The purpose of this study was to evaluate whether compliance and rehabilitative efforts were predictors of early clinical outcome of total hip resurfacing arthroplasty.A cross-sectional survey was utilized to collect information from 147 resurfacing patients, who were operated on by a single surgeon, regarding their level of commitment to rehabilitation following surgery. Patients were followed for a mean of 52 months . Clinical outcomes and functional capabilities were assessed utilizing the Harris hip objective rating system, the SF-12 Health Survey, and an eleven-point satisfaction score. A linear regression analysis was used to determine whether there was any correlation between the rehabilitation commitment scores and any of the outcome measures, and a multivariate regression model was used to control for potentially confounding factors.Overall, an increased level of commitment to rehabilitation was positively correlated with each of the following outcome measures: SF-12 Mental Component Score, SF-12 Physical Component Score, Harris Hip score, and satisfaction scores. These correlations remained statistically significant in the multivariate regression model.Patients who were more committed to their therapy after hip resurfacing returned to higher levels of functionality and were more satisfied following their surgery. By 2030, the demand for primary total hip arthroplasties is estimated to grow by 174% to 572,000 . Th. Th16]. In addition to the rehabilitation questionnaire, standard clinical outcome measures were collected at a mean follow-up of 52 months . Clinical assessments were made prior to surgery and at final follow-up utilizing the Harris hip objective rating system . FunctioAll data was collected using a Microsoft Access Database . Data was exported to SPSS version 13.0 software  for statistical analyses. All statistical comparisons were conducted using 95% confidence intervals where a p-value of less than 0.05 was considered significant. For each of the primary questions the following statistics were assessed: 1) Linear regression analysis and Pearson's coefficient were used to determine whether there was any correlation between the rehabilitation commitment scores and any of the outcome measures. A multivariate regression model was used to assess the influence of other factors including age, body mass index, medical comorbidities, diagnoses, and gender; 2) Multivariate regression analysis was used to assess the correlation of various factors with the level of commitment; 3) A Mann-Whitney Rank Sum test was used to compare the outcome scores between various patient populations. These results are shown in Table Overall, the level of commitment to rehabilitation was shown to predict each of the outcome measures assessed: SF-12 Mental Component Score , SF-12 Physical Component Score , Harris Hip score (r = 0.23), and satisfaction score. These correlations remained statistically significant in the multivariate regression model when controlling for age, body mass index, medical comorbidities, diagnosis, and gender. The overall mean rehabilitation commitment score was 8 points . The mean Harris Hip score improved from 56 points  prior to surgery to 92 points  at final follow-up. At final follow-up, the mean SF-12 mental component score (MCS), physical component score (PCS), and patient satisfaction were 56, 52, and 9 points, respectively.A comparison of the various clinical outcome measures between patients stratified by various demographic variables  revealed a significantly lower mean pre-operative Harris hip score in women compared to men, a significantly lower mean satisfaction level in men compared to women, and a significantly lower mean Harris hip score at final follow-up in non-obese patients  that make this type of analysis difficult. In addition, following their initial in-patient rehabilitation program, not all patients received physical therapy at the same institution. However, all patients reported similar conventional rehabilitation protocols to make this less of a factor subject to bias.Efforts have been made to develop standards regarding patient rehabilitation after conventional total hip arthroplasty. Youm et al  distribuIn addition to establishing standards for the participation in functional activities and rehabilitation protocols, the use of a comprehensive, multidisciplinary, inpatient rehabilitation regimen has been shown by Dohnke et al  to be imWhile the present study suggests that current rehabilitation protocols for hip resurfacing patients yield satisfactory results, it remains unclear whether these programs are optimal. The protocols were originally designed for total hip arthroplasty patients who often are older and less active than many resurfacing patients. In the present study, there were three patients who discontinued rehabilitation after reaching all functional goals by 6 weeks post-operatively. Based on their excellent results and accelerated progress, they were cleared by their physical therapist (AB) and surgeon (MAM) from any additional prescribed rehabilitation. These results were similar to those reported by Crow et al. who found that a multimodal treatment approach allowed a 43 year-old man to return to sports activity following bilateral resurfacing . Their rBased partly on the results of this study, we currently we allow considerable variation from the previously described protocol for patients who are treated with a hip resurfacing arthroplasty, with progression based on the ability to achieve certain functional goals, rather than using only time since the index arthroplasty, which has most often been used in the past. Thus, some patients can be treated in an individual manner based on their ability to achieve certain functional milestones. Our current rehabilitation goal by five weeks following surgery is for the patient be able to ambulate pain free using single point cane in the opposite hand, go up and down the a flight of stairs, flex their hip to 90 degrees, and abduct to 30 degrees. We avoid strengthening exercises of the hip that are associated with pain, and specifically avoid side lying hip abduction strengthening early because of our anterolateral surgical approach. If patients achieve these well-defined goals earlier than 5 weeks we recommend faster progression to full range of motion, including rotation. We also progress patients to weight bearing as tolerated without the use of an assistive device, and place patients on progressive resistive exercises to improve hip abductor and extensor strength as long as resistive exercise does not cause pain. Accelerated, rather than time based, rehabilitation performed in this fashion may reduce the total time spent in rehabilitation for a number of patients. This preliminary study suggests that in general, a major commitment to rehabilitation should be made by patients to achieve the best clinical outcomes. In addition, patients who remain stiff or have difficulty progressing may require additional, tailored rehabilitation regimens. Conversely, patients who rapidly regain excellent function and a high activity level following surgery may be able to avoid further rehabilitation once certain goals are met. However, further investigation and multi-center studies need to be performed to confirm and refine these conclusions.Based on the results of the current study, we suggest that increased body mass index may have a negative correlation with patient commitment to rehabilitation. Similar results were reported by Vincent et al. who examined whether obesity affected inpatient rehabilitation outcomes after total hip arthroplasty . In theiThe use of a comprehensive activity and rehabilitation tool such as the one reported in the present study may allow surgeons to predict the postoperative recovery course for patients for hip resurfacing as well as other arthroplasty treatments, and allow for a tailoring of rehabilitation treatments. Additionally, it may assist surgeons in providing guidance regarding which treatment modality may be most appropriate for a given patient. Further study is necessary to better define these potential benefits.The results of this study suggest that the level of commitment to rehabilitation influences outcomes with hip resurfacing, as we found that patients in our cohort who were more committed to their therapy returned to higher levels of functionality and satisfaction. The excellent early clinical outcomes following successful hip resurfacing in our cohort are similar to the results of other studies that have assessed modern hip resurfacing prostheses. We suggest that the importance of rehabilitation compliance should be stressed to resurfacing patients following surgery so that they can achieve maximal functional improvement and a healthier lifestyle.External financial support was received specifically in support of the database used in this study from Wright Medical Technologies . MAM is a consultant for Stryker Orthopaedics and Wright Medical Technologies.None of the other authors have any financial or non-financial competing interests to disclose.DRM, AB, TMS, MAM, MGZ designed the study. DRM, MGZ, TMS collected the data. DRM, MGZ, TMS analyzed the data. DRM, MGZ, AB prepared the manuscript. AB, MGZ, TM, DRM, MAM ensured the accuracy of the data and analysis. All authors have read and approved the final manuscript.


