PATHOGENESIS-RELATED (PR) genes. Mechanistically, NPR1 is the core of a transcription complex interacting with TGA transcription factors and NIM1-INTERACTING (NIMIN) proteins. Arabidopsis NIMIN1 has been shown to suppress NPR1 activity in transgenic plants. The ArabidopsisNIMIN family comprises four structurally related, yet distinct members. Here, we show that NIMIN1, NIMIN2, and NIMIN3 are expressed differentially, and that the encoded proteins affect expression of the SAR marker PR-1 differentially. NIMIN3 is expressed constitutively at a low level, but NIMIN2 and NIMIN1 are both responsive to SA. While NIMIN2 is an immediate early SA-induced and NPR1-independent gene, NIMIN1 is activated after NIMIN2, but clearly before PR-1. Notably, NIMIN1, like PR-1, depends on NPR1. In a transient assay system, NIMIN3 suppresses SA-induced PR-1 expression, albeit to a lesser extent than NIMIN1, whereas NIMIN2 does not negatively affect PR-1 gene activation. Furthermore, although binding to the same domain in the C-terminus, NIMIN1 and NIMIN2 interact differentially with NPR1, thus providing a molecular basis for their opposing effects on NPR1. Together, our data suggest that the Arabidopsis NIMIN proteins are regulators of the SAR response. We propose that NIMINs act in a strictly consecutive and SA-regulated manner on the SA sensor protein NPR1, enabling NPR1 to monitor progressing threat by pathogens and to promote appropriate defense gene activation at distinct stages of SAR. In this scenario, the defense gene PR-1 is repressed at the onset of SAR by SA-induced, yet instable NIMIN1.NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) is the central regulator of the pathogen defense reaction systemic acquired resistance (SAR). NPR1 acts by sensing the SAR signal molecule salicylic acid (SA) to induce expression of  PATHOGENESIS-RELATED (PR) genes  and benzothiadiazole-7-carbothioic acid S-methyl ester  genes . Some PRster BTH;. Furtherplay SAR , 1993. TNON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1). The gene was identified from Arabidopsis mutants compromised in chemical induction of PR genes and in resistance to fungal infection  and two NPR1 family members from tobacco (Nt) alter some of their biochemical capabilities in response to the SA signal molecule in a heterologous yeast system in absence of any other plant protein . One domain comprises the penta-amino acid motif LENRV (amino acids 429–433). The LENRV motif imposes SA sensitivity on NPR1 proteins from Arabidopsis and tobacco in yeast. The signature is altered in the non-functional nim1-4 mutant  proteins , a NIMIN homolog from rice, are able to suppress induction of PR genes and to cause enhanced susceptibility to bacterial pathogens in transgenic plants  analyses. The primers used and the sizes of fragments generated by PCR from plasmids carrying cDNAs for NIMIN3 and various control genes are listed in Table 1. NIMIN3 transcript levels were compared to expression of the NIMIN1, NIMIN2, and PR-1 genes. Unlike NIMIN1, NIMIN2, and PR-1, expression of NIMIN3 was neither induced by SA nor BTH . Moreover, jasmonate (JA), another plant defense signal, had no effect on either of the NIMIN genes (data not shown). However, we were able to detect NIMIN3 transcripts in several independent RNA preparations irrespective of whether they had been isolated from control or chemically induced plant tissue , suggesting that NIMIN3 may be expressed constitutively at a low level. To address this question, we isolated 1.4 kb of the NIMIN3 5′-upstream region and fused it to the β -glucuronidase (GUS) reporter gene. The chimeric gene was transferred to the tobacco genome, and GUS enzyme activity was determined in seven independent primary transformants, all containing intact copies of the reporter gene construct (data not shown). As compared to transgenic tobacco plants carrying analogous NIMIN1Pro::GUS or ProNIMIN2::GUS constructs . Reporter gene expression from the ProNIMIN3::GUS construct in the tobacco genome was not enhanced significantly by treatment of plants with SA , BTH (0.34 mM), methyl JA , or H2O2 . Likewise, gene expression from the NIMIN3 promoter was not elevated by elicitation of HR or by exogenous application of the phytohormones 2,4-dichlorophenoxy acetic acid , gibberellic acid (GA), indole-3-acetic acid (IAA), or 1-naphthalene acetic acid . As determined by histochemical staining, NIMIN3-mediated reporter enzyme activity is mainly localized in leaf tissue . Of note, NIMIN3 gene expression is independent from an intact NPR1 gene .Previously, we have shown that tivation . To furtFigure 1A had shown that NIMIN1 was expressed only after induction, just as PR-1, while NIMIN2 expression was occasionally observed prior to chemical treatment of plants. This finding was unexpected since the NIMIN2 promoter exhibits clear chemical induction in transgenic tobacco plants . Yet, NIMIN2 expression was clearly detectable in both npr1 mutants, although, in some experiments, NIMIN2 transcript levels appeared to accumulate to lower overall levels in npr1 than in wild-type plants . Our data are in conflict with another report.NIMIN1 and NIMIN2 is abolished in the npr1-1 mutant. To support our results, we verified the identity of the NIMIN2 RT-PCR products by digestion with restriction enzymes (data not shown). Hence, NIMIN2 expression, unlike NIMIN1 and PR-1 expression, may be either independent or only partly dependent on NPR1. Furthermore, the kinetics of gene induction turned out to be different between NIMIN1 and NIMIN2. Both genes are expressed transiently after SA application . Yet, NIMIN2 gene expression started immediately (0.5 h) after SA treatment, reached its maximum early (after 1 h) and was maintained at a high level for 24 h . Thus, NIMIN2 seems to be an immediate early SA responsive gene, as suggested previously for the tobacco NIMIN2a gene . This is clearly later than the onset of NIMIN2 expression, yet earlier than the onset of PR-1 induction. Notably, NIMIN1 expression appeared even more transient than NIMIN2 expression and was already shut down when PR-1 transcripts began to accumulate. The time course of NIMIN1 gene induction shown here is in accordance with previous results obtained by northern blotting . GUS enzyme activity was already switched on to high levels early after germination. In contrast, PR-1a promoter activation and accumulation of the endogenous PR-1 proteins occurred with significant delay . Thus, the kinetics of reporter gene activation from the NIMIN1 and NIMIN2 promoters in SA-treated tobacco seem to parallel the transcript accumulation patterns observed in Arabidopsis, i.e., NIMIN genes are induced by SA prior to PR-1 genes. The data indicate that the molecular cues for early induction during the SAR response are contained within the 1 kb 5′-flanking regions of NIMIN1 and NIMIN2, and that these cues are recognized in the heterologous species tobacco. Reporter gene expression from both the NIMIN1 and NIMIN2 promoters occurred in leaf and root tissue . Likewise, green fluorescent protein (GFP) expression from the 0.8 kb NIMIN1 promoter has been observed in roots, petioles, and leaves in transgenic Arabidopsis plants .The kinetics of gene induction were also monitored in tobacco seedlings containing s plants . This exNIMIN gene expression at different times during the SAR response, we have developed an in planta assay for NIMIN activity. The gene coding for GUS under control of the tobacco PR-1a promoter . One typical line (3 GUS units uninduced and 1100 GUS units after SA treatment) was propagated, and T2 plants were used for infiltration experiments with an Agrobacterium strain carrying the gene for GFP (mGFP4) driven by the Cauliflower mosaic virus (CaMV) 35S RNA promoter (Pro35S::mGFP4). Infiltration of Pro35S::mGFP4 Agrobacteria yielded GUS enzyme activities only slightly above the background levels of non-infiltrated control leaves, showing that agroinfiltration alone is not sufficient for efficient activation of the ProPR-1a::GUS reporter gene .To unravel the functional significance of PR-1a gene induction after agroinfiltration of N. benthamiana. It has been shown previously that overexpression of NIMIN1 suppresses SA-mediated PR gene induction and SAR in transgenic Arabidopsis plants . The excised leaf disks were then floated for 2 days on water or on a 1 mM SA solution. As controls, disks from non-agroinfiltrated leaves were incubated on water and SA. After floating, proteins were extracted from leaf tissue, and GUS reporter activity was determined. In other experiments, we have infiltrated the two halves of a single leaf with Agrobacterium strains harboring different constructs in order to allow an even more direct comparison between effects exerted by the respective NIMIN proteins. Consistent with what has been described for NIMIN1 overexpression in transgenic Arabidopsis plants, agroinfiltration of Pro35S::NIMIN1 bacteria suppressed SA-mediated PR-1a promoter activation to nearly background levels as compared to GUS levels observed in GFP expressing leaf disks floated on water . Quite surprisingly, NIMIN3 overexpression, too, clearly repressed GUS reporter gene induction from the Nt PR-1a promoter in N. benthamiana . Repression with NIMIN3 was, however, weaker than with NIMIN1 . The presence of NIMIN1 and NIMIN3 proteins in infiltrated N. benthamiana leaf tissue was monitored by immunodetection using specific antisera. NIMIN3 accumulated to high levels. The protein was readily detected in extracts from SA-floated leaf disks and also in extracts from agroinfiltrated tissue without SA induction . In contrast, we were not able to detect NIMIN1 expression in extracts from SA-treated leaf tissue. We therefore performed time course experiments monitoring NIMIN1 accumulation in twofold concentrated extracts from 1 to 4 days after agroinfiltration. Whereas GFP accumulated to high levels at 3 and 4 days post-inoculation , NIMIN1 protein was detected only faintly . The inability to detect high amounts of NIMIN1 in agroinfiltrated plant tissue is, however, not due to a low sensitivity of the anti-NIMIN1 serum we used. Detection of NIMIN1 and NIMIN3-Gal4 DNA binding domain (GBD) fusion proteins, which are expressed to similar levels in yeast .Next, we tested the influence of different NIMIN proteins on s plants . Howeverin yeast , was simN. benthamiana reporter line with Pro35S::NIMIN2 harboring bacteria did not repress SA-mediated induction of the ProPR-1a::GUS transgene . Expression of NIMIN2 in N. benthamiana leaf tissue was demonstrated by immunodetection using a specific antiserum