type 2 diabetic subjects [AR gene influence erythrocyte AR content and the development of diabetic complications. Unfortunately, however, a significant relationship between the AR content in erythrocytes and the prevalence of stroke was not observed in our study.Makiishi subjects . The resAR gene was limited to a single marker from the 5′ region of the gene. Thus, it cannot be excluded that this AR polymorphism is in linkage disequilibrium with an unidentified polymorphism strongly related to stroke risk. This could explain the lack of correlation between AR content in erythrocytes and the prevalence of stroke. Hyperglycaemia, hyperosmolality, and oxidative stress all regulate erthrocyte AR content [Our findings should be cautiously interpreted because there are some limitations to the present study. Our analysis of the  content –28, and AR gene were associated with an increased risk of stroke, and the significance of these genotypes remained high even after adjustment for other risk factors. In addition, the erythrocyte AR content was higher in the subjects with these genotypes. Therefore, it may be speculated that polymorphisms of the AR gene, concomitant with an increase in erythrocyte AR content, would increase vulnerability of brain arteries resulting in stroke. However, prospective intervention studies with aldose reductase inhibitors would be required to confirm the influence of the AR gene polymorphism and AR content on the development of stroke.There are several well-known conventional risk factors for macrovascular diseases  but little is known about inherited factors that could predispose to diabetic macroangiopathy. Among the conventional risk factors, in the present study, age and systolic blood pressure were higher in the subjects with stroke than those without stroke, although the significance was weak after adjustment for various risk factors. In contrast, the CT + TT genotype and the T allele of the AR gene with ischaemic heart disease. It can be speculated that brain vessels are more sensitive to polyol pathway-induced hyperosmolar stress than coronary vessels. Precise investigation will be required to clarify this issue.In contrast, it is noteworthy that the influence of this polymorphism on the development of ischaemic heart disease is less than that on stroke. There are important differences in risk factors for stroke and ischaemic heart disease. For stroke, hypertension is the most adverse risk factor, whereas for ischaemic heart diseases LDL cholesterol is the strongest. This suggests that there are differences in the pathogenesis of ischaemic heart disease and stroke, which might explain our lack of association of the AR polymorphisms in other populations. Although various studies have reported the relationship between diabetic microangiopathy and AR polymorphisms [It is important to replicate our results in other populations. No previous study has investigated the relationship between diabetic macroangiopathy and orphisms –12, consAR gene is associated with an increased risk of stroke in Type 2 diabetic patients. This observation might contribute to the development of further strategies for the prevention of stroke in Type 2 diabetic patients.In conclusion, we have found that the C-106T polymorphism in the promoter region of the


The rollator is a very popular walking aid. However, knowledge about how a rollator affects the walking patterns is limited. Thus, the purpose of the study was to investigate the biomechanical effects of walking with and without a rollator on the walking pattern in healthy subjects.The walking pattern during walking with and without rollator was analyzed using a three-dimensional inverse dynamics method. Sagittal joint dynamics and kinematics of the ankle, knee and hip were calculated. In addition, hip joint dynamics and kinematics in the frontal plane were calculated. Seven healthy women participated in the study.The hip was more flexed while the knee and ankle joints were less flexed/dorsiflexed during rollator walking. The ROM of the ankle and knee joints was reduced during rollator-walking. Rollator-walking caused a reduction in the knee extensor moment by 50% when compared to normal walking. The ankle plantarflexor and hip abductor moments were smaller when walking with a rollator. In contrast, the angular impulse of the hip extensors was significantly increased during rollator-walking.Walking with a rollator unloaded the ankle and especially the knee extensors, increased the hip flexion and thus the contribution of hip extensors to produce movement. Thus, rollator walking did not result in an overall unloading of the muscles and joints of the lower extremities. However, the long-term effect of rollator walking is unknown and further investigation in this field is needed. The rollator is a popular assistive walking device in most European and especially the Nordic countries . The