Haemophilus influenzae. Previous studies of the Rd KW20 genome identified the primacy of tetranucleotide SSRs in mediating phase variation (the rapid reversible switching of gene expression) of surface exposed structures such as lipopolysaccharide. The recent sequencing of the genomes of multiple strains of H. influenzae allowed the comparison of the SSRs (repeat units of one to nine nucleotides in length) in detail across four complete H. influenzae genomes and then comparison with a further 12 genomes when they became available. The SSR loci were broadly classified into three groups: (1) those that did not vary; (2) those for which some variation between strains was observed but this could not be linked to variation of gene expression; and (3) those that both varied and were located in regions consistent with mediating phase variable gene expression. Comparative analysis of 988 SSR associated loci confirmed that tetranucleotide repeats were the major mediators of phase variation and extended the repertoire of known tetranucleotide SSR loci by identifying ten previously uncharacterised tetranucleotide SSR loci with the potential to mediate phase variation which were unequally distributed across the H. influenzae pan-genome. Further, analysis of non-tetranucleotide SSR in the 16 strains revealed a number of mononucleotide, dinucleotide, pentanucleotide, heptanucleotide, and octanucleotide SSRs which were consistent with these tracts mediating phase variation. This study substantiates previous findings as to the important role that tetranucleotide SSRs play in H. influenzae biology. Two Brazilian isolates showed the most variation in their complement of SSRs suggesting the possibility of geographic and phenotypic influences on SSR distribution.Simple sequence repeat (SSRs) of DNA are subject to high rates of mutation and are important mediators of adaptation in  The genome sequence of Hi strain Rd KW20, the first completed for a free-living organism, revealed a high prevalence of simple sequence repeats (SSRs) (−4 per generation (−9) and, therefore, it has been suggested that their decreased prevalence reflects natural selection because the higher rates of mutation of these loci would be more often detrimental to fitness than beneficial. However, in some prokaryotes, predominantly host-adapted organisms, some SSRs are found in greater numbers than would be expected by chance  . SSRs ars (SSRs) . SSRs arneration  comparedy chance . Analysiresented . A strikresented .SSRs can be located in promoter regions or within open reading frames and changes in their length can result in the random, high frequency, reversible loss, gain or modulation of gene expression (phase variation). Since these regions of localised hypermutation, often termed ‘contingency loci’, can each independently result in altered gene expression, a repertoire of phenotypic variants is generated . ThroughHi pan-genome.The advent of the genomic sequencing of multiple strains of the same species has revealed that the genomic sequence of a particular strain may not reflect the diversity and variety of the entire species. The term ‘pan-genome’ has been used to describe the superset of genes of a species . The chaHi has been predominantly based on analysis of the strain Rd KW20 genome sequence. Whilst selective studies of other Hi strains have provided some evidence to suggest variation in the number, location and nature of the SSRs compared to that seen in the Rd KW20 genome .A database, named SSR_Hi genomes (herein termed the further 12 genome study) was then examined using the information from the initial four genome survey to guide analysis. Details of these additional genomes are given in HiSSRfinder script as described above. In order to determine which of the tetranucleotide SSRs identified in the 12 further genome sequences were equivalent to the tetranucleotide SSRs previously identified in the four genome study, each was compared to the SSR_Hi_4G database using the BLASTN program. Data and boxplot analysis of SSR data was performed using the R statistical package (http://www.r-project.org/) and Microsoft Excel. Transmembrane helicies were predicted using the TMHMM web server v2.0 , despite the differences in the origin, associated disease and date of isolation of the four strains , tetranucleotide (100%) and hexanucleotide (87%) SSRs occur within ORFs at approximately this frequency, whilst SSRs with repeat unit lengths of one, two, five and seven nucleotides were all found to be located within ORFs with a frequency of less than 88%. SSRs with longer repeat unit lengths were not included in this analysis due to their low frequency in the genomes.SSRs have previously been associated with hypermutation, as loss or gain of repeat units occur at high frequency due to replication slippage . Loss orHi.This suggests that the selective pressure against trinucleotide and hexanucleotide SSRs occurring within an ORF may not be as high as that against SSRs of other repeat unit lengths whose expansion or contraction would result in inactivation of an ORF by frameshift mutation. It is noteworthy that tetranucleotide SSRs are found exclusively within ORFs, consistent with the known importance of this class of repeat in mediating phase variable expression at contingency loci in 3.2Manual curation of each of the 223 SSRs allowed us to assess the likelihood of each SSR playing a role in modulating gene expression. Comparison of equivalent SSR loci (those located in the same relative genomic location) allowed the classification of each SSR into one of three categories: (1) SSRs that did not vary in length, sequence or position between the four genomes (invariable), (2) SSRs for which some variation between strains was observed but the variation was not considered likely to result in variation of gene expression (variable) and (3) SSRs that both varied in length and were located in regions consistent with mediating phase variation , the tetranucleotide, pentanucleotide and heptanucleotide repeat tracts were the only types of SSR considered to have a potential role in mediating phase variable gene expression in these four strains of Hi . The pot3.3Hi  to the phosphoethanolamine transferase gene, lpt3, of Neisseria meningitidis , suggesting that they have been acquired by horizontal transfer. The finding that multiple, distinct gene insertions have occurred in the same region of the bacterial genome in different strains may indicate that this is a hotspot for recombination.Two of the tetranucleotide SSR loci that we have identified in the four genome analysis have not previously been described as potential mediators of phase variation in ynthesis . In strangitidis . This isHi (The second novel tetranucleotide SSR locus contains a 5′CCAA tract associated with a putative glycosyltransferase (gene NTHi_1769 in strain 86-028NP). This SSR is present in all four genomes examined with between 8 and 16 repeat units and constitutes the first example of a 5′CCAA tract that is associated with a gene other than iron utilisation genes in Hi .3.4Hi strains investigated, six of these were located within ORFs (HI1287) is truncated due to the repeat), and has previously been implicated in the phase variable expression of this type I restriction-modification gene  are consistent with the expression of a putative LPS flippase, whilst the three unit SSR in R2866 is inconsistent with expression of this gene. It is noteworthy that these two potential phase variation-mediating pentanucleotide SSRs relate to gene functions (restriction-modification and LPS modification) whose expression has previously been reported to be phase varied by tetranucleotide SSRs.The second coding pentanucleotide SSR of interest (5′TCAGC) was found in a gene of the  the LPS . The two3.5Hi genomes, three of which we have designated as potential mediators of phase variation. Two of these heptanucleotide SSRs are located approximately 100 bp upstream of the hmw1a and hmw2a genes and have previously been described by hmw1a and hmw2a genes and that alteration of the number of repeat units present in these SSRs results in a modulation of gene expression. The exact mechanism by which these SSRs influence transcription from these genes remains to be determined but may involve modulation of transcription from two alternative start sites 17 and (5′TGAAAGA)23 units for hmw1a and hmw2a, respectively, but there are no equivalent loci or repeat tracts in the other two genomes.Four heptanucleotide SSRs were found in the survey of the four rt sites . Strain Hi.The third heptanucleotide SSR with a potential to mediate phase variation is the (5′AACAACC)1-7 tract situated only 13 bp upstream of a gene encoding a member of the TonB-dependent receptor family (PF0593) that has similarity to Fe transport proteins. One unit of the repeat is found in the genomes of strains Rd KW20 and R2846, seven in R2866 and six in 86-028NP. Rd KW20 and R2866 appear to have full length ORFs but the 86-028NP ORF is disrupted by a frameshift unrelated to the SSR. The observed variation in the length of this SSR, together with its position so close to the start of the downstream ORF, led us to postulate that it may mediate phase variation in 3.6Analysis of mononucleotide, dinucleotide, trinucleotide and hexanucleotide SSRs in the four genome study did not provide any evidence to suggest to us that these classes of repeat were associated with phase variable gene expression as detailed below.3.7Neisseria meningitidis . The (G)12 SSR was within the 5′ end of ORF ntHI0694. This gene shows homology with genes encoding methyltransferases of the FkbM family, some of which are involved in the biosynthesis of methylated sugars in Rhizobium etli LPS (Hi strains and suggests that 86-028NP LPS may be O-methylated. The (C)11 SSR was located 230 bp upstream of the acpP gene (ntHI0243). Members of the AcpP family are short proteins which are involved in the transfer of acyl groups and are considered house keeping proteins. In the three other genomes the tract at the same location contains five C residues.Examination of the further three genomes identified some anomalous mononucleotide SSRs. The first is an exceptionally long (A)34 tract identified in strain R2866. This SSR was located 120 bp upstream of the start of the ORF encoding the autotransporter adhesin Hia, which is an autotransporter protein containing the YadA domain and is believed to bind vitronectin and aid survival in human serum . This SSetli LPS . This ge3.8Hi. A (TA)9-11 tract, located in the promoter region of two divergently transcribed genes, hifA and hifB was shown to control fimbriae biogenesis in some strains 4ATTA sequence. The threshold value set for dinucleotide SSRs in this study was five, therefore this tract was not identified as an SSR; further discussion of this locus is found later in this paper.Phase variation mediated by dinucleotide repeats has been documented previously in  strains . The hifEight dinucleotide SSR loci were identified in this four genome analysis, all of which were found to be of the threshold value of five repeat units in length. Five were located within coding regions. In a similar fashion to the variation observed for many of the mononucleotide SSRs, seven of the dinucleotide SSR loci were found to have sequence variations that did not alter the overall length of the sequence between strains and