directed against Nt NIMIN2a-maltose binding protein (MBP) which exhibits cross-reactivity with Arabidopsis NIMIN2 . Thus, albeit similar to each other and possessing similar NPR1 interaction motifs, NIMIN2 and NIMIN1 seem to fulfill different, even opposing, functions in the SA signal transduction pathway.Likewise surprisingly, agroinfiltration of the NIMIN genes in N. benthamiana is able to suppress induction of endogenous PR-1 genes. N. benthamiana (Nb) carries a gene for a basic PR-1 protein. The amino acid sequence for the basic PR-1 protein is co-linear with N. tabacum acidic PR-1 proteins except for a 19 amino acid-long extension at the C-terminus of Nb PR-1. In the co-linear region, the identity (similarity) between the basic Nb PR-1 protein and Nt PR-1a is 64% (87%). Consequently, using an antiserum raised against Nt PR-1a, we were able to detect a PR-1-related protein exhibiting a slightly higher molecular weight than the acidic Nt PR-1 proteins in extracts from N. benthamiana leaf disks floated on 1 mM SA (data not shown). SA induction of this protein was clearly suppressed in N. benthamiana tissue overexpressing NIMIN1 or NIMIN3, but not in tissue overexpressing NIMIN2 .We also tested whether transient expression of At NIMIN genes and differential effects of NIMIN proteins on PR-1 induction strongly suggested that NIMINs serve unique functions at specific time points during the SAR response in Arabidopsis, an assumption fully consistent with our previous observation that NIMIN3 and NIMIN1/NIMIN2 bind to physically separate regions of At NPR1  screen with the At NPR1 bait . Truncated NIMIN1 or NIMIN2 including their NPR1 interaction motif were expressed as fusions with the Gal4 transcription activation domain (GAD), and full-length NIMIN1 or NIMIN2 were expressed from the Met25 promoter, which is repressed in presence and de-repressed in absence of methionine. NPR1 was expressed as GBD fusion. The interactions of NIMIN1 or NIMIN2 with NPR1 were disrupted in presence of NIMIN2 or NIMIN1, respectively . Furthermore, complex formation between NPR1 and NIMIN1 was clearly dependent on the concentration of NIMIN2 . Together, the data suggest that NIMIN1 and NIMIN2 may compete for the same binding site on NPR1.Initially, we tested whether NIMIN1 and NIMIN2 can bind together to NPR1 in Y3H assays. Both proteins possess similar NPR1 interaction motifs by which they bind to the C-terminus of NPR1 . Hence, NIMIN3 binding to NPR1 seems to inhibit NIMIN1/NIMIN2 interaction, and simultaneous binding of NIMIN1, NIMIN2, and NIMIN3 to NPR1 may exclude each other.Next, we asked whether NIMIN1 or NIMIN2 can bind to NPR1 in presence of NIMIN3 which interacts with NPR1 via a site distant from the NIMIN1/NIMIN2 binding site . NIMIN1 Arabidopsis, regarding both the sequence and its position within the amino acid chain . To test whether both NIMIN1 and NIMIN2 bind to this region in At NPR1 and whether binding occurs in a similar fashion, we introduced mutations F507S and F508S into At NPR1. Nt NPR1 F505/506S is no longer able to interact with Nt NIMIN2a or Nt NIMIN2c .In tobacco NPR1, binding of NIMIN2 proteins occurs in the region from amino acids 494 to 510 . The dom NIMIN2c . Similar50 ≈ 20 μM SA;Arabidopsis NPR1 with TGA factors is not diminished with SA . Quite surprisingly, however, ternary complexes comprising NIMIN2 proved to be stable in presence of SA . Thus, although possessing similar NPR1 interaction motifs and binding to the same site in the C-terminus of NPR1, NIMIN1, and NIMIN2 can form complexes with NPR1 and TGA factors exhibiting differential sensitivity to SA, implying that these two NIMIN proteins interact differentially with NPR1 in transcription complexes on PR gene promoters.We then analyzed the relations of NPR1 with NIMIN1 and NIMIN2 in ternary protein complexes including TGA transcription factors. We have shown previously that SA administered to growth medium impairs formation of NPR1–NIMIN1 and NPR1–NIMIN2 complexes in Y2H assays, and that the sensitivity of loss of protein–protein interaction is very similar for both NIMIN1 and NIMIN2  motif at their C-terminus, and NIMIN1 and NIMIN2 possess a common motif for interaction with a domain in the C-terminus of Arabidopsis and tobacco NPR1. On the other hand, NIMIN1 and NIMIN3 have been reported to share a conserved PA/SFQPEDF signature . It is of interest, however, to note that binding of At NPR1 to NIMIN3 occurs within the 60 amino acid-long C-terminal half including the EDF motif  have been described in the databanks, underscoring our assumption that the EDF domain may have functional relevance also in tobacco. The mechanism by which the EDF domain in NIMIN proteins could suppress PR-1 gene induction remains, however, elusive. Alternatively, suppression of PR-1 induction in N. benthamiana by NIMIN3 and NIMIN1 may occur via the C-terminal LxLxL/EAR motif which has been implicated in recruiting the transcriptional co-repressor TOPLESS . While NIMIN3 represses inadvertent PR gene activation in unchallenged plants, NIMIN2 is induced at low tissue levels of SA to relieve NIMIN3 repression by binding to the NPR1 C-terminus. This process may allow activation of early SA- and NPR1-dependent genes, e.g., NIMIN1. Interaction of NIMIN2 with the NPR1 C-terminus does not, however, appear to be sufficient to activate substantial expression of the late SAR gene PR-1. NIMIN2 action on NPR1 is transient and is followed by NIMIN1 replacing NIMIN2. NIMIN1 suppresses activation of NPR1-dependent SAR genes. NIMIN1 action on NPR1 seems even more transient than NIMIN2 action, and instability of NIMIN1 protein would be a crucial prerequisite for relief of PR-1 gene repression. In this scenario, late SAR genes would be activated through direct action of SA on NPR1  Heynh. Col-0 genomic DNA using primers N3-P2 (5′-TTAAGCTTATACGGGACATAGTGCACAGCC) and N3-P1 (5′-AAGGATCCTGAACCGCTCTCTCTTCCTTCC). N3-P1 primes immediately upstream of the ATG translation start codon of NIMIN3. The resulting 1.4 kb fragment was ligated to HindIII/BamHI cleaved pBin19/35SPro::GUS from which the 35S RNA promoter had been removed.The ic genes . The NIMNPR1. To this clone, the N-terminal At NPR1 sequence was added as a 1.4 kb BamHI/DraIII fragment, and the complete mutant sequence was ligated to BamHI/SalI cleaved pGBT9 and pGAD424.To map the NIMIN1/NIMIN2 binding site in At NPR1, Phe-507 and Phe-508 were mutated to Ser using overlap extension PCR . The priAll clones generated by PCR amplification were verified by DNA sequence analysis.Figure 2B, RT-PCR assays were conducted to give approximately equal amounts of reaction products in order to enable direct comparison of NIMIN1, NIMIN2, and PR-1 transcript accumulation at different time points after treatment of Arabidopsis with SA. To this end, RNAs were diluted 1:20 for RT-PCR amplification of NIMIN2 transcripts.RNA isolation and RT-PCR analyses were performed as described byN. tabacum L. cv. Samsun NN) by Agrobacterium tumefaciens was performed according toProPR-1a::GUS, Pro35S::GUS, ProNIMIN1::GUS, and ProNIMIN2::GUS have been described earlier  and for determination of SA-induced GUS activity in time course experiments , seeds from transgenic tobacco were sown on MS medium with 400 μg ml-1 kanamycin or on selective medium supplemented with 0.3 mM SA.Transformation of tobacco  of 0.5 for all strains. Agrobacteria were incubated for 2–3 h at room temperature before agroinfiltration. To suppress post-transcriptional gene silencing, the bacterial suspensions were mixed with an equal volume of a strain carrying the p19 suppressor from Tomato bushy stunt virus , when strong GFP fluorescence was observed. At this point of time, bacterial titers were similar in leaf tissue agroinfiltrated with strains Pro35S::mGFP4, Pro35S::NIMIN1, or Pro35S::NIMIN2 . Furthermore, co-overexpression of mGFP4 and NIMIN1 produced the same levels of GFP fluorescence and of GFP protein accumulation as overexpression of mGFP4 alone .The pBin19 gene constructs were transferred by triparental mating to nt virus . Four too::mGFP4  were usein situ were performed as described previously  gels.Determination of GUS enzyme activity and histochemical localization of GUS activity eviously . GUS actNIMIN genes in N. benthamiana, two leaf disks each were punched out from non-infiltrated control or from agroinfiltrated leaf tissue at 4 or 5 dpi. Disks were floated for 2 days on water or on 1 mM SA and thereafter extracted with 150 μl GUS lysis buffer. The SA-induced reporter gene expression from the PR-1a promoter was compared in non-agroinfiltrated leaf tissue and in tissue infiltrated with Pro35S::mGFP4 and Pro35S::NIMIN chimeric genes. The same extracts were used for immunodetection of protein accumulation.To determine GUS enzyme activity after transient expression of E. coli expressed and purified proteins NIMIN1-GST, Nt NIMIN2a-MBP, and NIMIN3 according to standard procedures. PR-1 protein accumulation in N. benthamiana was detected with a specific antiserum against Nt PR-1a. For detection of GFP and GUS proteins, rabbit polyclonal antisera were used as recommended by the manufacturers . To analyze accumulation of NIMIN1 at different times after agroinfiltration , four leaf disks were harvested directly from each infiltrated tissue and extracted with 150 μl GUS lysis buffer yielding twofold concentrated extracts. SA induction of the GUS reporter protein and of an endogenous N. benthamiana PR-1 protein was compared in tissue infiltrated with Pro35S::mGFP4 and Pro35S::NIMIN chimeric genes. Equal extract volumes were loaded in each lane of an SDS gel. The loading of SDS gels for immunodetection of protein accumulation was checked by staining the nitrocellulose filters with Ponceau S (0.1% in 5% acetic acid). Alternatively, unspecific bands reacting with the antisera used are marked for demonstration of equal gel loading.Immunodetection of proteins separated by SDS gel electrophoresis was performed as described earlier . SpecifiLacZ reporter gene activities are given in Miller units. Most plasmids used in the protein–protein interaction assays have been described (Yeast two-hybrid and yeast three-hybrid analyses in absence and presence of SA were conducted as reported earlier . LacZ reescribed . pGAD10/escribed .