exaThe main purpose of using a rollator is to improve the walking performance and minimize the risk of falling. Studies have shown that the walking performance in elderly subjects measured in terms of distance, cadence and velocity is improved when they walk with a rollator . FurtherHowever, knowledge about how the rollator affects the walking pattern is limited. To our knowledge no studies have investigated the biomechanical differences between walking with and without a rollator except from one study that observed a reduction in the vertical ground reaction force during rollator walking . Such inIt is unclear whether an unloading of certain muscle groups and joints during walking would impair the functional ability during other types of daily physical activities and movements like sit-to-stand, short walking distances, stair climbing, balance control during standing/squatting etc. One study concluded that the use of walking-aids combined with a high activity level may protect against falls in elderly subjects . Thus, iAccordingly, the purpose of the present study was to investigate the biomechanical effects of walking with a rollator on the walking pattern of healthy subjects. The reason for studying a group of healthy subjects was that it was both unethical and difficult to ask actual rollator users to walk without their rollator.Seven healthy women (age: 34.7 (range: 25–57) years, height: 1.70 (range: 1.64–1.78) m, weight: 64.7 (range: 55–75) kg) participated in the study. None of the subjects had any history of injuries or musculo-skeletal dysfunctions in their lower extremities. All subjects gave their informed consent to participate in the experiments which were approved by the local ethics committee.The subjects were fitted with fifteen small reflecting spherical markers (12-mm diameter) according to the marker set-up described by Vaughan et al. . The marThe subjects were allowed to practice walking both with and without the rollator to become familiar with the movements and the pre-determined walking speed. The speed was controlled by photocells, which made it possible to teach the subjects to approach 4.5 km/h.Five video cameras (Panasonic WV-GL350) operating at 50 Hz were used to record the movements. The video signals and the force plate signals were synchronized electronically with a custom-built device. The device put a visual marker on one video field from all cameras and at the same time triggered the analogue-to-digital converter which sampled the force plate signals at 1000 Hz. The subjects triggered the data sampling and synchronization when they passed the first photocell.The video sequences were digitized and stored on a PC. Sixteen non-coplanar points on a standard calibration frame (Peak Performance 5) were digitized to calibrate each of the video sequences. The calibration frame was placed in the middle of the walkway and covered both force plates. Three-dimensional co-ordinates were then reconstructed by direct linear transformation using the Ariel Performance Analysis System (APAS).Prior to the calculations, the position data were digitally low-pass filtered by a fourth order Butterworth filter with a cut-off frequency of 6 Hz, and the 1000 Hz force plate signals were downsampled to 50 Hz to fit the video signals.Internal flexor and extensor joint moments about the ankle, knee and hip were calculated using a three-dimensional inverse dynamics approach described by Vaughan et al. . FurtherThe peak values as well as the angular impulses of the ankle, knee and hip moment were calculated and used as input parameters for the statistical analyses.The angular position of the ankle, knee and hip joints was calculated to describe the movements in the sagittal plane. In addition, the angular position of the hip movement in the frontal plane was calculated. Zero degrees defined the anatomical position (foot at 90° to leg) and positive values reflected ankle dorsiflexion, knee hyperextension, hip flexion and hip abduction.The average angle as well as the range of motion (ROM) , i.e. thMATLAB was used for all calculations.Data obtained from the left leg during the stance phase were analyzed. Six gait cycles were normalized and averaged for each subject and situation  and without  a rollator, respectively). Normalization was performed by interpolating data points to form 500 samples for each gait cycle. The joint moments were normalized to body mass. Ensemble averages were then calculated for rollator (n = 7) and normal walking (n = 7) using the mean value for each individual subject.A Student's t-test for paired data was used to identify statistically significant differences between rollator- and normal walking in selected kinematic and kinetic variables of the walking patterns. All results are presented as means (SD). The level of significance was set at 5%.There was no difference in the walking speed between rollator- (4.49 (0.05) km/h) and normal walking (4.51 (0.03) km/h) (p = 0.731).The joint angular kinematics were significantly different between rollator- and normal walking Fig. , Table 1The joint moments were significantly different at each joint between the two situations Fig. , Table 2The peak hip abductor moment in the first half of the stance phase was significantly smaller during rollator-walking than during normal walking Fig. , Table 2The present study demonstrated significant differences between normal and rollator-walking patterns. The study included seven healthy subjects between the ages of 25 and 57 years who were able to walk with and without a rollator at identical walking speeds (4.5 km/h), which is important when comparing joint moment curves -15.The main findings of the present study showed that walking with a rollator resulted in a remarkable reduction in the knee extensor moment and thus an unloading of the quadriceps muscle during the stance phase. There were also small but significant reductions of the ankle plantarflexor and hip abductor moments. In contrast, the angular impulse of the hip extensors and the duration of the hip extensor moment were increased during rollator-walking. The hip joint was generally more flexed throughout the whole stance phase during walking with the rollator, while the ankle and knee joint were less dorsiflexed/flexed. In addition, the ankle and knee ROM in the sagittal plane along with the hip ROM in the frontal plane were decreased during rollator-walking.These results confirm that although the weight of the trunk was supported by the rollator, this did not result in an overall reduction of the joint moments around all three joints in the lower extremities. The unloading of the ankle and knee joints during rollator-walking seemed to be partly compensated by an increase in the hip extensor moment, which probably was needed to push the rollator in a forward direction and keep up its horizontal velocity.The increased hip flexion throughout the whole stance phase was due to the increased forward flexion of the trunk during rollator-walking. The increased hip flexion could possibly explain the increased hip extensor moment during rollator-walking. This concurs with other studies that have observed increased hip flexion along with an increase in the hip extensor moment during walking .The sagittal ankle, knee and frontal hip joint ROM's were reduced and the knee and ankle joints were less flexed during rollator-walking. During normal walking the time period between heel strike and peak knee flexion in the first half of the stance phase reflects the weight acceptance and energy absorption controlled by the knee extensors . During The rollator is a common and popular walking-aid among elderly and disabled subjects ,2,17. RoThe rollator is definitely a very effective walking-aid that supports the body, improves the walking performance in terms of distance, cadence and velocity  and in mThe rollator-walking pattern in healthy subjects was characterized by increased hip flexion, decreased ankle dorsiflexion and knee flexion, and reduced the ankle and especially the knee joint moments significantly, while the contribution from the hip extensors to produce movement was increased. However, the functional consequences of these changes and the long-term effects of rollator-walking are unclear and further investigation in this field is needed.TA and EBS designed the study. TA, PKL, GP, LHN performed the experiments. TA was responsible for the data analysis, calculations and drafted the manuscript. PKL, GP, LHN, participated in the data treatment. EBS contributed to the discussion and the interpretation of the results. TA, PKL, GP, LHN and EBS shared the discussion. All authors read and approved the final manuscript.


The differential diagnosis of metastatic mammary adenocarcinoma and adenocarcinomas from other primary sites can be challenging, particularly in tumors that are poorly differentiated and negative for Estrogen/Progesterone receptors (ER/PR). With progression of disease, Androgen receptors (AR) are preserved with higher frequency than ER/PR in metastatic mammary carcinoma. This study was undertaken to evaluate the diagnostic significance of AR expression in adenocarcinoma of breast and other morphologically similar adenocarcinomas.Formalin-fixed paraffin-embedded tissue sections of 113 primary adenocarcinoma of various sites  were immunostained with monoclonal antibody for AR. Except for well differentiated lobular carcinoma of breast (5) and bronchoalveolar carcinoma of lung (10), majority of the tumors were moderately to poorly differentiated. Tumors immunoreactive for ≥ 10% of nuclei were considered AR positive. However, AR immunoreactivity in the cytoplasm only was also recorded.56% (19/34) mammary carcinoma and 20% (2/10) adenocarcinoma of ovary were positive for AR. Remaining 69 adenocarcinomas did not