so would not cause frameshifts consistent with phase variation. For example, a (CA)5 repeat conserved in the genomes of strains Rd KW20, R2846 and 86-028NP was found to be replaced with CACG(CA)3 in strain R2866.3.9Eighteen trinucleotide SSRs were identified in this study, the majority of which, (15/18), were located within coding regions. All of these 15 SSRs consisted of no more than four repeat units and where variation in the repeats was observed between strains, it either resulted in a reduction in length of the SSR or disruption of the sequence whilst maintaining the same length. The three trinucleotide SSRs that were found in non-coding regions showed greater variation in overall length but were not within identified promoter or other regulatory regions.3.10HI0525 in strain Rd KW20, which encodes a phosphoglycerate kinase involved in central metabolism and the influence of this SSR on the expression of this ORF is unknown.Ten hexanucleotide SSRs were identified within the four genomes, eight within ORFs and two in non-coding regions. Variation in coding hexanucleotide repeats can lead to altered amino acid sequence but not phase variable gene expression. The coding region hexanucleotide SSRs identified in this study were either conserved or, like the mononucleotide and dinucleotide variations discussed above, showed changes in sequence but not length and thus were inconsistent with modulating phase variable expression. Of the two non-coding region associated hexanucleotide repeats, one is conserved across all four strains and is present downstream of the closest ORFs, whilst the other 5′TTAAAA SSR is present as three repeat units in Rd KW20, two units in 86-028NP and as two units plus an interrupted third repeat unit in R2866 and R2846. This SSR is situated 19 bp from the start codon of 3.11hmw2C gene. As discussed above, variations in heptanucleotide SSR associated with the hmw2A loci are thought to modulate gene expression but the function of this nonanucleotide SSR is not known. An octanucleotide (5′ATTATTTG) SSR however, was found in multiple strains, varying in length between 1 and 6 repeat units. It was found to be located between the divergently transcribed cmkB and pdxS genes which encode a cytidylate kinase 2 and a pyridoxal biosynthesis lyase, respectively, (designated HI1646 and HI1647 in strain Rd KW20). They are both suggested to play roles in metabolism and so it is uncertain whether this SSR would actually be utilised in modulating their expression.Of the limited number of SSRs with repeat unit lengths greater than seven nucleotides that were identified in the four genomes study, most were found in only one strain. These include a nonanucleotide SSR found in the genome of strain 86-028NP. This (5′GTTTTCTTA)19 SSR was found to be located 92 bp upstream of the 3.12Hi genomes was ongoing, 12 further Hi genomes were sequenced and the resulting full or partial sequences made publicly available 9 SSR is found 225 bp upstream of a gene encoding a putative DNA repair enzyme, formamidopyrimidine-DNA glycosylase MutM, in strain F3043. The equivalent position in other es in Hi .Hi (including HI0223 in strain Rd KW20) but without the associated SSR. The function of this gene is unknown but it may contribute to LPS expression in strains F3031 and F3043.The second novel tetranucleotide SSR identified from strains F3031 and F3043 is a 5′CAAT SSR contained within the 5′ region of an ORF that encodes a putative glycosyltransferase with homologies to glycosyltransferase family 8 (PFAM01501). Homologues of this gene are found in other strains of The third of the four additional novel tetranucleotide SSR loci contains (5′CAAT)21 and was found only in strain F3043. It is located within the 5′ end of an ORF that encodes a putative S-adenosylmethionine (SAM)-dependent methyltransferase and shows some homology to HI0096 in strain Rd KW20. SAM-dependent methyltransferases have been implicated in various cellular processes including protein trafficking and sorting, signal transduction, biosynthesis, metabolism, and gene expression.htpX . HtpX has not been investigated in Hi but in E. coli it is part of the membrane-localised proteolytic system and may play a part in the degradation of unstable membrane proteins 32 SSR located 58 bp upstream of a gene encoding an adenine specific methylase homologue (EcoRI) and 202 bp upstream of the divergently transcribed proteins .3.15Hi biogroup aegyptius strains F3031 and F3043, which are associated with unusual clinical symptoms and have the highest number of tetranucleotide SSR, display a similar distribution of SSR lengths to all other strains.In total, 199 tetranucleotide SSRs associated with 28 different loci and consisting of nine different repeat unit sequences have been identified in the complete genome collection. The distribution of tetranucleotide SSR length, and the relationship between length of tetranucleotide SSR and strain are shown in 3.16Hi are located within ORFs and, with only two exceptions, are located immediately adjacent to or just downstream of the translational start site. In this position, any frameshift due to variation in length of the SSR, would result in a peptide being made from the incorrect reading frame and a premature stop to translation. The location of tetranucleotide SSRs within the 5′ region of the ORFs limits the encoded tetrapeptide repeat to the N-terminus of the respective protein. The two exceptions to this pattern are the 5′GCAA tetranucleotide SSR located in the middle of the oafA gene that has been previously described (pgt1) in the genomes of strains R2846, 86-028NP, 3655 and PittEE. These repeats may modulate the protein function rather than control ON/OFF switching of its expression.As noted previously, the tetranucleotide SSRs identified in the four genome study of escribed , and a 5Tetranucleotide SSRs may constitute a substantial proportion of the coding region of a gene and thus the repeat unit sequence will have a significant influence on the amino acid composition of the encoded protein. The constraints that this may impose, in terms of permissible tetranucleotide sequences, have not been well characterised although in silico analysis of the repeat sequences identified in the 16 Hi genomes analysed was performed and hydrophilic amino acids are over represented in the SSR encoded peptides, compared with their frequency in the normal proteome. Of the eight tetranucleotide repeat sequences found within ORFs in Hi, five encode hydrophilic peptides with no net charge , one encodes a hydrophobic peptide with no net charge (5′TTTA) and two encode hydrophilic peptides with a net positive charge (5′GCAA and 5′CAAA). The high proportion of hydrophilic peptides encoded by the tetranucleotide SSRs and their frequent N-terminal location suggests that they are likely to be surface exposed and have the opportunity to ‘loop out’ of the folded protein structure and thus be less likely to interfere with the tertiary structure of the protein and, therefore, its function. The exception is a 5′TTTA tetranucleotide SSR which encodes a hydrophobic peptide within a putative drug/metabolite exporter (HI0687 in strain Rd KW20). Transmembrane helices predictions . Another observation of this study was that although previously SSRs of a particular tetranucleotide repeat unit sequence have been associated with genes of related function, e.g. 5′CCAA tracts with genes encoding iron utilisation proteins  genes containing 5′CCAA SSR that show considerable variation in length. Hi lacks most of the genes of the heme biosynthetic pathway and requires hemoglobin/hemoglobin–haptoglobin-binding proteins to capture heme-containing compounds required for growth , partial or fully duplicated hifA loci and duplicated lic1A genes (strain PittGG).One feature of certain tetranucleotide SSR, noted during the course of this study was their interruption by an imperfect repeat unit. All of the genomes in this study were found to contain between one and four related, hemoglobin/hemoglobin–haptoglobin-binding  instead of dinucleotide SSRs located either 63 or 93 bp upstream of hifA 14, was 3.5 kb within the 7.3 kb putative full length ORF rather than in the promoter region, and a frame shift had occurred which is consistent with this being caused by variation in the length of the SSR. In the further genome analysis, an octanucleotide SSR was found associated with hmw loci. This SSR contained twelve to fifteen copies of a 5′GCATCATC repeat and was identified 200–213 nucleotides upstream of the hmw1a and hmw2a loci of strain F3043 and F3031.The heptanucleotide SSRs associated with the HI1369 gene (encoding a putative TonB dependent iron ligand gated channel). Thirteen units of the 5′AACAACC repeat are found in 22.4.21, and eight repeat units in strain R3655 which results in a truncated ORF due to a frameshift.A further novel heptanucleotide SSR with the potential to mediate phase variation was identified in strains 22.4.21 and R3655, within an ORF which is a homologue of the An octanucleotide SSR identified in the four genome study as containing one, four or six copies of a 5′ATTATTTG unit 12 bp upstream of a gene encoding a pyridoxine biosynthesis protein, was also identified in seven strains of the further twelve genome collection , Brazil (two strains) and the United States of America (13 strains). A majority of the novel SSRs identified were in the F3031 and F3043 genome sequences (the Brazilian strains) and it remains unknown whether the population/geographical structure of Hi and has characterised and compared 28 tetranucleotide SSR loci (9 of them previously unreported) across 16 strains. Additionally, this study has identified a number of previously unrecognised mononucleotide, dinucleotide, pentanucleotide, heptanucleotide, and octanucleotide SSRs as potential mediators of phase variation that will be the focus of future research efforts. Thus, the utility of whole genome sequences in the investigation of the biology of pathogenic bacteria has been confirmed and, further, the analysis of multiple genomes has revealed non-intuitive subtleties in the population structure concerning the distribution of SSRs across the Hi pan-genome.In conclusion, this study has reaffirmed the primacy of tetranucleotide SSRs as mediators of phase variation in