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phimosis is nonretraction of prepuce. It is normally seen in younger children due to adhesions between prepuce and glans penis. It is termed pathologic when nonretractability is associated with local or urinary complaints attributed to the phimotic prepuce. Physicians still have the trouble to distinguish between these two types of phimosis. This ignorance leads to undue parental anxiety and wrong referrals to urologists. Circumcision was the mainstay of treatment for pathologic phimosis. With advent of newer effective and safe medical and conservative surgical techniques, circumcision is gradually getting outmoded. Parents and doctors should a be made aware of the noninvasive options for pathologic phimosis for better outcomes with minimal or no side-effects. Also differentiating features between physiologic and pathologic phimosis should be part of medical curriculum to minimise erroneous referrals for surgery. It is aPenile formation starts from 7th week of gestation and is complete by 17th week . The intAround 96% of males at birth are noticed to have a nonretractile foreskin. This is due to naturally occurring adhesions between prepuce and glans and due to narrow skin of prepuce and “frenulum breve.” This is physiological phimosis. The foreskin gradually becomes retractable over a variable period of time ranging from birth to 18 years of age or more. This is aided by erections and keratinisation of the inner epithelium . Thus prBut medical doctors are not trained enough to distinguish between these two types of phimosis , 19–22. Physiologic phimosis is the rule in newborn males. The prepuce is adhered to glans and this separates over time. Enthusiastic attempts to retract foreskin in physiological phimosiscauses microtears, infection, and bleeding with secondary scarring and true phimosis. Poor hygiene and recurrent balanitis (infection of glans penis), posthitis (inflammation of foreskin), or both could lead to difficulty in retraction of foreskin and consequent true phimosis. Diabetes mellitus predisposes to these infections due to high glucose content of urine, which is conducive for bacterial proliferation –27. PathThe incidence of pathological phimosis is 0.4 per 1000 boys per year or 0.6% of boys are affected by their 15th birthday. This is much lesser than physiological phimosis, which is common in younger children and decreases with age . PhysiolPhimosis in boys and adults can vary in severity. Meuli et al. have graded severity of phimosis into following 4 grades , namely,Diagnosis of phimosis is primarily clinical and no laboratory tests or imaging studies are required . These mWhen a child is brought with history of inability to retract the foreskin, it is important to confirm whether it is physiologic or pathologic. Management depends on age of child, type of nonretraction, severity of phimosis, cause, and associated morbid conditions.When it is certain that phimosis in the child is not pathologic, it is vital to reassure the parents on normalcy of the condition in that age group. They should be taught how to keep the foreskin and its undersurface clean and hygienic. Normal washing with lukewarm water and gentle retractions during bathing and urination makes the foreskin retractile over time . Mild soTopical steroids have been tried in cases of phimosis since more than 2 decades. Overall, studies using topical creams for phimosis have yielded dramatic results. Efficacy figures range from 65 to 95% . MechaniIn order to alleviate the concern over side-effects of topical steroids, nonsteroid anti-inflammatory ointment, diclofenac sodium thrice daily was evaluated and found to be 75% efficacious as compared to petroleum jelly, which was effective in none of the cases used . 0.1% esIf a patient has concomitant balanitis or balanoposthitis, depending on the etiology, he may be treated with topical antibiotics or antifungals . Proper In this, gentle preputial retractions are carried out by a doctor on an outpatient basis. This nonsurgical adhesiolysis is found to be effective, cheap, and safe treatment for phimosis , 62–64. These invasive measures are to be reserved for recalcitrant phimosis that fails to respond to medical management.It is a conservative alternative to traditional circumcision which is fraud with many complications, problems, and risk , 69–88. In this case, the phimotic foreskin is totally excised. Circumcision is one of the oldest elective operations known in humans. It started as a religious/ritual sacrifice . But graProlonged antibiotic therapy, intralesional steroid injection, carbon dioxide laser therapy, and radial preputioplasty alone or with intralesional injection of steroid have all been described as therapies for phimosis, but there are no proper randomised controlled trials of their efficacy and long-term outcomes.Phimosis needs to be differentiated from non-retractile prepuce, which is the rule in young children. Doctors should be taught on distinguishing these two types of phimosis in order to avoid parental anxiety and needless referrals to urologists for circumcisions. Newer nonsurgical modalities such as topical steroids and adhesiolysis are effective, safe, and cheap for phimosis in children. Parents should be made aware of these measures to treat phimosis. If surgery is indeed needed, conservative plastic surgical techniques should be performed rather than the traditional circumcision. This would help the patients, their family, and the healthcare as well as the society at large.


The androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily of ligand-inducible DNA transcription factors, and is the major mediator of male sexual development, prostate growth and the pathogenesis of prostate cancer. Cell and gene specific regulation by the AR is determined by availability of and interaction with sets of key accessory cofactors. Ski-interacting protein  is a cofactor shown to interact with several NRs and a diverse range of other transcription factors. Interestingly, SKIP as part of the spliceosome is thought to link mRNA splicing with transcription. SKIP has not been previously shown to interact with the AR.The aim of this study was to investigate whether SKIP interacts with the AR and modulates AR-dependent transcription. Here, we show by co-immunoprecipitation experiments that SKIP is in a complex with the AR. Moreover, SKIP increased 5α-dihydrotestosterone (DHT) induced N-terminal/C-terminal AR interaction from 12-fold to almost 300-fold in a two-hybrid assay, and enhanced AR ligand-independent AF-1 transactivation. SKIP augmented ligand- and AR-dependent transactivation in PC3 prostate cancer cells. Live-cell imaging revealed a fast  translocation of AR from the cytoplasm to the nucleus upon DHT-stimulation. Förster resonance energy transfer (FRET) experiments suggest a direct AR-SKIP interaction in the nucleus upon translocation.Our results suggest that SKIP interacts with AR in the nucleus and enhances AR-dependent transactivation and N/C-interaction supporting a role for SKIP as an AR co-factor. Pre-mRNA Processing 45) [SNW/SKIP proteins, which include human Ski-interacting protein SKIP; SNW1, NCOA62) and its yeast homologue the essential splicing factor Prp45  ,4. SKIP sing 45) . It alsosing 45) , and prosing 45) . A recensing 45) . This apThe fundamental effects of SKIP in transcriptional regulation are supported by its co-regulatory effect on nuclear hormone receptors, including estrogen receptor , the VitThe androgen receptor (AR) is a member of the nuclear hormone receptor superfamily that regulates male sexual development, and is a major player in the pathogenesis and progression of prostate cancer -22. Uponin vitro, suggesting that N/C-interaction is functionally important in vivo[The AR contains distinct structural domains also found in other nuclear receptor superfamily members. The amino (N)-terminal transactivation domain, which is also termed ligand-independent activation-function-1 domain (AF-1), is followed by a DNA-binding domain (DBD) that is linked via a hinge region to a carboxy (C)-terminal ligand-binding domain (LBD), which contains the activation function-2 domain (AF-2) ,26. Ligat in vivo. The AR t in vivo. Importat in vivo. Examplet in vivo,32.While AF-1 mediates hormone-independent constitutive transactivation when artificially isolated, the AF-2 is inactive in the absence of hormone, but required for strong, ligand-dependent activity in androgen-dependent prostate cancer cells -36. ThisIn this study, we demonstrate that SKIP interacts with the AR. We show that SKIP acts in multiple ways, by augmenting AR AF-1-dependent activity as a classical type I co-activator, while it also enhanced AR N/C-interaction and AR-dependent transcription in prostate cancer cells.SKIP modulates the transcriptional activity of a variety of nuclear hormone receptors. We therefore tested, whether SKIP can also act as a co-regulator of androgen receptor (AR), which is critically involved in prostate cancer ,22. To aAR transcriptional activity is modulated by ligand-mediated interdomain interaction of its N- and C-termini ,30. To aIn the absence of SKIP, the AR N- and C-terminal, ligand-dependent interaction resulted in a significant increase in relative luciferase activity by 12-fold  MMTV-luc and the prostate-specific antigen (PSA) PSA-luc luciferase reporter plasmids in a prostate cancer cell line. We used the AR-negative PC3 cell line, where we overexpressed WT AR. In these cells SKIP overexpression augmented basal ligand-independent MMTV reporter activity ~6-fold. Additional stimulation with the AR ligand DHT (0.01 to 10 nM) increased activity ~18-fold over control levels , without any apparent dose-dependence  imaging. We transiently co-expressed AR, which was C-terminally tagged with ECFP (AR-ECFP) as fluorescent donor, and SKIP, N-terminally tagged with EYFP (EYFP-SKIP) as an acceptor in BHK cells Figure . The EYFIn order to gain further insight into the temporal characteristics of this interaction, we followed the DHT induced nuclear translocation of AR-ECFP to EYFP-SKIP positive structures in live BHK cells. We used sensitised acceptor emission FRET imaging with fully crosstalk corrected FRET images . When stIn conclusion, FRET-experiments provide additional evidence that a direct and transient interaction of AR and SKIP occurs within the nucleus, which increases within minutes after DHT-stimulated nuclear translocation of AR.In this study we provide evidence that SKIP acts as a co-regulator of AR transcription. Using mammalian one- and two-hybrid-experiments, we showed that SKIP augments AR AF-1 ligand-independent transcriptional activity and AR ligand-dependent AF-1 and AF-2  transcriptional interaction. Moreover, SKIP augmented AR-dependent transcription with two ARE-containing reporters in prostate cancer PC3 cells. Immunoprecipitation and FRET-experiments showed that AR interacts