show nuclear immunoreactivity for AR in ≥ 10% nuclei; however, 52% (36/69) showed variable cytoplasmic immunoreactivity.Significant proportion of mammary carcinomas and some ovarian carcinomas express AR in the nuclei of more than 10% tumor cells. If metastatic tumor with unknown primary in a female is AR positive, breast and ovary are the most likely primary sites. Cytoplasmic immunoreactivity alone without nuclear immunoreactivity for AR was non-specific for this differential diagnosis. Over the years with vast advances in the chemotherapeutic regimes specific for certain primary sites of tumors, the correct identification of the origin of tumor is of utmost importance for the determination of appropriate therapy and prognosis. In women, mammary carcinoma is a leading cause of death in several regions of the world . It is aThe primary site of poorly differentiated metastatic adenocarcinoma may not always be discernible by morphology alone. Immunohistochemistry has proved to be a useful adjunct for this purpose. Commonly applied panels of immunomarkers for breast carcinoma including ER, PR, GCDFP-15 (Gross Cystic Disease Fluid Protein-15), and lactoferrin while very useful -6 may bePrevious studies have shown that AR is retained more often than ER/PR in metastatic mammary carcinoma ,10, beinThe tumors that are included in our study are primary adenocarcinomas from breast, lung, stomach, pancreas, liver (cholangiocarcinoma), colon, ovary, endometrium, and cervix.A total of 113 cases were obtained after a computerized search from Anatomic Pathology files at Froedtert Hospital/Medical College of Wisconsin from 1996 to 2001. The neoplasms studied included the following: 34 mammary carcinomas , 10 ovarian carcinomas, 7 uterine endometrial carcinomas, 6 uterine cervix adenocarcinomas, 11 liver and bile duct cholangiocarcinomas , 7 pancreatic adenocarcinomas , 6 gastric carcinomas , 9 colon carcinomas , and 23 adenocarcinomas of the lung  including 10 of the broncho-alveolar type. Majority of the tumors except for lobular carcinoma of the breast and broncho-alveolar carcinoma of the lung were of a moderately to poorly differentiated grade. All of these tumors were primary tumors of their respective organs. Out of the 56 adenocarcinomas , 19 tumors belonged to males and 37 to females.2O2 solution following which antigen retrieval is performed with DAKO pH 10.0 citrate buffer at 95°C for 20 minutes. After allowing it to cool down, non-specific binding was quashed with DAKO protein block. The tissue was then immunostained for 45 minutes with monoclonal antibodyAR441  corresponding to amino acids 229–315 of the human androgen receptor at a dilution of 1:100. The signal was visualized with DAKO LSAB+. Then they were placed in DAKO DAB+ for 7 minutes. Lastly hematoxylin counter stain was performed using Harris Hematoxylin. Tumors were considered positive for AR if 10% or greater than 10% of nuclei were immunoreactive. Cytoplasmic reactivity alone with AR was interpreted as negative, but was recorded for analyzing the results. Formalin fixed paraffin-embedded tissue sections of prostate served as a positive control.These specimens were fixed in 10% formalin and embedded in paraffin blocks. Sections of 3.5 micron in thickness were cut and mounted on DAKO silanized slides to accommodate alkaline epitope retrieval. They were then dried and depariffinized. The endogenous peroxidase activity was blocked using 50/50 methanol/HOf the 34 breast carcinoma cases, 29 were of infiltrating ductal type (IDC) and 5 of infiltrating lobular type (ILC). All of the IDC were assigned Bloom and Richardson grade 3. Nuclear immunoreactivity was observed in 56% (19/34) of the total breast cases. Out of the 29 cases of IDC, 14 were positive for AR. All 5 ILC were positive for AR. The staining pattern for AR was patchy in many of the tumors with proportion of tumor nuclei exhibiting immunopositivity for AR varying from 40–100%. The intensity also varied from weak to strong. In the ILC group, 4 cases displayed positive nuclear immunostaining with strong intensity, and in 1 case 75% of the nuclei stained positive with moderate to strong intensity. Simultaneous or isolated cytoplasmic staining with AR, ranging from weak to strong was noted in 62% (21/34) of these breast carcinomas.The group of 10 ovarian tumors included 5 papillary serous adenocarcinomas, 3 endometriod carcinomas, 1 mixed papillary mucinous and serous adenocarcinoma, and 1 mixed endometriod – papillary serous adenocarcinoma. Immunoreactivity with AR was noted in 2 of these tumors, a papillary serous adenocarcinoma and the other mixed endometriod – papillary serous adenocarcinoma. Moderate to strong staining was noted in 80% and 95% of the tumor nuclei in these 2 tumors respectively. Nine out of the ten ovarian tumors displayed variable cytoplasmic immunoreactivity with AR.Out of the 10 cases selected initially as