Drosophila, and Caenorhabditis elegans, FoxO transcription factors mediate cellular responses to stress, including oxidative stress and dietary restriction. Although FoxO genes have been identified in early-arising animal lineages including sponges and cnidarians, little is known about their roles in these organisms.In the face of changing environmental conditions, the mechanisms underlying stress responses in diverse organisms are of increasing interest. In vertebrates, Hydra. We find that Hydra FoxO is expressed at high levels in cells of the interstitial lineage, a cell lineage that includes multipotent stem cells that give rise to neurons, stinging cells, secretory cells and gametes. Using transgenic Hydra that express a FoxO-GFP fusion protein in cells of the interstitial lineage, we have determined that heat shock causes localization of the fusion protein to the nucleus. Our results also provide evidence that, as in bilaterian animals, Hydra FoxO activity is regulated by both Akt and JNK kinases.We have examined the regulation of FoxO activity in members of the well-studied cnidarian genus These findings imply that basic mechanisms of FoxO regulation arose before the evolution of bilaterians and raise the possibility that FoxO is involved in stress responses of other cnidarian species, including corals. Drosophila (dFOXO) and C. elegans (daf-16), and four are present in mice and humans . In Drosophila, C. elegans, and mammalian cells, FoxO proteins increase resistance to oxidative stress Drosophila, C. elegans, and mammals C. elegansDrosophila, C. elegans and mammals, they increase levels of enzymes that detoxify reactive oxygen species (ROS) Drosophila undergoing dietary restriction C. elegans and mammals, FoxO proteins increase resistance to DNA damage C. elegans, DAF-16 regulates transcription of genes involved in pathogen resistance and acts together with the transcription factor heat shock factor 1 to upregulate transcription of small heat shock protein genes In bilaterian animals, members of the FoxO family of transcription factors are well-known for their roles in cellular responses to environmental and physiological stress. A single FoxO gene is present in Activity of FoxO proteins is regulated by post-transcriptional modification, including phosphorylation. In response to insulin/IGF-1 and other growth factors, Akt and the related serum- and glucocorticoid-inducible kinase (SGK) phosphorylate FoxO proteins (with the exception of FoxO6) at three conserved sites. These phosphorylations promote FoxO binding to 14-3-3 proteins and localization to the cytoplasm Hydra. The Hydra magnipapillata genome has been sequenced Hydra can be produced Hydra has been examined in some detail A substantial amount is known about the role of FoxO proteins in the stress responses of bilaterian animals. However FoxO genes have also been identified in members of earlier-evolving metazoan lineages, such as sponges Hydra species are well characterized Hydra polyp consists of two tissue layers, an ectoderm and an endoderm. At one end of the body is a mouth surrounded by a ring of tentacles. At the other end is a basal disk by which the animal adheres to the substrate. In the adult, cells divide continuously in the body column. Cell division in the body column causes displacement of epithelial cells into the tentacles and basal disk Hydra routinely reproduce asexually, with cells from the body column displaced into buds. The continuous growth and production of buds by adult Hydra can be explained in part by the presence of three types of multipotent stem cells. These are ectodermal stem cells, endodermal stem cells, and interstitial stem cells. Interstitial stem cells are located between epithelial cells in the ectodermal layer and give rise to neurons, secretory cells, gametes, and nematocytes, the stinging cells unique to cnidarians FoxO in H. vulgaris and the closely related H. magnipapillata and have examined cellular localization of a FoxO-GFP fusion protein in transgenic animals. We find evidence for significant parallels in regulation of FoxO between Hydra and bilaterian animals.Cell and tissue dynamics in H. magnipapillata genome identified a single FoxO gene. The predicted H. magnipapillata FoxO protein . Phylogenetic analyses including sequences of FoxO forkhead domains from diverse animals were also performed  FoxO is expressed in interstitial cells, we treated H. magnipapillata with HU. Counting cells from macerated control and treated animals showed that treatment reduced the ratio of interstitial cells to total cells present by 85 to 90%, depending on the experiment, but did not reduce the ratio of nematoblasts, nematocytes, or neurons to total cells. . We characterized FoxO-GFP localization in these cell types.To examine the regulation of FoxO cellular localization in  lineage . In inteH. magnipapillata FoxO protein suggest that Akt and/or SGK may be involved in regulating Hydra FoxO activity. Because PI3K activity leads to activation of Akt and SGK, we treated transgenic animals with an inhibitor of PI3K, LY294002 Hydra FoxO localization. We found that inhibitor treatment significantly increased the percentage of interstitial lineage cells showing nuclear localization of FoxO-GFP , Akt, and SGK. Phosphorylation of FoxO by Akt and SGK promotes its cytoplasmic localization, reducing FoxO transcriptional activity FoxO-GFP , suggestHydra, we examined FoxO-GFP localization in transgenic animals that were fed daily for two weeks and then starved for two, three, four, or ten days. We found no significant difference in the percentage of interstitial lineage cells showing fusion protein nuclear localization between control animals and starved animals .To determine whether dietary restriction leads to FoxO nuclear localization in Hydra FoxO localization, transgenic animals were incubated for 90 minutes at 33°C. These conditions cause synthesis of HSP70 in Hydra vulgarisTo address the effect of heat shock on HydraHydra, as in bilaterian animals, JNK plays a role in increasing FoxO nuclear localization under conditions of thermal stress.Finally, we asked whether the stress-associated kinase JNK promotes the nuclear localization seen in response to heat shock. Transgenic animals were treated for 24 hours with either of two inhibitors of JNK, SP600125 Hydra. FoxO genes have been found in members of early-evolving animal phyla, including sponges, placozoans, and cnidarians We have identified a single FoxO gene in a member of the cnidarian genus Hydra FoxO is strongly expressed in interstitial cells in the body columns of adult animals. Hydra interstitial cells include multipotent stem cells which maintain populations of stinging cells, secretory cells, and neurons in the adult Hydra vulgaris do not show senescence–increased mortality with age Hydra and its asexually produced offspring. In addition, because Hydra germ cells are not segregated early in development and instead arise from interstitial stem cells Clytia hemisphaerica. However, it is not yet clear how similar cell and tissue dynamics are in these two species. CheFoxO in C. hemisphaerica is expressed throughout the polyp as well as in the larval and medusa stages of the life cycle not present in Hydra. As in the Hydra polyp, expression in both polyp and medusa is strongest in tissues characterized by cell proliferation FoxO expression has been examined in another cnidarian, the marine hydrozoan Hydra FoxO transcriptional activity is negatively regulated by the PI3K/Akt/SGK pathway. Like other FoxO proteins, the predicted H. magnipapillata FoxO protein includes three Akt/SGK phosphorylation sites. Treatment with a PI3K inhibitor significantly increased nuclear localization of the FoxO-GFP fusion protein  nauplii cultured from cysts for 48 hours at room temperature.H. magnipapillata were incubated in 10 mM hydroxyurea for 24 hours and then in medium without hydroxyurea for 12 hours. This cycle was repeated two additional times To reduce the interstitial cell population, H. magnipapillata 105 genome sequence in the Metazome database using tblastn with DAF-16 and dFOXO protein sequences as queries. DNA encoding H. magnipapillata FoxO was isolated from first strand cDNA by PCR using the primers GCGAGATATGTTTTTAAATGTCAGTGC and TCCATATAGAACTTTCCTGAGTTCATTAG. cDNA was produced from H. magnipapillata poly-A RNA using the Invitrogen GeneRacer Kit.To identify FoxO genes, we searched the assembled Phylogenetic analyses were based on the predicted amino acid sequence of the forkhead domain. Sequences were aligned using ClustalX FoxO coding sequence was amplified from H. magnipapillata 105 cDNA using the primers CCCAGATGCAAAAGCAGGGAAATC and GCTTTACTGGTCTAAGTCGCTCGG. This PCR product was cloned into the Promega pGEM-T Easy Vector. To produce the digoxygenin-labeled antisense probe, the plasmid was linearized by digestion with SalI and in vitro transcription was performed using the Roche DIG RNA Labeling Kit and T7 RNA polymerase. To produce the sense probe, the plasmid was linearized with NcoI and transcription was performed using the SP6 polymerase. Whole mount in situ hybridization was performed as described in A 992 base pair long portion of the H. magnipapillata 105 FoxO coding sequence fused in frame with a sequence encoding enhanced green fluorescent protein, under the control of the Hydra β-actin promoter. Using PCR, an NheI site was added to the 5′ end of the FoxO cDNA and a HpaI site was added to the 3′ end. The resulting cDNA was cut with NheI and HpaI, and then cloned into the expression vector pHyVec4 (GenBank accession DQ385853), which had been cut with NheI and SmaI. pHyVec4 is a modified version of the hoT G plasmid FoxO coding sequence within the construct was checked against the NCBI trace archive sequences for H. magnipapillata 105. Plasmid DNA for embryo injection was purified using the Qiagen Endo-free Gigaprep kit and resuspended in sterile deionized water.We produced an expression construct with the H. vulgaris strain AEP at the one to eight cell stage as described in Wittlieb et al. Hydra. The majority of experiments were conducted with a line termed in[act:FoxO-GFP]1, in which a subset of cells of the interstitial lineage expressed the fusion protein. Specifically, the cells which expressed the protein were precursors to stenotele nematocytes, mature stenotele nematocytes, and ganglionic neurons. Fusion protein localization was examined in all of these cell types. The number of cells of each cell type scored was approximately equal in the control and treated animals in each experiment. Fusion protein localization following heat shock was also examined in the line ec[act:FoxO-GFP]1, in which ectodermal cells expressed the fusion protein.The FoxO-GFP expression construct was microinjected into embryos of 1-Methyl-1,9-pyrazoloanthrone  in hydra medium containing 0.0125% DSMO. Animals treated with the JNK inhibitor AS601245  were incubated in the dark in 2 uM AS601245 in hydra medium containing 0.15% DMSO. They were first incubated for 24 hours at 18°C and then placed at 33°C for 90 minutes. Control animals were incubated at the same temperatures in 0.15% DSMO/hydra medium. Animals subjected to starvation were fed daily for two weeks and then starved for two, three, four, or ten days. Control animals were fed daily but starved for one day before fixation to reduce background fluorescence. In heat shock, LY294002, SP60025, four-day starvation and ten-day starvation experiments, three treated and three control Hydra were used for each experiment. In AS601245, two-day starvation and three-day starvation experiments, five treated and five control Hydra were used. For all treatments, the percentage of cells showing nuclear localization in control and treated animals were compared using the Mann-Whitney U test.Animals subjected to heat shock were incubated at 33°C for 90 minutes, conditions known to increase levels of HSP70 To determine FoxO-GFP localization, animals were processed as follows. Following experimental treatments, animals were fixed for 1 hour in 4% paraformaldehyde in hydra medium and then washed for 10 minutes in PBS, for 30 minutes in 5 µg/mL DAPI in PBS, and for 5 minutes in PBS. Fixation and washes were performed at 4°C. Cells expressing the fusion protein were visualized at 1000× using a Nikon Eclipse 80i microscope and photographed using a SPOT RTke digital camera. Localization was examined in nematoblasts, nematocytes, and neurons.