with SKIP and that the AR translocates to the nucleus within minutes after ligand stimulation to interact with SKIP.FRET detects proximities of 3–7 nm for ECFP/EYFP-FRET pairs , which iOur FRET data are supported by our one- and two-hybrid data, which suggest that SKIP can activate both the N- and C-terminus of AR. Therefore, FRET-fluorophores on the C-terminus of AR and N-terminus of SKIP may come into very close proximity, despite the relatively large size of both proteins. The strong increase of FRET upon DHT-stimulated nuclear translocation of AR-ECFP to EYFP-SKIP positive structures that resemble nuclear speckles, can be explained by the fact that an excess of the acceptor, EYFP-SKIP, leads to high FRET-levels in a molecular complex, where the donor-acceptor-ratio is 1: >1 .Nuclear speckles form a functional compartment within the nucleus that include splicing factors. Active gene transcription coupled with pre-mRNA splicing is thought to occur at the periphery of the splicing factor compartment -51. It wIt is of interest that the AR AF-1 domain has been shown to interact within nuclear speckles with another splicing factor ANT-1 (homologous to yeast splicing factor Prp6p) . This diInterestingly, the SKIP-EYFP signal disappeared completely ~16 minutes after DHT mediated translocation of AR to the nucleus, suggesting AR-mediated degradation or masking of SKIP. Loss of the SKIP-EYFP signal started soon after the maximum FRET was reached, raising the possibility that a regulated degradation is triggered at high AR concentrations. The AR has been previously linked to proteasome-mediated degradation, by its interaction with ubiquitin protease USP10 , the ubiAR transcriptional activity is dependent on its N-terminal and C-terminal domains, which if artificially isolated can act in a ligand-independent (AF-1) and ligand-dependent (AF-2) manner, respectively ,34. ImmuMoreover, our two-hybrid data indicate that SKIP bi-functionally interacts with the N- and C-termini of AR, or at least facilitates this N/C- interaction. Interestingly, Saitoh et al.  observedIn conclusion, our data suggest SKIP as a novel AR co-activator that enhances its AF-1 function and interdomain-interaction, as well as AR-dependent transcription. The diverse network of interactions shown for SKIP, in particular with nuclear hormone receptors, explains its impact on several signalling pathways involved in growth and development, including possibly in AR-dependent prostate cancer cell growth.H. sapiens: embryonal kidney cells); PC3 (origin: H. sapiens: prostate cancer cells) were maintained in medium with 10% foetal calf serum and plated 24–36 hours before transfection into 24 well plates at a density of 2×104 cells (HEK293) or 2.5×104 cells (PC3) per well so that at time of transfection cells were about 75% confluent. Cells were transfected overnight with Fugene 6 (Roche) following the manufacturer’s instructions, using 1.5 μl Fugene per 0.6 μg of total plasmid DNA per well. The ligand DHT in DMEM/F12 for HEK293 cells of RPMI for PC3 cells was added to medium supplemented with 2% charcoal stripped FCS for 24 hours. Medium was removed and cells were washed once with ice cold PBS and then lysed with 2× Promega lysis buffer. Luciferase assays were performed in triplicate with the Firefly luciferase assay kit (Promega) and measured on a luminometer (Berthold LB953 Autolumat), as described previously [HEK293 cells  culture5E1bTATA-luciferase reporter [′-TTT GAA TTC ATG GCG CTC ACC AGC TTT TTA CCT GC-3′ and 5′-TTT GTC GAC TAT TCC TTC CTC CTC TTC TTG CC-3′), phosphorylated by T4 polynucleotide kinase, and cloned into the SmaI site of pEYFP-C1 (Clontech). The AR-pECFP-N1 plasmid was as previously described [SKIP-pSG5, SKIP-pM, SKIP-pCGN were previously described ,8. All mreporter  were prereporter . The MMTreporter . The vecreporter . pCMV-ARreporter ,64. All escribed .Mesocricetus auratus, golden hamster) that were cultured and transfected as previously described. BHK cells were chosen as they were easy to transfect and showed good expression of the constructs. Donor dequenching experiments were carried out, using a LSM 510 Meta confocal microscope (Zeiss). Fluorescent 12 bit images were recorded in the donor-  and acceptor-  channels before and after bleaching of EYFP (at 514 nm with 100% laser transmission at 50% output in a ROI circumscribing nuclear speckles using 200 iterations). Average bleaching of the EYFP-signal was 98.3±0.5% (sem). The average apparent donor dequenching FRET-efficiency was calculated on individual EYFP-SKIP positive speckles, as: E=1-Dbefore/Dafter, where Dbefore/after are the background corrected average donor channel signals before and after bleaching, respectively. On each recorded image 3 different SKIP-positive speckles were analysed. Sensitized acceptor emission FRET imaging was carried out essentially as described [norm=(TL-TL0)/(TL988-TL0). To determine the translocation half-time, τ0.5, where 50% translocation occurred, a monoexponential function was fitted: TLnorm=1-exp(−t/τ), with τ0.5= − τ ln0.5.FRET experiments were performed in BHK-21 cells , calculated FRET- , and the acceptor-images . DHT was added in frame 1 (time given in middle panel) at 10 nM. The donor images show a DHT induced increase in AR-ECFP translocation from the cytoplasm to the EYFP-SKIP positive nuclear speckles. This leads to a transient increase in FRET on the speckles, suggesting an increased number of AR/SKIP interactions. The FRET signal subsequently decreased, as the signal of EYFP-SKIP diminished.Click here for file


Variability in reporting and classification methods in previous published data of the final dispositions in the rehabilitation of wild raptors makes use of this data limited in trying to audit the quality of the rehabilitation process. Crude as well as stratified disposition rates are needed if quality auditing of the rehabilitation process is to be adequately performed.Final dispositions of 6221 hospitalized wild raptors admitted at a wildlife rehabilitation centre (WRC) of Catalonia during 1995–2007 were analyzed. These dispositions were calculated as the euthanasia (Er), unassisted mortality (Mr), release (Rr) and captivity rates (Cr)., time to death (Td) for dead and euthanized raptors, and length of stay for released (Tr) raptors was estimated. Stratified analyses by main causes of admission and clinical signs were performed.10 = 0-P90 = 59) for both trauma and non-trauma categories, and Td  = 36 days for the orphaned young group . The median Td in the unassisted dead birds was 2 days for all the categories (P10 = 0-P90 = 31). Finally, the median Tr in the centre was variable among categories.The disposition for the total population were: Er  = 30.6%, Mr = 19.1%, Rr  = 47.2%, and Cr  = 3%. By main causes of admission, Er was higher in the trauma category (34.2%), whereas Mr was found similar between trauma (37.4%) and non-trauma categories (34.8%). The highest Rr was observed for the orphaned group (77.9%). Furthermore, Cr was low in all the categories (<4%). By clinical signs, the highest Er was found in animals suffering musculoskeletal (37.9%) or skin (32.3%) lesions; Mr was high in infectious/parasitic diseases (66.7%) and in case of neurological symptoms (64.5%). The euthanized birds had a median Td  = 1 day , by causes and clinical presentation, as well as Td and Tr, to allow meaningful auditing of the rehabilitation process quality. Rehabilitation of wild raptors is a complex process that includes both veterinary care of the injured bird and physical recovery and reconditioning of this animal for subsequent release in the wild Data published from wildlife rehabilitation centres (WRC) have been mainly focused on the causes of admission Quality assessment is one of the strategic elements for the improvement and transformation of the modern human health system The main objective of the present study was to analyze the outcomes of the rehabilitation of wild raptors in a WRC, adopting the four categories of the final disposition, the time until death and the length of stay as indicators of the quality audit of the rehabilitation process before release back to the wild.A retrospective study was performed using the original medical records of birds of prey admitted at the Wildlife Rehabilitation Centre (WRC) of Torreferrussa from 1995 to 2007. The centre is under the direction of the governmental Catalan Wildlife-Service. Samples were collected in compliance with the Ethical Principles in animal research guidelines in wildlife rehabilitation centres. The rehabilitation centres directly depend on the individual regional government wildlife services in Spain. Management and protocols were established according to the guidelines approved by each regional government according to legislation Overall data about species, gender, age, date of admission, date of death or release, and primary cause of admission were included in the analyses. Classification of primary morbidity causes, criteria for sexing and ageing, as well as the geographical and demographical characteristics of the population were the same as those reported in a previous study r), unassisted mortality rate (Mr), release rate (Rr), and captivity rate (Cr). In addition, Rr was analysed taking into account the season of admission and the season of release.The final disposition was divided into four categories adapted from Cooper (1987) The final disposition was first analyzed based on the primary cause of admission grouped as trauma, non-trauma and orphaned young categories. It was then analyzed according to the main clinical signs of the animals at the time of the admission. This clinical presentation was based on the International Statistical Classification of Diseases and Related Health Problems-ICD-10  d; difference between the date of admission and the date of the death) for euthanized and for dead animals, and length of stay in the centre for the released raptors  were also evaluated. In order to study the cases with longest Td, the percentiles 10 (P10), 75 (P75) and 90 (P90) of this variable were selected as a cut-off point.Additional parameters such as time until death (Td) and the length of stay at the centre for the released raptors (Tr).Quality indicators of the rehabilitation process conducted at the centre were evaluated based on different outcome variables following guidelines used in human medicine 50). Percentiles 10, 75 and 90  were provided for the descriptive analysis of the dispositions Td and Tr. Comparisons of the median were evaluated using the U-Mann-Whitney and Kruskal-Wallis test. Chi-square (χ2) or Fisher exact tests were used for comparisons between the Er, Mr, Rr and Cr and sex, age and order co-variables.Descriptive statistics, normality test and inferential analyses were done at 95% confidence levels with SPSS Advanced Models ™ 15.0 . Median , a total of 7553 raptor admissions were reported at the WRC. After a critical review of all the admissions, 1332 cases were excluded for not fulfilling the inclusion criteria . Thus, the final population of this study was 6221 individuals distributed in the following orders: 3241 Strigiformes and 2980 Falconiformes.The age distribution demonstrated that 46.3% (2884/6221) of birds were within their first year of age, 32.3% (2009/6221) were >1 calendar year and 21.3% (1328/6221) were of unknown age. Most of the animals, 59.4% (n = 3695), were classified as undetermined gender, 21.9% (n = 1363) of raptors were sexed as female and 18.7% (n = 1163) as males.r = 30.6% (1903/6221), Mr = 19.1% (1191/6221), Rr = 47.2% (2939/6221), Cr = 3% (188/6221)  .Stratifying by the primary cause of admission, 49.7% (3092/6221) of birds were classified into the trauma category, 15.7% (976/6221) in the non-trauma and 34.6% (2152/6221) in the orphaned young category. The euthanasia rate was notably higher in the trauma category (34.2%) compared to the non-trauma (9.2%) or orphaned young (2%) , and mai2 = 6.6; p = 0.0098) and orphaned young  categories.In the subgroup of animals with known sex and age, the unassisted mortality rate was higher in males than in females, in both non-trauma (χr) was higher in those animals suffering lesions at the skin level (32.3%), mostly affected by extensive wounds and electric burns, or at the musculoskeletal system, basically due to fractures and luxations (37.9%)  and digestive disorders (5.3%). The unassisted mortality rate (Mr) was elevated in raptors with infectious/parasitic diseases (66.7%), mainly trichomoniasis, or with neurological symptoms like depression, ataxia and paralysis (64.5%). The highest rate of release was observed in the apparently healthy animals (88.9%), mostly represented by young orphaned birds and birds belonging to the fortuity category, including birds found inside buildings or other human structures. The Rr was also high for animals with behavioural abnormalities (57.3%) and in animals in the endocrine/nutritional/metabolic (54.1%) category when this comprised birds with low body condition and weakness as main general symptoms. Finally, the captivity rate was elevated in those animals with behavioural abnormalities (15.9%) and respiratory distress (10.5%) (The euthanasia rate (E (37.9%) . By cont (10.5%) .d = 1 day  for the trauma  and non-trauma  categories, and Td = 36 days for the orphaned young group  . On the other hand, the median time of stay in the centre was highly variable among categories, presenting the trauma the longest times (Tr = 115) compared to non-trauma (Tr = 58) and orphaned young (Tr = 59) groups (The group of euthanized birds had a median T0 = 596) . Interes) groups .r was statistically different among seasons , with raptors admitted in spring presenting stays of 85 days , 53 days  if admitted in summer, 113 days  if admitted in autumn and 130.5 days  if admitted in winter.Taking into account the season of the admission because it is of relevance for the decision of approving the release of rehabilitated animals, the median TNo statistically significant differences were observed among the final dispositions during the 12 years of the study in the overall group. However, in the traumatic category, a significant decrease in the unassisted mortality rate was observed .Historically, wildlife programs were developed as a consequence of the concern of modern society with both animal welfare and the negative impact of human activities in wildlife population. Rehabilitation of birds of prey and owls has led to the development of many of these programs due to the sensitivity of wild birds to human threats, the unfavourable status of many species and the public interest in these predators A detailed description of primary causes of admission has been thoroughly reported From the data it is evident that less than half of raptors admitted to rehabilitation in Catalonia were successfully released. 52.8% of raptor admissions resulted in euthanasia, mortality or permanent captivity. Only 47.2% of birds were successfully returned to the wild. Nevertheless, an estimation of the final dispositions based on the main causes of admission or the clinical entities is essential in order to compare the results. The most simplistic and realistic classification is that consisting of two groups: 1) healthy young birds requiring rearing, 2) injured and ill birds, including those that have been kept illegally in captivity. Orphaned young birds represent an important part of the admissions to the WRC Literature on the dispositions of bird of prey rehabilitation is variable, making comparison between studies difficult. Most studies emphasise the release rate Euthanasia is an essential option in all wildlife rehabilitation, based on both animal welfare and optimization of economical resources Mortality rate has been used as a quality indicator parameter in human medicine r was due to infectious diseases, particularly trichomoniasis. It has previously been reported that the majority of cases demonstrating lesions produced by Trichomonas spp affecting the oral cavity and choanal slit, have a poor prognosis r had an approximate 30% value in the three most prevalent causes of trauma. Most of those cases suffered severe trauma with multiple body systems affected. Finally, the unassisted mortality rate found in our young orphaned group (18.9%) was similar to other reports (16.1%) Our data demonstrated a similar rate of mortality for trauma (37.4%) and non-trauma (34.8%) cases. In the non-traumatic group, the higher Mr was over 50% when the nervous, respiratory or digestive systems were primarily affected or in cases of general systemic infectious or parasitic disease. The Mr was higher in birds with integument and musculoskeletal conditions. On the other hand, the higher Mr in animals apparently healthy on admission or with nutritional and metabolic conditions is suggestive of captivity-related complications and requires further investigation. In the authors' opinion, the present classification focusing on clinical signs allows a more accurate assessment of the rehabilitation protocols than those based on the primary cause of admission. Both classifications are useful; clinical classification allows a veterinary perspective, while the primary cause of admission allows an assessment of environmental causes and problems, and should be included in the analysis of dispositions of the rehabilitation of wild birds of prey.According to the classification of clinical signs, MThe release rate in our study was higher in the orphaned young group, followed by fortuity and captive birds that were mainly affected by minor health conditions. The overall release rate of trauma cases was 24.3% (ranging from 1% of electrocution cases to 61.5% of birds suffering impacts with buildings). The release rates of gunshot, collision with vehicles and unknown trauma were very similar to those previously reported Circaetus gallicus, Pernis apivorus and Otus scops were maintained at the centre until the next spring migration. As a general rule, the length of stay must be as short as possible in order to reduce the risk of captive-related complications, infectious and parasitic disease, and behavioural abnormalities Length of stay is a quality indicator parameter frequently used in human medicine The parameter time to death provides direct insight into the initial assessment and prognostication, the overall rehabilitation process, as well as the validity of veterinary protocols. This complements understanding of the mortality and euthanasia rates. In all time dependent variables we have included the extreme values because they highlight the real daily work of the rehabilitation centre, with birds remaining in captivity for unknown reasons. Interestingly, the median time to euthanasia was 1 day. That means that the decision is taken at the moment of the admission, resulting in optimization of welfare and financial resources. On the other hand, the median time of death was 2 days even for the young orphaned group. This fact suggests that special care and a complete clinical evaluation should be performed on all young birds, despite their apparently healthy appearance.r and Er over the P90 of the Td, as an indicator of undesirable or unexpected dispositions. The decision of euthanasia over 59 days was mostly taken due to complications related to trauma or musculoskeletal conditions. In our protocols, at 59 days most birds are in outside enclosures undergoing active flight conditioning. At this stage the decision to euthanize is taken in birds with musculoskeletal problems as well as those demonstrating abnormal behaviour incompatible with release to the wild.In our work, we paid attention into the MFinally, a significant decrease in the unassisted mortality rate was observed in the traumatic category. This finding could be consequence of the improvement of both diagnostic and therapeutic protocols applied in the last years. The optimization of protocols for identifying specimens that are non-viable, has permitted the early euthanasia of these animals, avoiding unnecessary animal suffering and improving the management efficiency of resources.In conclusion, the basic outcome research of the rehabilitation process of wild birds of prey and owls should include the four final disposition rates , but also the parameters time until death (Td) and length of stay at the centre (Tr). The reports should also include the overall rates and the stratified analysis according to the cause of admission and the clinical entities. Moreover, both Td and Tr should be estimated by the overall group, but also stratifying by final decision and cause of admission and clinical entities. These six parameters are measurable items that should be considered as a baseline indicators for quality audits. Our results could represent a reference of a large amount of parameters related with the outcomes of the wildlife rehabilitation process that could be adapted by other centres as a start-point for further comparison. Finally, consensus of the professionals involved in rehabilitation of wild birds of prey is essential in order to develop evidence-based clinical guidelines and recommendations that will lead to an improvement of the rehabilitation procedure.