colon adenocarcinoma, one tumor displayed AR immunoreactivity in scattered malignant glands but was negative in the poorly differentiated regions of the tumor. This tumor belonged to a male patient. Due to morphology unusual for colon carcinoma, further investigation with Prostatic specific antigen (PSA) immunomarker was performed to determine if this tumor had possible prostate origin. PSA immunoreactivity was observed in areas showing few well-differentiated malignant glands consistent with prostatic adenocarcinoma. The tumor was predominantly poorly differentiated and these poorly differentiated cells did not show AR immunoreactivity. Patient had a past history of prostatic adenocarcinoma. The rest of the colon adenocarcinoma cases showed non-reactivity of the nuclei with AR although variable cytoplasmic staining was noted in 7 of the 9 cases.Amongst the 7 cases initially selected as gastric carcinoma, six did not display nuclear immunoreactivity for AR; however, one case belonging to a female patient showed moderate to strong positive nuclear staining with AR in 60% of the nuclei. Further immunohistochemical studies were performed which demonstrated immunoreactivity of the tumor cells for ER (20–30% nuclei with moderate intensity), PR (90% nuclei with strong intensity) and Cytokeratin 7. These tumor cells were negative for Cytokeratin 20, consistent with metastatic mammary carcinoma. Patient had a past history of mammary carcinoma. Cytoplasmic staining was noted in 3 out of the 6 gastric carcinoma cases.Remaining 54 adenocarcinomas, including pancreatic adenocarcinomas (7), liver and bile duct cholangiocarcinomas (11), uterine endometrial carcinomas (7), uterine cervix adenocarcinomas (6), and pulmonary adenocarcinomas including broncho-alveolar type (23), did not show nuclear immunonegativity for AR. However 48% (26/54) of these tumors demonstrated variable cytoplasmic reactivity. Out of all 69 non-mammary/non-ovarian adenocarcinomas, 52% (36/69) showed variable cytoplasmic immunoreactivity.Androgen receptors are nuclear proteins that are functionally critical to several organs and tissues . They arAR expression has been noted in some benign and malignant tumors. As noted earlier, majority of breast carcinomas evaluated express AR as determined by IHC and biochemical studies ,11-14. IIn our study there was a clear dominance of AR expression in mammary carcinoma compared to other adenocarcinomas. AR expression in 56% (19/34) of the mammary carcinomas is in concordance with previous studies. Whilst 48% (14/29) of the IDC's were immunoreactive for AR, ILC cases demonstrated consistent AR positivity at 100%. The higher frequency could be attributed to lower grade of the ILC's as compared to the poorly differentiated IDC's chosen in our study. It is noteworthy that 20% of ovarian carcinomas were positive with immunoreactivity for AR in more than 10% nuclei.As specifically evaluated in this study, the nuclear immunostaining pattern is important during interpretation. Only nuclear immunostaining pattern was observed in mammary carcinoma and in a few cases of ovarian carcinoma. All other adenocarcinomas are negative for nuclear immunoreactivity. However, cytoplasmic immunoreactivity was not uncommon in many of these non-mammary/non-ovarian carcinomas.Two cases  showed positive immunoreactivity for AR in nuclei in the tumors initially selected in the non-mammary adenocarcinoma group. After further evaluation with additional immunomarkers and clinical correlation, these tumors were consistent with metastatic adenocarcinomas. The primary sites respectively were breast in a female and prostate in a male.Addition of AR in the immunopanel for immunohistochemical evaluation of unknown primary in women is strongly recommended. Silanized slides are recommended to avoid floating and washing away of the tissue sections during alkaline epitope retrieval step. Depending on the clinical correlation, including the status of ovaries and breast, AR positivity favors primary from mammary carcinoma and sometimes ovarian carcinoma.In summary, AR immunoreactivity in 10% or more nuclei is consistent with AR positive tumor, which is strongly suggestive of breast and in some cases ovarian primary. This study suggests that AR is a potentially useful immunomarker in evaluating metastatic adenocarcinoma of unknown primary in females. It is important to assess the true nuclear immunoreactivity in tumor cells. Cytoplasmic immunostaining alone is non-specific for differential diagnosis of these primary sites.The author(s) declare that they have no competing interests.JM designed, carried out entire study and prepared manuscript. VS conceived, designed and provided expertise in assisting in data collection and manuscript preparation as senior author and mentor. RK participated in the design and coordination of the study. All authors read and approved the final manuscript.