Starting from either the syn- or the anti-α,β-epoxy alcohol, stereospecific reactions generate two separate diastereoisomeric series of this motif. The route is a significant improvement on an earlier six step strategy.A three step route to single diastereoisomers of the vicinal trifluoromethyl motif is described. The route starts from either  Selective fluorination is an important strategy for the design of performance molecules in medicinal chemistry programmes and in organic materials –2. To dasyn- or anti-α,β-epoxy alcohols A was envisaged, each step incorporating a fluorine atom in a stereospecific manner. Conversion of the free hydroxyl group to fluorine would generate α-fluoro-epoxides B. Epoxide ring opening with an HF source could then provide difluoroalcohols C. Insertion of the third fluorine would reasonably be achieved by fluorination of the free hydroxyl group of C to generate D. Such a strategy offers a three step route to the vicinal trifluoroalkane motif and would avoid the use of TBAF, reducing the risk of elimination reactions competing with fluorine substitution, a problematic aspect of the last step in the earlier route shown in A three step strategy, as illustrated in ® mediated deshydroxyfluorination reactions ®  was added to a solution of 12b  in DCM (3 mL) at RT. The reaction mixture was then heated at 40 °C for 1 h, and the reaction was quenched by the addition of silica gel. Solvents were removed under reduced pressure, and the product was purified over silica (hexane 5 /DCM 3 /Et2O 1). The title compound was recovered as a colourless oil .Deoxo-FluorD20 = −2.8 . 1H NMR : δ (ppm) 7.78 ; 7.35 ; 7.31–7.21 ; 4.88 ; 4.73–4.38 ; 4.42 ; 4.26 ; 3.21 ; 3.01 ; 2.46 . 13C NMR : 145.2 (C ar); 135.1 (C ar); 132.3 (C ar); 129.9 (2 CH ar); 129.3 (CH ar); 128.9 (CH ar); 128.0 (CH ar); 127.2 (CH ar); 90.1 ; 87.0 ; 86.0 ; 67.4 ; 36.3 ; 21.7 (CH3). 19F NMR : −198.80 to −199.23 ; −200.27 to −200.69 , −215.48 to −215.78 . 19F {1H} NMR : −198.61 ; −200.03 ; −215.18 . νmax/cm−1 1365, 1261, 1267, 1208, 1190, 1177, 1151, 749. m/z (ES+) = 394.95 ; HRMS (ES+) found 395.0906 for C18H19F3NaO3S, requires 395.0905.[α]®  was added to a solution of 12a  in DCM (1 mL) at RT. The reaction mixture was then heated at 40 °C for 1 h and the reaction was quenched by the addition of silica gel. Solvents were removed under reduced pressure and 17a was purified by preparative TLC (hexane 7/Et2O 3) and recovered as a colourless oil .Deoxo-FluorD20 = +1.75 . 1H NMR : δ (ppm) 7.74 ; 7.37–7.22 ; 5.06–4.43 ; 4.73–4.38 ; 4.35 ; 4.27 ; 3.15–2.97 ; 2.45 . 13C NMR : 130.1 (CH ar); 129.3 (CH ar); 128.9 (CH ar); 128.0 (CH ar); 127.3 (CH ar); 21.7 (CH3). Quaternary carbons and carbons coupled bound to fluorine were not observed, even with an extended number of scans (12000 scans). 19F NMR : −195.84 to −196.29 ; −201.07 to −201.51 , −212.23 to −212.63 . 19F {1H} NMR : −196.1 ; −201.29 ; −212.43 . νmax/cm−1 1360, 1269, 1208, 1190, 1146, 983, 910, 740. m/z (ES+) = 394.96 ; HRMS (ES+) found 395.0903 for C18H19F3NaO3S, requires 395.0905.[α]File 1Experimental methods and full characterisation and spectral data of all prepared compounds.


Arsenic is known as a toxic metalloid, which primarily exists in inorganic form [As(III) and As(V)] and can be transformed by microbial redox processes in the natural environment. As(III) is much more toxic and mobile than As(V), hence microbial arsenic redox transformation has a major impact on arsenic toxicity and mobility which can greatly influence the human health. Our main purpose was to investigate the distribution and diversity of microbial arsenite-resistant species in three different arsenic-contaminated soils, and further study the As(III) resistance levels and related functional genes of these species.Acinetobacter, Agrobacterium, Arthrobacter, Comamonas, Rhodococcus, Stenotrophomonas and Pseudomonas. Five arsenite-oxidizing bacteria that belonged to Achromobacter, Agrobacterium and Pseudomonas were identified and displayed a higher average arsenite resistance level than the non-arsenite oxidizers. 5 aoxB genes encoding arsenite oxidase and 51 arsenite transporter genes  were successfully amplified from these strains using PCR with degenerate primers. The aoxB genes were specific for the arsenite-oxidizing bacteria. Strains containing both an arsenite oxidase gene (aoxB) and an arsenite transporter gene (ACR3 or arsB) displayed a higher average arsenite resistance level than those possessing an arsenite transporter gene only. Horizontal transfer of ACR3(2) and arsB appeared to have occurred in strains that were primarily isolated from the highly arsenic-contaminated soil.A total of 58 arsenite-resistant bacteria were identified from soils with three different arsenic-contaminated levels. Highly arsenite-resistant bacteria (MIC > 20 mM) were only isolated from the highly arsenic-contaminated site and belonged to Soils with long-term arsenic contamination may result in the evolution of highly diverse arsenite-resistant bacteria and such diversity was probably caused in part by horizontal gene transfer events. Bacteria capable of both arsenite oxidation and arsenite efflux mechanisms had an elevated arsenite resistance level. The toxin arsenic in soil and aqueous environments is considered as one of the prominent environmental causes of cancer mortality in the World, especially in Bangladesh, India and China. In recent years, chronic intake of groundwater with high levels of arsenic has caused endemic arsenicosis in several provinces of China and new cases of arsenicosis are continuously emerging . DevelopBacteria have developed different strategies to transform arsenic including arsenite oxidation, cytoplasmic arsenate reduction, respiratory arsenate reduction, and arsenite methylation . The priPseudomonas ; (ii) Among the strains belonging to Pseudomonas or Agrobacterium, the MICs of arsenite oxidizers were higher than the non-arsenite oxidizers ; (iii) The average MIC of the 5 arsenite oxidizers (20 mM) was higher than the 53 non-arsenite oxidizers (12 mM); (iv) A total of 12 highly arsenite-resistant bacteria  whose MICs exceeded 20 mM were only obtained from highly arsenic-contaminated TS soil; (v) Only intermediate (10 mM < MICs ≤ 20 mM) and low (MICs ≤ 10 mM) MIC levels of arsenite-resistant bacteria were identified from the intermediate and low arsenic-contaminated soils; (vi) Strains of the highly arsenic-contaminated TS site had higher arsenite resistance levels (average MIC = 14 mM) than the strains of the intermediate (average MIC = 11 mM) and low arsenic soils (average MIC = 8 mM).The MIC range for arsenite of the 58 strains was from 2 mM to 34 mM Fig. . The numACR3(1) and ACR3(2) were amplified in strains of the high and intermediate arsenic-contaminated soils only. In contrast, aoxB and arsB were found in strains isolated from all three kinds of arsenic-contaminated environments  and an arsenite transporter gene (ACR3 or arsB) showed a higher average arsenite resistance level than the strains possessing arsenite transporter genes only [aoxB/ACR3 (20.25 mM) ≈ aoxB/arsB (20 mM) > arsB/ACR3 (14.29 mM) ≈ ACR3 (14.13 mM) > arsB (10.1 mM)].A total of 5 arsenite oxidase genes and 51 arsenite transporter genes were successfully amplified from 38 strains using PCR with degenerate primers. The aoxB sequences were amplified from all of the five arsenite oxidizers. No aoxB sequences were amplified from any of the 53 non-arsenite oxidizers. The deduced amino acid sequence of aoxB from Achromobacter sp. SY8 showed 95% identity to AoxB from Achromobacter sp. NT-10 (GenBank accession no. ABD72610) , Shewanella sp. TS29, Delftia spp. TS12, TS30, TS33, TS41 and Pseudomonas sp. SY4 [arsB and ACR3(2)], Stenotrophomonas spp. TS28, SY1, SY2, Aeromonas spp. TS26, TS36 and Pseudomonas sp. TS44 [ACR3(1) and ACR3(2)] p group had 12 sequences from γ-Proteobacteria and Actinobacteria; The Acr3(2)p group contained 21 sequences from α-, β-, and γ-Proteobacteria p, 81%–90% aa identities had 10 ArsB, 5 Acr3(1)p and 11 Acr3(2)p, aa identities over 90% included 6 ArsB, 7 Acr3(1)p and 4 Acr3(2)p. The aoxB appeared to be vertically transferred when comparing the phylogeny of 16S rRNA genes with those encoding aoxB. In contrast, certain inconsistency occurred when comparing phylogenetic trees based on 16S rRNA genes and arsenite transporter genes. Phylogenetic discrepancies could be detected in 8 ACR3(2) and 1 arsB  sequences; (ii) Stenotrophomonas spp. TS28, SY2, SY1 belonging to γ-Proteobacteria using 16S rDNA analysis were assigned to α-Proteobacteria based on Acr3p(2) sequences; (iii) Comamonas sp. TS32, TS35 and Delftia sp. TS33 were shown to belong to β-Proteobacteria, but were assigned to the γ-Proteobacteria clade using Acr3(2)p sequences; (iv) LY4 belonged to α-Proteobacteria based on the 16S rRNA gene, but its ArsB was in γ-Proteobacteria clade  genes were from the strains isolated from the highly arsenic-contaminated TS soil.The arsenite oxidase gene rsB Fig. : (i) AerAcinetobacter, Comamonas, Pseudomonas, Stenotrophomonas, Delftia, Agrobacterium, and Bacillus as the major genera of arsenite-resistant bacteria in the four arsenic-contaminated soils. Among them, Acinetobacter, Agrobacterium, Bacillus, and Pseudomonas species were commonly found at other arsenic-contaminated sites . Related reports also found the presence of multiple sets of arsenic resistance genes and operons in one strain, especially the arsenite transporter genes. Pseudomonas putida KT2440 contains two operon clusters (arsRBCH) for arsenic resistance . This indicates that arsenite transporter genes may be horizontally transferred and increasingly present in a microbial population under conditions of long-term elevated arsenic stress. It is important to note that HGT occurred in somewhat closely related species in this study, however, this does not detract from the suggestion of HGT and it is likely that the HGT events occurred between these closely related species. Martinez et al. . The sterile CDM medium containing the same amount of NaAsO2 was used as an abiotic control. The arsenite oxidizing phenotype was also detected using the molybdene blue method with a spectrophotometer   and 2.5 units of Taq DNA polymerase , and adding ddH2O to a final volume of 50 μL. The PCR program consisted of an initial 5 min denaturation step at 94°C; 30 cycles of 1 min at 94°C, 1 min at 50°C, 1.5 min at 72°C; and a final extension step at 72°C for 5 min.Total DNA of each strain was extracted using standard molecular genetic methods. Nearly full-length 16S rDNA of the bacteria was amplified by PCR using universal primers Uni-27F and Uni-1492R   and ACR3(2) genes were performed using three pairs of degenerate primers  . DNA sequencing analysis was performed using ABI 3730XL DNA analyzer by Sunbiotech company . All sequences were analyzed by BlastN (for 16S rRNA gene) and BlastX (for deduced AoxB and ArsB/Acr3p) searching tools . All seq2, incubated with shaking at 28°C for one week and the OD600 values were determined. The initial screening for MICs was performed with 5 mM, 10 mM, 15 mM, and 20 mM of NaAsO2. Subsequent determinations were performed with 1 mM NaAsO2 intervals over the appropriate range. The sensitivity of MIC detection was 1 mM.The MIC, defined as the lowest concentration of arsenite that inhibited growth in CDM broth, was performed with all arsenite-resistant bacteria. Triplicate samples of each single colony were inoculated in 3 mL CDM broth supplemented with increasing concentrations of NaAsOEU073067–EU073124 for 16S rRNA genes, EF523515, EU311944–EU311947 for aoxB, and EU311948–EU311999 for arsB/ACR3.The nucleotide sequences are posted in the NCBI GenBank database. Their accession numbers are: All authors participated in the design of the study and data analyses. LC carried out samples collection, bacterial isolation and drafted the manuscript, participated in molecular genetic studies. GL carried out molecular genetic studies and construction of phylogenetic trees. CR participated in the design of the experiments and helped to draft the manuscript. GW is the Principal Investigator of the funded projects. She coordinated the study and helped to draft the manuscript. All authors read and approved the final manuscript.