Herbicides, namely 4- butyrate (DPBA) and 2-(3-chlorophenoxy) propionate (CPPA), were intercalated simultaneously into the interlayers of zinc layered hydroxide (ZLH) by direct reaction of zinc oxide with both anions under aqueous environment to form a new nanohybrid containing both herbicides labeled as ZCDX. Successful intercalation of both anions simultaneously into the interlayer gallery space of ZLH was studied by PXRD, with basal spacing of 28.7 Å and supported by FTIR, TGA/DTG and UV-visible studies. Simultaneous release of both CPPA and DPBA anions into the release media was found to be governed by a pseudo second-order equation. The loading and percentage release of the DPBA is higher than the CPPA anion, which indicates that the DPBA anion was preferentially intercalated into and released from the ZLH interlayer galleries. This work shows that layered single metal hydroxide, particularly ZLH, is a suitable host for the controlled release formulation of two herbicides simultaneously. Nanomaterials such as two-dimensional 2D) nanosheets have recently gained much attention due to their unique physical and chemical properties . ExcelleD nanoshe1−xM(III)x(OH)2](A−n)x/n·yH2O where M(II) and M(III) are the divalent and trivalent metal cations, respectively. An− is the exchangeable anion and y represents the water content of the interlayer region. HDSs are identical to LDHs, only the metal hydroxide inorganic layers are composed of two divalent metal cations [2+, Cu2+, Zn2+ and Ni2+ and can be represented by the general formula, M(II)(OH)2−x(A−n)x/n·yH2O. In this structure, the OH− anions on the brucite hydroxide layer are substituted by water molecules and counter anions [Layered double hydroxides (LDHs) and hydroxy double salts (HDSs) have been studied extensively and are recognized for their anion-exchangeable properties ,6. LDHs  cations . Layered cations  such as r anions –12.The gallery structure of LDHs is expandable, if the guest anion is larger in size than the interlayer anion present in the LDHs, or the spatial orientation of the guest warrants the expansion. Various guest anions can be intercalated into the LDH’s gallery structure, thus a variety of hybrid nano-layered materials can be designed ,13. IdenTo our knowledge, unlike LDHs, the use of ZLH as matrices ,18,19, pThis paper aimed at the formation of phase-pure, well-ordered ZLH-intercalated nanohybrids by the intercalation of phenoxy herbicides-type active agents, namely 4- butyrate (DPBA) and 2-(3-chlorophenoxy) propionate (CPPA) simultaneously into the ZLH interlayer using a simple direct reaction of zinc oxide (ZnO) with the guest anions in an aqueous environment. In our previous study, we reported the successful intercalation of both herbicides anions into LDHs by an anion-exchange method. Our results showed that the loading and release percentages of DPBA were found to be higher than CPPA . In thisAll solutions were prepared using deionized water. The ZLH nanohybrid was prepared by the direct reaction of ZnO with the guest anions in an aqueous environment. About 1.00 g of pure commercial ZnO was reacted with 100 mL deionized water and mixed with 50 mL aqueous solution of 0.1 M DPBA and 0.1 M CPPA. The solution mixture of ZnO and the anions were stirred for 2 h before aging for 18 h at 70 °C in an oil bath shaker. The slurry was centrifuged, washed with deionized water, dried in an oven for 24 h and kept in a sample bottle for further use and characterization.max = 220.0 nm and 230.0 nm for CPPA and DPBA, respectively.Simultaneous release of CPPA and DPBA from the ZCDX nanohybrid was studied by adding a 0.4 mg sample into 3.5 mL of 0.001–0.004 mol/L sodium carbonate aqueous solution. The quantity of phenoxyherbicides released into the solution was measured at the preset time at λα radiation (λ = 1.5405 Å) at 40 kV and 30 mA, 2°·min−1. The FTIR spectra were collected in a Perkin-Elmer 1752X spectrophotometer using the KBr disc method, in the range 400–4000 cm−1. Thermal analyses (TGA-DTG) were performed using a Mettler Toledo instrument at a heating rate of 10 °C·min−1 between 35 °C and 1000 °C, under nitrogen flow of about 50 mL·min−1. A field emission scanning electron microscope (FESEM), Carl Zeiss Supra 40VP model was used to study the surface morphology of the materials.Powder XRD patterns were recorded at room temperature with a Shimadzu XRD-6000 using filtered CuKmax = 220.0 nm and 230.0 nm for CPPA and DPBA, respectively. The nanohybrid was dissolved into 2.0 M sodium carbonate aqueous solution so that both anions could exchange with CO32− and OH− anions in the aqueous solution and release completely from the ZLH interlayers. Since the wavelengths of the pure herbicides is relatively close, there is an overlap between the two peaks  represents the concentration of 100% release of anions and b is the path length (1 cm). As shown in DPBA at λ1 and ɛDPBA’ at λ2.where λThe PXRD spectra and basal spacing for the ZLH nanohybrid intercalated with dual herbicides, CPPA and DPBA, labeled ZCDX together with single intercalation of both anions separately via direct reaction with ZnO are shown in -deposition mechanism, was adopted for the formation of the ZCDX nanohybrid [2 on the surface. The dissociation of Zn(OH)2 in the aqueous environment resulted in the release of Zn2+ and OH−. These two species then reacted with CPPA, DPBA and H2O in the solution to produce the layered ZLH-CPPA-DPBA nanohybrid compound.Several studies has reported on the preparation of LHS by the hydrolysis of divalent metal salts and a metal oxide such as ZnO, followed by the inclusion of guest molecules into the host interlayers ,7,12. Innohybrid ,29. ZnO The LHS can be described as morphologically having a plate-like structure, as reported elsewhere –12,15. F2+ located at the top and bottom of the octahedral sheet [al sheet ,7,11,17 al sheet  and the Considering several factors such as charge density of the layer, anion dimension and assuming that the layer structure remains intact after the intercalation of both anions ,15, thenThe thermal decomposition behavior of ZnO and ZCDX nanohybrid was investigated using TGA-DTG studies. It is known that the LHS thermograms are generally described by two thermal events which are similar to that for organo-LDHs ,12. The The TG curve of ZCDX shows two major weight losses at temperature maxima of 118.2 °C and 295.3 °C. The first stage of weight loss with 7.3% is ascribed to the removal of the surface and intercalated structure of water molecules, leading to the decomposition for dual herbicides of the nanohybrid at 295.3 °C with 49.1% weight loss. This shows that the ZCDX nanohybrid is thermally more stable than CPPA and DPBA in their salt form . DPBA ha−1 is due to the Zn–O sublattice stretching vibration [−1 are attributed to the OH stretching vibration of COOH for CPPA and DPBA anions, respectively. Intense peaks at around 1700 cm−1, for CPPA (1705 cm−1) and DPBA (1714 cm−1) are ascribed to C=O stretching vibration of the protonated COOH group of herbicides. Strong bands at 1471 and 1467 cm−1 are due to the stretching vibration of the aromatic ring, C=C and bands at around 1200 cm−1 are due to C–O–C antisymmetric and symmetric stretching.The presence of the two herbicides in the ZLH interlayers was also elucidated by FTIR spectroscopy, which supported the PXRD results . Figure ibration ,27. Simi−1 are due to the stretching vibration of the C=O of the COOH group and C=C stretching of the aromatic ring, as mentioned earlier, indicating that the two herbicides were successfully intercalated into the ZLH interlayers [−1 is due to the antisymmetric and symmetric carboxylate stretching of the anion. At the lower wavenumber, the disappearance of the broad ZnO peak in the nanohybrid confirmed that all the ZnO had reacted with the CPPA and DPBA, resulting in the formation of the nanohybrid, which concurs with the PXRD data.As expected, ZCDX has an absorption spectrum similar to the herbicide anions. However, some bands are slightly shifted due to the interaction of both the anions and host layers. Weak bands at around 1700 and 1400 cmerlayers . A stronThe loading percentages of CPPA and DPBA were analyzed using a UV-Vis spectrophotometer and the data were calculated using simultaneous equations, as stated earlier. The data show that the ZCDX nanohybrid is composed of CPPA and DPBA with percentage contribution of 16.22% and 83.78%, respectively. The percentage represents the ratio of CPPA and DPBA intercalated into ZLH. The simulation data of both single intercalations of ZC and ZD nanohybrids at different percentages  shows th32− and OH− anions in the aqueous solution is higher than the CPPA and DPBA towards the ZLH interlayers; therefore, both herbicides anions can be ion-exchanged with the carbonate [max = 220 and 230 nm, respectively. Generally, the release rate is initially rapid, and then slows until it reaches equilibrium. The percentage release of CPPA and DPBA increased when the ZCDX was placed in a higher concentration of sodium carbonate, as shown in The simultaneous release of CPPA and DPBA from the nanohybrid lamellae into the release media was performed in various concentrations (0.001–0.004 mol/L) of sodium carbonate aqueous solution. The affinity of COarbonate ,22,25 an− atoms bonded with Zn2+ ions compared to only one Cl− atom in the CPPA [Previous work revealed that release behaviors of different drugs from the same host are quite different . Such bethe CPPA , as propr2) for the pseudo second-order kinetic gave the best fit for both CPPA and DPBA, as shown in t1/2 values for the DPBA and CPPA which are 286 and 543 min, respectively, confirmed that the former is easier to release than the latter from the nanohybrid, when both anions are present simultaneously. In most of controlled release formulation, a large amount of anions  were released at the very beginning of the release time before the release rate reaches a stable profile. This is due to the swelling of the porous structure followed by the dissolution of the anions. This phenomenon is referred to as “burst release” [The data of the cumulative release of the guests were fitted to several kinetic models and plotted in release” , which hIn conclusion, the monophasic, well-ordered ZCDX nanohybrid containing two herbicides, CPPA and DPBA, was found to be composed of a higher loading of DPBA compared to CPPA between the ZLH inorganic interlayers with percentage contribution of 83.78% and 16.22%, respectively. Simultaneous release of CPPA and DPBA from ZCDX into sodium carbonate aqueous solution was found to be controlled by pseudo second-order kinetics. The release rate of both CPPA and DPBA was found to be different, suggesting that the anionic guest molecules sizes and the interactions between the host and guest could control the release kinetics. The present results demonstrate the potential application of a layered single metal hydroxide, particularly zinc layered hydroxide as the host for the preparation of a nanohybrid compound with tunable controlled release property containing two herbicides simultaneously.