A new treatment to determine the Pareto-optimal outcome for a non-zero-sum game is presented. An equilibrium point for any game is defined here as a set of strategy choices for the players, such that no change in the choice of any single player will increase the overall payoff of all the players. Determining equilibrium for multi-player games is a complex problem. An intuitive conceptual tool for reducing the complexity, via the idea of spatially representing strategy options in the bargaining problem is proposed. Based on this geometry, an equilibrium condition is established such that the product of their gains over what each receives is maximal. The geometrical analysis of a cooperative bargaining game provides an example for solving multi-player and non-zero-sum games efficiently. Some simple games, such as the bargaining problem n-player game, there is always a solution for the optimal strategy for each person. This strategy guarantees their average payoff or loss is maximized or minimized under the assumption that the opposing player also uses an optimal strategy. This solution corresponds to the equilibrium point (EP) of the game, and may be non-deterministic, in that the optimal choice of strategy to use in any given play of the game is chosen randomly according to a probability distribution over all possible strategy choices.Pareto-optimality is concerned with the logical analysis of optimizing a set of strategies in a real life situation involving the interaction of more than one individual, that can be mathematically modeled as a game In this paper, the focus is on the analysis of non-zero-sum games. Another classic non-zero-sum game is “the Prisoner's Dilemma” utility gains of all possible actions are computed and plotted as a set of alternatives on a two-dimensional graph. The convex point at the vertex of the possible set of solutions corresponds to the optimal utility gain for both persons. For an n-players game, this treatment can be extrapolated to a set of alternatives on a multi-dimensional graph.The notion of an EP is the key ingredient in a game, and is what we aim to obtain. There may be more than one EP; and for a two-person zero-sum game, it is simply the set of all pairs of opposing good strategies utility can be observed in many games. In the simplest bargaining scenario presented by Nash, two cooperative individuals have a certain list of goods to barter trade utilities to each party, a point of equilibrium in trade will be reached at a certain point of time. Coal will naturally be more valued within Village 2 due to limitation, but a huge quantity of cattle will essentially not be produced if greater efforts are aimed at producing coal. It is better to trade cattle for coal since this is a less expensive alternative in terms of value that pertains to the good. Village 1 that trades coal for cattle perceives and thinks likewise. A certain equilibrium trading quantity of the two goods can be reached in a mutual trade agreement depending on the utility value of goods to each village. This equilibrium corresponds to the payoff of the two villages.The theory of utility value of an item to an individual is dictated by state of mind at a particular time. It varies according to the events that occurred to the person previously, and is evidently a random psychological parameter. Analogously, the security level of performing a specific task can also be linked to this utility value. In practice, we are all dictated by this value in determining our actions or performing any type of task assigned to us. For instance, a shopper may decide to buy a tin of cookies instead of a box of chocolates because the cookies appeal more to that person in terms of taste, price or aesthetic property, and hence, has a higher utility value in comparison. This utility value may change when the shopper tires of eating cookies after a few days and desires a taste of chocolate the next time. Desire or utility of a grocery to a shopper may also vary based on available quantity at its time of acquisition.From a psychological perspective, the It is difficult to construct a perfect model of a game that is a reflection of reality. There will always be numerous outcomes to consider even for the simplest games. For example, when volumes of items are divided for exchange, its value to each player may change. There are usually too many variables such as the bargaining abilities of the players, the norms of the society, and the variation in utility of the items over time for the formal theory to be accommodated utility gains are maximized. We assume two players - Player 1 and Player 2 who are in a position to barter goods but have no money to facilitate the exchange cooperative games in which all players have identical interests.For the bargaining problem, we illustrate how, as a special case, the two persons can perform barter trade such that their iS of mixed strategies iσ pertains to Player i, for utility gains for the players. For each pair of mixed strategies  from , the payoffs for the deployment of these strategies are denoted by Π1 and Π2 respectively. Such payoff of each mixed strategy pair corresponds to a point in the convex polytope of the super set strategy alternatives In the two-player bargaining situation, a compact convex metrical space utility gain iG is achieved is known as Pareto-optimal. Note that gain is the excess of payoff after bargaining over the initial payoff before strategy choices are chosen. Nash has shown that obtaining the maximum of the product of the two utility gains (G1 and G2) from the set of alternatives, known as the Nash product G1G2, will attain the Pareto-optimal solution for the bargaining situation. Pareto-optimality is a non-zero sum Nash game equilibrium point that determines the Pareto efficiency of the outcome. It is also worthwhile mentioning that there may be more than one equilibrium point, and this set of points can be defined as the equilibrium point (EP).An outcome is in equilibrium if there is no other possible agreement that allows both players to have higher payoffs simultaneously utility gains is maximized at its vertex. In practice, we aim to optimize the Nash product 1G2G.The combinatorial plot for this bargaining situation is illustrated in The solution is a Pareto-optimal outcome, in which the joint profits by all parties are maximized. It may be worthwhile noting that the Nash equilibrium is not unique as multiple or even an infinite number of strategies that pertain to the Nash equilibria exist. In this game theoretical setup, all items are discrete, and the discreteness of the payoffs increases the complexity of attaining the solution.Based on a multi-player bargaining situation, establishing the payoff matrix containing the super set of strategy choice alternatives is computationally expensive. It is to be noted that proving the existence of the Pareto-optimal equilibrium point and finding the solution set at the equilibrium are different tasks. In general, computation of a Pareto-optimal equilibrium point in mixed strategies of a finite game poses a numerical challenge for the following reason. For multiple players, determining the Pareto-optimal equilibrium in mixed strategies amounts to solving a system of multivariate polynomial equations of high order utility of strategy executions geometrically is used to answer the fundamental question in the Pareto-optimal equilibrium solution. Obtaining information regarding the strategy options and payoffs to every player, and with respect to other players, is crucial to the computation of the Pareto-optimal EP. This information can be represented spatially in a geometrical framework. It turns out that by using the spatial game setup, the extraction of the subset of strategy alternatives from the superset can be achieved and the convergence to the Pareto-optimal solution using less computation can be obtained. Such a concept forms the main basis of this paper, and we will examine the game theoretical geometry in greater details with the support of case studies.The concept of representing n-player games as payoff vectors such that points exist in an n-dimensional utility space utility space for n-person game models to date. In the spatial game model that they have created, outcomes of the games have been assumed to lie in some low-dimensional Euclidean space such that utilities to the players are defined in terms of distance from their most preferred, or ideal points utility is a decreasing function of the distance between the achieved outcome and the ideal point The game theoretical studies by von Neumann and Morgenstern (1980) is based on the representation of all outcomes of m-dimensional spatial game is described as a collection of n points iP, i = 1,…,n, in m-dimensional Euclidean space mR. Point iP is player i's ideal point. The convex hull of the points mR are the items in the game that we have discussed. The players are to choose among all the items in the game what they wish to own. It is assumed that a player may be most satisfied with an item at iP, however failing to obtain that, the next possible closest item will be chosen.For example, their n>2). The fundamental concepts of representing utility as a distance in space will be presented.The next section will focus on presenting a more refined technique for determining the Pareto-optimality of a bargaining problem for multi-players  that are presented as vertices of this spatial volume. For example, based on three players, the items will lie within an enclosed triangle. With four players, the items are enclosed by a tetrahedron with four vertices. Based on a five-player game, the positioning of items is within a space enclosed by a pyramid. In general, n number of players will result in an n-polyhedron defined by the ideal position of all players representing its vertices. The distance between player-to-player decreases as more players participate because for an item x, its normalized and relative distances to all the players  add up to a unitary value. Therefore, the space enclosed by the polyhedron becomes smaller as the number of players increases.The convex polytope of a multi-player game can be simplified to a three dimensional spatial representation with the player-to-player distances defined to be constant as shown in We introduce the following notation:i,xu represents the utility of item x to player i;i,xd represents the normalized distance of item x to player i with respect to other players;utility. The inverse proportionality function is used. Here, the distance of item x to player i is defined to be the inverse of i,xu. Next, based on every item, its normalized distance is given by the ratio of distance for player i to the sum of distances for all players. The following equation presents the geometrical parameter asThe distance of an item to the player is defined as a decreasing function of the normalized distances to every player in ascending order. Here, a list of items ordered in terms of priority for player i is denoted as normalized distance contains information regarding the priority of the item to one player with respect to the others based on the geometrical treatment presented in Section 3.1.1. The priority of an item is an indication of its value or importance to the player, and is the inverse of this normalized distance. Therefore, assigned items in terms of priority is defined here as a possession list of items in descending order of importance to a player.The items are arranged spatially based on their priorities to a player shifts the equilibrium away from the Pareto-optimality. On the contrary, assignment of items with higher priorities converge the solution set towards an EP. Assignment of items to a player by traversing them from the highest priority to the lowest one enhances computational resources. Therefore, instead of using a combinatorial technique to obtain the optimal product of gain for all players, a more efficient assignment approach based on priority of items can be applied. Items can be spatially arranged in terms of value for every player in the game. An item priority to a player is based on the normalized distance with respect to all players.Combinatorial analysis for all alternatives is computationally expensive. Assigning items of lower In summary, the method of assigning items focuses on considering a set of alternatives that lies close to the real solution set. In a way, it eliminates the redundant computation of alternatives that deviates from the EP. In Section 4, we demonstrate that the EP solution set lies within the reduced set of alternatives.n players, such assignment of items is performed n times.It is computationally expensive to consider all bargaining alternatives from a combinatorial set. This section describes a technique for evaluating a bargaining game via item assignment to each player. It analyzes the game based on multiple cases of item assignment by taking into account one player that pertains to each case. For utility is related to priority. Since priority is the inverse of normalized distance of a bargain item, a property that relates to the payoff per priority is introduced here. To achieve this, we will discuss this indicator from a mechanical perspective. For a mechanical lever system, the moment Q is defined as the product d, where d is the (perpendicular) distance between Q and the line of action L of Q is given by The ixutilitynormalized distanceutility-distance product of an item value (which is related to its importance in terms of payoff to the player) is based on distance d from a player spatially and in vector form. We denote this entity asn players, using each player as a pivot point, the importance of an item can be weighing about n number of pivots such that their utility-distance product vectors have the same magnitude.Assume two players ). Here, i,xP represents an item x in the order of priority for a player. Cumulative values of utility-distance products are determined based on the list k items in descending order of priority. Here, x in this list. The possession list of every player is initialized to null. We define i respectively. The items are traversed in the high to low priority direction, and assigned to player i's possession list until the cumulative utility-distance products equals utility-distance products up to a point of equilibrium (Equation (6)). The item that corresponds to the pivot location is hereby denoted as i forms a strategy set iσ. Repeating such an assignment for n players forms n strategy sets, {σ1, σ2, …, nσ}, whereby this n-tuple of strategies forms the negotiation set of the game. Pareto-optimal equilibrium is attained when no player can increase their payoff any more by changing strategy and assuming that none of the other players is going to change their strategies.For utility gain of a typical transaction is analogous to the payoff of executing a particular strategy. The proposed case studies will only work for relative payoff values for the players. In this paper, we denote the payoff as a function of item x, whereby Πi (i and k number of items. To limit the EP to only one solution (assuming that no two or more items are similar), the optimality condition is defined in this paper as a function of the product of gains.The multi-player bargaining scenario is a cooperative game; information is shared and every member trades in such a manner that their strategies will mutually benefit all the players in the game. The existence of a transferable commodity is assumed to be present. From the bargaining perspective, the items of transaction are analogous to the strategy choices, and the utility gains are positive provided that the initial total payoff value of any one list does not exceed that of the created solution possession list. Assume that the initial payoff for Player i is Πi′. Then for n players, the product of payoff gain is given byi such that Πi is greater than Πi′, positive payoff by all persons is achieved. Negative gain situations can be prevented by first assigning the items starting from those with higher priorities in the ordered list based on normalized distances until their aggregate payoff values equal or exceed the initial payoff value. The remaining items are then assigned again starting from the one with the next highest priority.We have assumed that the items are being assigned based on non-initialized possession lists. The A primitive setup of a two-village bargaining problem is defined here, in which Villages 1 and 2 possess goods to perform barter trade , with thutility sums for the villages arenormalized distances and utility-distance product for Villages 1 and 2 (denoted as i = 1 and 2 respectively) are presented.The initial values of the utility-distance products of the goods are listed in priority for Village 1 that is ordered from left to right. For Village 2, the cumulative operation starts in the opposite direction. The geometrical treatment gives x = 3). An EP set is determined here: all goods of higher priority to Village 1 up till before utility gains are established using a different data set. An example using a data set with a significantly different total utility values between the two players can illustrate the problem.The equilibrium condition isutilities for initial goods of possession. If one village has a final utility sum that is lower than the initial total utility value, then the product of utility gain is negative. This usually occurs when one village has a significantly larger possession list in comparison with the other one. Let us consider the case of having a larger difference in sum of utilities for initial goods. Assume that Villages 1 and 2 have different goods as shown by It is assumed that Villages 1 and 2 have goods in their initial possession list with a difference in sum of utility gains with optimality by assigning of goods a priori. Referring to the priority ordering, and listing the cumulative utility values for Villages 1 and 2, some of the goods can be assigned a priori in The initial values of the utility sums for the villages areutility-distance products for Village 1 and 2 to give utilities and gains areutility-distance products for the goods of bargain that can be initialized a priori.Here, a possession list that pertains to Village 1 comprises of Iron, Salt, Steel, Wine, and Mineral, while that of Village 2 consists of Coal only. The remaining goods are Cattle, Oil, and Gas. These goods can be assigned by determining the n players. In general, this method facilitates an ordered priority list for every player when determining the equilibrium point. This concept can be extended to the calculation of bargaining solution for multiple villages involved in the exchange of their produce limited by our assumptions. As only one village needs to be considered at a time for good assignment, we are able to determine the assignment of bargaining goods towards attaining EP and maximization of gains.The geometrical framework for a bargaining game by two players can be generalized to in = 3). The normalized distances and utility-distance products of initially possessed goods are presented in utility values of the goods for each village spatially on a two-dimensional plane. utility-distance product of each item to the respective player. The goods are positioned at various loci of the enclosed triangle. Here, the locus is based on the distances from item to each player vertex.Suppose that three villages  demonstrates that equilibrium can be attained by balancing the utility-distance product values of all goods among the three villages. Payoff in terms of utility is arbitrarily set to present a barter trade scenario here as a case study. In practice, the game is based on an arbitrary number of players in order to demonstrate the fundamental principles of the technique without incurring further complexities that may arise if more players are used.The result of assignment is displayed in The solution to a bargaining problem is reflected as the Pareto-optimal EP of the set of bargaining alternatives. The combination of possible sets escalates when the number of strategy choices and players increases. To reduce the number of alternatives considered in a combinatorial set, the concept of using spatial distance to represent the significance of the item to the player in a non-zero-sum game can be implemented.utility of items is introduced. Then, this technique can extract a smaller set of alternatives from the super set of strategy alternatives and enables the Pareto-optimal EP to be obtained from this reduced set. The attainment of the solution is determined by the nature of the strategy choices. Instead of using multivariate polynomial functions, the geometrical approach reduces the computational expenses involved in determining EP. For instance, the concept of shifting the bargaining outcome towards Pareto-optimal equilibrium is by geometrically positioning items of higher priority to one player with respect to the others using a shorter relative distance.To allow assignment of items while considering only one player at a time, the concept of using the geometrical distance to represent utility can be used to derive the Pareto-optimal EP in non-zero-sum games. This method relies on the concept of spatial distribution of items respective to its level of significance to the players involved. The efficiency of calculating the EP for n- players in a game has been greatly improved, but there are a few limitations that have yet to be resolved such as the definition of an optimality equation in the bargaining game. This is a very important tool as the EP of a large-scale game is the main objective of almost all the real games in the world.Spatial representation of items based on their