Collaboration may be understood as the execution of coordinated tasks  by groups of users, who cooperate for achieving a common goal. Collaboration is a fundamental assumption and requirement for the correct operation of many communication systems. The main challenge when creating collaborative systems in a decentralized manner is dealing with the fact that users may behave in selfish ways, trying to obtain the benefits of the tasks but without participating in their execution. In this context, Game Theory has been instrumental to model collaborative systems and the task allocation problem, and to design mechanisms for optimal allocation of tasks. In this paper, we revise the classical assumptions of these models and propose a new approach to this problem. First, we establish a system model based on heterogenous nodes , and propose a basic distributed mechanism so that, when a new task appears, it is assigned to the most suitable node. The classical technique for compensating a node that executes a task is the use of payments (which in most networks are hard or impossible to implement). Instead, we propose a distributed mechanism for the optimal allocation of tasks without payments. We prove this mechanism to be robust evenevent in the presence of independent selfish or rationally limited players. Additionally, our model is based on very weak assumptions, which makes the proposed mechanisms susceptible to be implemented in networked systems . Selfish behavior is becoming a subject of great concern and practical importance to network designers In this paper we revisit the study of communication systems with selfish users (or players), reevaluating and relaxing the above-mentioned common assumptions. In particular, we propose the problem of analyzing and designing a fair collaborative system under a very weak set of game theoretic assumptions. In this general context, we propose mechanisms to be used to implement this collaborative system with provable properties, like the fairness of the system and the truthfulness of its users. The mechanisms proposed can be applied to such varied technologies as social and crowd computing, Web 2.0, P2P, opportunistic networks, and cloud systems.As mentioned, we abstract the problem to be solved as the fair execution of tasks in a decentralized collaborative system. The main challenge when creating collaborative systems in a decentralized manner is dealing with the fact that system nodes may behave in selfish ways, trying to obtain the benefits of the tasks but without participating in their execution.  We assume that all nodes have an interest on having the tasks done. However, establishing fair mechanisms for sharing the generated work-load is not immediate.  It would, therefore, be desirable that each node could take the responsibility of the execution of a balanced fraction of the tasks.opportunity of execution. Let us assume that each node has some capacity for timely execution of a given task. This capacity may vary over time and with the type of task. For example, at a given time, a node may have free bandwidth but have full utilization of its CPU, while theits situation could be the opposite at another time. Hence, at a particular moment, a node may have greater ability to perform tasks involving communication, while at a later time itits situation may change to prefer tasks that are more intensive in CPU computation.The objective is to establish some kind of protocol to share the task execution costs. For this, we need to consider the concept of ability or This opportunity or ability is related with the notion of task execution cost. In other words, we define the cost as some kind of metric measuring the capability of executing a particular task at a given time. Hence, the cost varies from one task to another . In Game Theory, closely related to cost, there is the notion of utility. We define the utility as the cost savings associated with a work not done. Hence, given that all nodes are interested in the execution of the tasks, a node gets more utility whenever it avoids running tasks, by letting other nodes running them.private information. To obtainFor obtaining the private information of a node, the basic mechanism is to directly ask for it and expect the node to declare its value correctly.Clearly, when trying to formalize a model based on these notions, a number of problems arise. First, node's costs are only known by the node itself. For external entities it would be difficult to audit or check if a given particular node has more or less CPU capacity. In Game Theory, this concept is called For us, each node is a computing elementnode that belongs to a user who can alter her node's behavior for her own benefit . Whenever this happens, we claim that the user acts in a selfish way. This selfishness is one of the factors that may distort the internal workings of a distributed application. The loss of system performance produced by selfish nodes is a parameter to consider and it is called price of anarchy Quid Pro Quo Mechanism (QPQ). The name comes from a Latin expression commonly used by lawyers and which may be translated as “This for that” or “A thing for another”. This expression is often used when someone does a job and waits for anand equivalent compensation in exchange. We used this expression since it reflects the spirit of the algorithm: due to the lack of payments in our model, the nodes work for others with the hope that others will work for them in the future.Therefore, the problem we face consists of designing a system capable of assigning tasks to nodes so that all the tasks are executed, and the total cost incurred is minimal. When the behavior of nodes is guaranteed to be fair, this is just a simple optimization exercise. However, when nodes may choose whether to be selfish the problem becomes much more complex. In this paper we propose an algorithm that, based on Game Theorybasing on game theory principles, solves this problem. We have called this algorithm As described above, the problem addressed in this paper is the allocation of task executions to potentially selfish users. This problem has been extensively studied in the literature. One important related work was carried out by Rosenschein et al. linking mechanism) which, instead of offering incentives or payments to players, limits the spectrum of players' responses to a probability distribution known by the game designer. In that paper, the authors proved that a linking mechanism is valid when the players' possible decisions are distributed following discrete probabilities. Additionally, the authors show that a linking mechanism can also be used for repeated games. Even though the work of Jackson et al. is very relevant to the problem we consider, it does not offer a method for the construction of mechanisms when the game is based on unknown continuous probability distributions, as assumed here. A second work that explores the idea of linking mechanism is due to Ferenc Our work falls naturally into the large area of mechanism design without money .In this paper, we face the problem of task allocation relaxing these (and other) common hypotheses, so that the obtained results can be applied in real environments. Hence, the contributions of this paper are twofold. First, to the best of our knowledge, this is the first work proposing a linking mechanism solution without prior knowledge of the distribution of the players' decisions, and without a payment system among them. Second, we generalize and improve previous works in the area to provide algorithms which are susceptible of being applied in the context of repeated task execution allocation in real communication and computing systems, even in the presence of selfish or non-rational users.As we previously claimed, we do not want to restrict our mechanism to a set of unrealistic hypotheses. Instead, we establish a number of requirements that our model must satisfy. These requirements should provide the appropriate flexibility to guarantee the applicability of our results in real environments.Abstract utility metrics: We assume, as an abstract notion, that the cost of executing a task for a node  depends on its interest on the task, its opportunity or ability to execute it, or its degree of willingness to cooperate. We need to accept that each node may measure this parameter in its very own metric and units. Hence, for example, a node may decide on the cost of a task according to the occupation of its CPU, but another one may prefer to make it depend on its available bandwidth. In a real scenario, the number of factors that can influence the execution cost of a task can be extremely large. In this direction, ourout model must enable each node to define, in a flexible way, how costs (and utilities) are measured.No payment system: Payments are, in its most basic interpretation, a way of exchanging costs. Many existing mechanisms base their incentive schemes on the existence of payments. For payments to be possible, it is necessary that all players manage a common currency reference . However, given our previous requirement, it is not clear how we can find that shared currency reference in our model. If a node measures its costs in terms of, for example, reputation, it can hardly “pay” to another node that measures its costs on CPU units. Hence, in our work, we assume that payments are not possible.Players' rationality: In Game Theory, most of the existing algorithms require players to be perfectly rational. This means that a player, using the available information, should always be capable of selecting the best strategy (the one that maximizes her utility). However, this is a controversial hypothesis which is suffering much criticism. Accepting this assumption means that players are capable of mathematically calculating all alternatives, which in some cases requires solving complex (NP-hard) problems. Clearly, this is not always feasible for all players. Hence, we commit ourselves to proposing mechanisms suitable for finding quasi-optimal task allocation, even in the presence of rationally-limited players.Incentive to participate: In relation to players rationality, even in the case in which we are able to find global quasi-optimal task allocation, it is possible that the behavior of rationally-limited users may harm the benefit of other players. In this direction, we add a stronger requirement. We force to ensure an incentive to participate in the game to all nodes, independently of whether they are rational or not.No central entity: A final requirement we impose is the capability of the system to work without the existence of any kind of central entity. This means that the proposed mechanisms must be susceptible of being implemented following completely distributed schemes.The rest of the paper is structured as follows. First we provide a formal definition of the problem and define basic terminology. Then, we present a basic linking mechanism, and evaluate the issues that need to be faced to make it suitable for our problem. Next, we present the QPQ mechanism, and formally prove its properties. Finally, we describe how QPQ could be used in real environments and present some conclusions.To establish a formal framework for the problem, let us provide some definitions.Definition 1 (Problem). The problem of the assignment of tasks is a tuple It is important to remark some aspects of the above definitions. First, we assume that the set of tasks is not known beforehand. Tasks appear one by one in a sequence of time steps, which drives our discrete time evolution. Hence, the arrival of a new task dictates the start of new a round of our repeated game. We assume that tasks are independent among them and that the execution of a task does not influence the cost of the subsequent ones. Moreover, we force that one task must be completely executed by the time the next task is issued. For simplicity, we assume that the mechanisms take negligible time to coordinate the allocation of the tasksto coordinate the allocation of the tasks take negligible time (with respect to the time step). Finally, we assume that every node that is assigned a task by the allocation mechanism actually executes the task.Hence, as tasks are issued, each node Given that all players enjoy the result of any task executed in the system, we can define the utility of a player as the savings obtained by not executing some tasks . That is, the utility We define reliable and concurrent. In particular, in the mechanisms we propose all players exchange their values Finally, we assume that communication between players is As mentioned above, a linking mechanism is applicable to repeated games where the decision  of players is restricted to a particular known set. In our problem, the decision is the cost As it can be observed, for each task, each player estimates the cost of computing the task and publishes it. Publication means broadcasting a message with the cost to all players . By assumption, a player send its costs before it receives any of the others , and all of the costs are correctly received at each player (reliability). Then, the algorithm assigns the task to the player that publishes the lowest cost. If players publish their real costs, this will produce that the total utility is maximized. However, this kind of approach could drive selfish users to publish fake costs in order to avoid executing tasks. For this reason, we add an acceptance test. When a published cost is not considered acceptable, then the system generates a random value for the cost of that node on the round. The implementation of this acceptance test will be discussed later, however it is important to remark that it contains the linking part of the mechanism . Just as an example, we can imagine that if we force that nodes must publish costs between Algorithm at Digging into these problems, it is easy to understand that one of their causes is the fact that, given our requirements, each player has the right of measuring her costs on her preferred metric.  