Anopheles gambiae and An. arabiensis with the Ifakara Tent Trap-model B (ITT-B) correlate better with human landing catches than any other method but fail to reduce the proportion of blood-fed mosquito caught, which indicates that users are exposed to bites during collection. An improved C model (ITT-C) was developed and evaluated by comparing with ITT-B in semi-field and full-field conditions in southern Tanzania. The sensitivity of the ITT-C was approximately two times that of the ITT-B: relative rate  = 1.92 (1.52–2.42), 1.90 (1.48–2.43), and 2.30 (1.54–3.30) for field populations of An. arabiensis, Culex spp., and Mansonia spp., respectively. The ITT-C caught 73% less blood-fed An. arabiensis than the ITT-B in open field experiments and none in semi-field experiments, which confirmed that the C design is a safe trapping method. Validation of ITT-C by comparison with human landing catches and parasitologic measures of human infection status may be necessary to confirm that this design produces consistent and epidemiologically meaningful results.Catches of  Anopheles vectors of human malaria all have significant limitations, particularly where densities of malaria-transmitting mosquitoes are low.In the drive to eliminate malaria, mosquito sampling measures are crucial for monitoring changes in human exposure to infections and the effect of vector-control interventions.A new device for sampling malaria vectors in Africa, called the Ifakara Tent Trap-design B (ITT-B), has recently been developed and evaluated as a means to catch malaria vector mosquitoes under conditions of low and high mosquito densities in Tanzania.This study reports an evaluation of the mosquito sampling properties of an improved C model of the Ifakara Tent Trap (ITT-C), compared with ITT-B to confirm that this new version is comparably efficacious and successfully prevents operator exposure to mosquito bites.Anopheles arabiensis is the dominant malaria vector in the area.Plasmodium falciparum malaria transmission with an entomologic inoculation rate exceeding 500 infectious bites per person per year, in spite of high coverage with mainly untreated bed nets.21The field study was conducted in Lupiro village in the Kilombero River Valley in Tanzania. Detailed description of the area is found elsewhere,2 chambers of a screen house at the Ifakara Health Institute,21The semi-field system or screen house is an enclosed structure with walls of mosquito netting and a polyethylene roof located within the natural ecosystem of the target vector.www.ajtmh.org).The Ifakara B and C traps were the only traps used. Although the ITT-B design has been described in detail,pm to 7:00 am.Four outdoor catching stations were selected approximately 50 meters apart and aligned approximately 100 m from the main rice irrigation area on one side and approximately 15 meters from local houses on other side. Each collector was assigned to and remained associated with a specific sampling station throughout the experiment to control for the effect of differences in individual attractiveness and of a particular station. Two pairs of Ifakara B and C traps were allocated to all four catching stations and a cross-over experimental design was implemented in which each trapping method was exchanged between the two adjacent catching stations on each experimental night. This experiment was conducted for 10 nights  at a time when there was no rainfall. Mosquitoes were collected by both techniques from 8:00 An. gambiae sensu stricto were released from the central release point at 7:00 pm each night and mosquitoes were collected from 7:00 pm to 7:00 am for four nights .Two sampling stations approximately 16 meters apart inside a screen houseAn. gambiae sensu lato ) for An. gambiae s.l., which appeared to be normally distributed, was treated as the dependent variable with an identity link function.Although the goal of this study was to test whether the ITT-C is an exposure-free mosquito sampling method, it was also essential to confirm that it is as sensitive as the ITT-B. Using SPSS version 15 software , we applied generalized estimating equations to quantify the influence of trap design upon mosquito catches by treating station and date as subject and within-subject variables, respectively. The logarithmically transformed catches  or not , with trap design as an independent categorical factor in the model.19Binary logistic regression analysis was used to test for differences in the distribution of abdominal status of mosquitoes from the ®; Roche, Basel, Switzerland) the recommended first-line treatment of malaria in Tanzania.Prior to any field work, research clearance was obtained from the Ifakara Health Institute Ethics Review Committee and the Medical Research Coordination Committee of the National Institute of Medical Research in Tanzania (Reference nos. NIMR/HQ/R.8a/Vol.IX/279 and 324). Informed consent was obtained in writing from all participants before initiation of the study and re-confirmed on each experimental night. These volunteers were screened for malaria parasites by microscopy during recruitment and after finishing the experiment. Those persons who were found to be malaria positive were offered treatment free of charge with artemisinin-lumefantrane  and 3% (11) were An. arabiensis and An. gambiae sensu stricto, respectively. This finding implies that An. arabiensis is the main malaria-transmitting vector in this locality. Therefore, the results presented relating to the An. gambiae s.l. species complex overwhelming reflect the response of this particular sibling species to these traps.Of 366 successfully amplified specimens of An. gambiae s.l. than the ITT-B in the field and none were caught with the ITT-C in the semi-field experiment .The ITT-C caught 73% less blood-fed periment . The obsperiment , these rWe demonstrated that modifying the ITT-B improved this prototype beyond our primary target of preventing operator exposure from mosquito bites. The ITT-C sampled twice as many mosquitoes as the ITT-B, which suggests that it may yield mosquito catches more or less equivalent to that of the human landing catches based on previous comparisons of the latter two methods.The high proportion of blood-fed mosquitoes caught with the ITT-B matches observations in previous studies.A pilot community-based surveillance system using ITT-B in urban Dar es Salaam has already proven to be representative, affordable, and effective in terms of mosquito catch and species composition.28Any alternative mosquito sampling tool, apart from being safe and sensitive, must also yield epidemiologically representative estimates of human exposure to mosquito bites and pathogen transmission.


All tumours were stained for PSA protein expression. Significantly more tumours exhibited AR amplification following the development of hormone resistance  compared to matched hormone-sensitive tumours from the same patient  (P=0.0085). The level of AR expression was significantly higher in hormone-resistant tumours compared to matched hormone-sensitive tumours from the same patient . AR expression levels in hormone-resistant tumours with and without AR amplification were not significantly different. However, an increase in AR expression was seen with the development of AR amplification in paired tumours. The rate of AR gene amplification and/or an increase in AR protein expression during androgen resistant is too low to wholly explain the development of androgen resistance. Alternative mechanisms for modulating the function of the AR, or other signalling pathways, must be considered as key factors in the development of hormone-resistant prostate.This study examined androgen receptor (AR) gene amplification and protein expression in 102 matched paired hormone sensitive and resistant tumours from 51 patients. AR gene amplification and X chromosome copy number were assessed by fluorescent  Prostate cancer is the second most frequent cause of male cancer-related deaths  were retrospectively selected for analysis; all tumours had patient identification removed including block number and hospital number and were coded in order to make the database anonymous. In 20 cases, AR fluoresence μm) cut from archival formalin-fixed, paraffin-embedded tissue were placed on aminopropyltriethoxysilane (silane)-treated slides. The slides were pretreated on a VP2000 robotic slide processor , as previously described14 using dual labelling .Sections . Amplification was defined as an AR : X ratio greater than 1.5 (μg ml−1) or AR  was used as the primary antibodies. In each case, an isotype-matched antibody was used as a negative control in which no staining was observed. Positive control sections were included in each IHC assay. Bound antibody was visualised by the streptavidin–biotin  method including diaminobenzidine as a chromogen (DAKO).Prostate-specific antigen and AR protein expression was determined in paraffin-embedded tissue sections by IHC with a standard immunoperoxidase procedure. Antigen retrial was performed by microwaving under pressure for 5 min in TE buffer. A purified immunoglobulin fraction of either a rabbit antiserum to human PSA  and AR MHS or PSA MHS and AR MHS and Gleason sum were calculated using the Spearman rank test.The Fisher's exact test was used to compare FISH results pre- and post-hormone resistance. Prostate-specific antigen protein expression and AR protein expression are shown as median and interquartile ranges. Paired Student's P<0.001)  (P=0.46) had increased copies of the X chromosome (P=0.075) showed evidence of increased AR gene copies    with oveFour samples did not express AR protein. In one of the hormone-resistant cases, the previous hormone-sensitive biopsy showed both AR amplification (2.9) and a mean AR histoscore of 140. Following the development of androgen resistance, the AR amplification ratio did not change (3.09); however, no AR expression was detected in either of the two post-treatment biopsies from this patient. PSA expression remained unchanged in the transition from hormone-sensitive to hormone-resistant disease .AR expression levels in hormone-resistant tumours with and without AR amplification are not significantly different , the level of PSA expression was significantly lower in hormone-resistant tumours compared to matched hormone-sensitive tumours P=0.018; .P=0.0085); however, this is only observed in a proportion of cases. Based on our results and those from previous studies between 70 and 80% of cases with hormone relapse must therefore involve mechanisms other than amplification of the AR. While patients with AR gene amplification may represent a readily identifiable, and possibly treatable, group more research should be focused on alternative mechanisms of hormone escape. This is especially relevant as we have also identified a patient with AR amplification in the hormone-sensitive tumour, which responds fully to therapy, suggesting that AR amplification does not preclude a response to androgen deprivation therapy. In the light of this observation it is possible, though at present unproven and to our view unlikely, that AR amplification does not mediate hormone resistance.Androgen receptor gene amplifications are uncommon in hormone-sensitive tumours and are present in 20–30% of hormone-resistant tumours (P=0.019) and that 80% of cases in which AR amplification was observed also exhibited an increase in AR expression. However, an increase in AR expression was also seen in 35% of cases that did not develop AR amplification. This suggests that although an increase in AR expression is associated with AR amplification, an increase in expression may also be due to alternative mechanisms, for example, decrease in protein degradation or an increase in protein stabilisation. Almost half of all patients do not show any increase in AR expression during the development of hormone-resistant disease. We also identified a patient with AR amplification in both hormone-sensitive and hormone-resistant tumours and three cases with loss of AR protein expression at the development of hormone resistance, as observed by It is hypothesised that AR gene amplification is involved in the development of hormone-resistant prostate cancer due to amplification, resulting in an increase in AR protein expression (P=0.018), this decrease was marginal with considerable overlap between values for pre- and post-hormone-resistant tumours (While PSA expression was significantly lower in hormone-sensitive tumours compared to hormone-resistant tumours ( tumours . The sigThis is the first study to our knowledge that has correlated AR gene amplification, AR protein expression and PSA protein expression in matched hormone-sensitive and hormone-resistant prostate cancer. We have confirmed that AR gene amplification is associated with the development of hormone resistance in 20% of patients and this is related to an increase in AR expression in the majority of cases. We have also demonstrated that this is not the only mechanism by which AR expression is increased and also that in almost 60% of cases, AR expression remains unchanged or decreases. Therefore, in these cases, hormone resistance must be due to alternative mechanisms of AR activation or may be due to mechanisms that can stimulate AR responsive genes independent of the AR.