For this reason, cost comparisons cannot be easily made. Additionally, there is a second aspect that must be addressed. In the literature about linking mechanisms, authors assume that instances of the game (rounds) are simultaneous in time. In this case, defining the acceptance function over the set of values is easier. However, in our case, tasks are issued , over time. Then, from the point of view of the designer, it is not clear how to determine the acceptance of a value by comparing with a certain probability distribution. The issue is even worse given the fact that this distribution is not known by the designer. To solve all these problems we propose a novel solution based on applying a transformation over the utility function.Probability Integral Transformation (PIT). Our idea is to use the known fact that any cumulative probability distribution function has in itself a uniform distribution Given that the utility is defined as the work not done by a node, we may use as utility function of a node its probability distribution of costs. Once this is done, we may modify Algorithm at Definition 2  Let the Probability Integral Transformation (PIT) defines a new random variable As mentioned above, our interest in the PIT is due to the following lemma.Lemma 1 (PIT follows uniform distribution) Let  be a continuous random variable with CDF , then  follows a uniform distribution on interval . That is, the random variable  defined by the probability integral transformation  is a normalized uniform distribution.Generalized Distributional TransformMoreover,Note that Definition 3  Let The modified distribution functionis defined asProofs of these properties can be found in Coming back to Algorithm at Based on these arguments, it is clear that the PIT provides a mechanism for comparing  node costs. However, we may wonder if the proposed transformation is valid, in the sense that it may not preserve the preferences of the player. To solve this issue, it suffices to notice that, what we are doing is changing the space of preferences. Therefore, the PIT somehow means that, instead of asking the user “How much does it cost to execute the task?”, we inquire for something like “What percentage of tasks do you prefer to this one?” At the end of the day, and for our objectives, these questions are requesting the same information, but the latter is normalized in the interval Although from an analytic point of view we assume that players can compute the PIT perfectly, in a practical set up players do not need to consider any a priori distribution of probability. They can simply generate costs using their particular distribution and apply the PIT using the successive generated samples. This process uses what in statistics is know as the Empirical Cumulative Distribution Function (ECDF). We will review this concept later, when we analyze the practical formulation of QPQ.Goodness-of-Fit (GoF) tests.Once we know the properties of the PIT, it is clear how we can implement the acceptance test for the linking mechanism. The idea is that any player applying correctly the PIT on her real cost distribution, must generate a uniform distribution on the unit interval on her published normalized cost values. Hence, from the point of view of the mechanism designer, the problem consists inon determining whether these published values follow or not that uniform distribution. There are a wide range of tests that allow checking that. These tests are called Continuing with this argument, we propose to implement the acceptance test of our algorithm by using some GoF test on the declared transformed sequence of costs published by the player. Whenever a player is honest and she declares the values by applying the PIT transformation on her own distribution, these values will be uniformly distributed in the unit interval. In that case (with high probability) the GoF tests will accept the samples. More important, this process has an error which tends to zero when the number of samples (rounds) increases for any reasonable value of the threshold. For the study of our analytic results, we assume that GoF tests are perfect and this error is zero. In the case that a dishonest player tries to avoid the execution of tasks, one possible strategy is to generate increasing cost values, so that the PIT transformed values are close to the unit. However, this type of behavior is quickly detected by the test. An open question is how to establish a punishment to this and any other player whose GoF test comes out negative. One possibility is to force the node to execute the task. Unfortunately, this policy would force fair players to execute tasks in cases of false negatives.Another possibility, inspired byon previous works on linking mechanisms, is to reject the value declared by the player and generate a new random value according to the normalized uniform distribution. Additionally, we require that no central entity existsexist on the system. For these reasons, we propose to use a deterministic (repeatable) random generator that any of the remaining nodes can use to calculate the new value.  At a first sight, this strategy may seem as a very poor punishment, given that there is always a chance that a player emerges victorious of a lie. However, later in this paper we will prove that this is not only enough to discourage dishonest players, but also a crucial ingredient to guarantee that our mechanism is strategy-proof.Quid Pro Quo (QPQ) mechanism. The details can be observed in Algorithm at After describing the different ingredients of our solution, we are able to propose the final algorithm, which we call the Note that we use In the following sections, we formally study the expected harm (or reduction of benefit) that dishonest behavior causes on QPQ. Intuition says that the loss due to a dishonest player should be comparable to having that player executing tasks at random. Indeed, we show below that, independently of their behavior, nodes may never expect a profit of less than the one obtained through a mechanism in which tasks are randomly assigned. This property is very useful in case the node is not capable of accurately evaluating theits costs .Another important aspect is that QPQ guarantees a minimum benefit to the entire system, even if one or more players are non-rational or rationally limited. In this sense, we will show that the best strategy for any player is to act as if the rest of the players were rational and fair. That is, incorrect behaviorbehaviors of some players does not alter the strategy of correct players. In the next section, we prove all these claims in a formal way.Our QPQ algorithm is strongly inspired by the work of Jackson and Sonnenscheinet al. QPQ is optimal in the sense that it minimizes the total work done when all players are honest.For any player, the rest of the players can be seen as a single aggregated player. For each task, the aggregated player's cost is the smallest of its members'. These costs follow a Beta distribution.The best strategy of a player is independent of the behavior of the rest.The strategy that optimizes the utility of a player is being honest. In Game Theorygame theory terminology, this means that QPQ is strategy-proof.Each player always obtains a positive expected utility, which is determined by the number of players.An irrational or rationally-limited player always obtains a positive profit.The system is fair in the allocation of tasks and in normalized effort. That is, all the players will run the same number of tasks and perform a similar normalized effort (in expectation).When the number of player is high enough, QPQ ensures very attractive performance.aggregated player. We evaluate the performance of a node playing against a “fictitious” node that aggregates the responses of all the other nodes. This aggregated player behaves by publishing at each round the minimum of all the normalized costs of the players in the aggregation. This approach is compatible with all the assumptions of the model and is helpful because it significantly simplifies the analysis.To address the mathematical analysis of the algorithm we will assume that the PIT and GoF steps are perfect. In fact, with a large number of samples, these processes have errors close to zero. Another aspect that will simplify our analysis, is the idea of To make our notation clear,clearer, given a task, we use With this notation in mind, we can prove that, for any player We argue here that all players' normalized costs follow an independent uniform distribution on Proposition 2The set of final normalized costs  considered in Line 10 of Algorithm at  are drawn from independent and identical distributed (iid) random variables, with uniform distributions on .The QPQ algorithm is optimal in the sense that, if all players are honest, it minimizes the total normalized work done, as shown in the following proposition.Proposition 3Assume that all players are honest. For a given set  of tasks, there is no mechanism  such that , where  is the random variable associated with the normalized work done by player  when using mechanism .Proof. The proof is straightforward using contradiction. Assuming that such mechanism It is assumed that players' normalized costs have independent uniform distributions on Proposition 4The costs  of the aggregated player of  i.i.d. players (with uniform distribution on ) follow a  distribution, with probability density probability function  and CDF .Proof. Recall that the cost of an aggregated player is the minimum of the normalized costs of the players in the aggregation. The CDF Every rational player knows that the rest of players follow uniform and independent distributions. The question a selfish rational player makes is which is the best strategy for obtaining the greatest possible benefit. If a player uses a distribution other than the uniform, her values will be rejected by the GoF, and will be re-generated from a uniform distribution. However, a player could lie following a uniform distribution that is not independent of her actual values. Note that QPQ does not know about true normalized costs (they are private) and uses for the assignment decision the declared value or the random value assigned by the system if a lie is detected. In both cases, the aggregated player observes a random variable Let us first quantify the expected work done by honest players.Proposition 5The expected normalized work  done by an honest player  is .Proof. Recall that we assume that player However, publishing dishonest values independent ofon the real distribution drives to making an amount of normalized work of Proposition 6When a player  publishes dishonest non uniform values or values independent of her true normalized uniform distribution, it performs in expectation  work.Proof. The values From this result, Proposition 5, and Corollary 7Assume a player  publishes dishonest non uniform values or values independent of her true normalized uniform distribution. Given that , it holds that . Hence, since the sum of the expected work and expected utility is , players obtain higher expected utilities by being honest than by publishing dishonest normalized costs.In essence, the previous result shows that, unless the player cheats declaring values Proposition 8The total expected normalized work assigned to an aggregate player  (aggregating  nodes), with costs , does not change when a player  (not in the aggregation) declares dishonest values .Proof. Given that □In summary, an aggregate player Proposition 9The total expected normalized work assigned to player , does not depend on the strategies of the other  players, and therefore on the perceived strategy of the aggregate player .This allows us to prove that the optimal strategy for a player is to be honest.Theorem 10A player  never does more normalized work (in expectation) by being honest. That is,Proof. For the sake of contradiction, let us suppose this proposition is false. Hence, there is some set of tasks for which, if Note that the normalized work done by ana honest player, as calculated above, is equal to Theorem 11For each player , the real expected utility iswhere the real cost of player  is a continuous random variable with support , probability density function , and CDF .Proof. Let The following result, combined with Proposition 2, will be used to show that all players execute, on expectation, the same number of tasks, even when some players are non-rational or dishonest.Proposition 12Let  be  continuous and i.i.d. random variables, then:Proof.Hence, QPQ not only offers best utility guarantee to honest rational players, but it also offers good properties in environments where nodes have difficulty in estimating costs. This is because, even in environments where the nodes are non-rational, QPQ divides the work fairly and optimaly with respect to the declared normalized costs. Clearly, non-rational players run major efforts, but it is always under completely random task assignments. In other words, the extra cost of non-rational players is caused by their own ignorance, not by the wickedness of the other players. Then, given that players are assigned tasks by choosing the smallest value from a set of i.i.d. random variables (Proposition 2), QPQ ensures that the expectation of the number of tasks executed by each node is Corollary 13In QPQ, players will execute in expectation a proportion of  of the tasks, and thus a total of  of tasks.Proof. Declared values follow continuous and independent identically distributed (uniform) random variables in efficiency as the ratio between the utility of an equilibrium  and the utility of some optimal solution.Finally, we think that it could be interesting to define some ratio that measures the efficiency of the QPQ mechanism. Following concepts similar to the “price of anarchy” Obviously, the player's normalized utility must be between Although this case has null probability, we propose to use this value for our definiton of measure of efficiency.Definition 4 (Measure of efficiency) We define the measure of efficiency of an algorithm Hence, we can compute the efficiency of QPQ asIn this section, our objective is analyzing what are the restrictions for QPQ to be implemented in real environments. From the above sections, we may claim that the computation and communication capabilities required by the algorithm are affordable with current technology. We do not claim that implementing such capabilities would be an easy task, since there are many technological challenges that should be addressed to do it. Other previous works solve some of them However, going beyond the required communication and computation capabilities, we may see that a number of issues arise. The first of all is on the definition of selfishness itself. This paper is mainly focused on detecting and neutralizing users publishing values not coming from the PIT of their real costs. However, one can claim that other non-cooperative harmful behaviors are possible such as, for example, not executing tasks at all, or executing them incorrectly. Hopefully, most of these evil conducts can be easily avoided using a two step scheme. First, by detecting such behaviors  random generator that any of the remaining nodes can use to calculate the new value. One possibility for generating the random value is to use a hash function over the published normalized cost of other nodes. Alternatively, it is possible to request a random value to each player  and apply the hash function on them. Even another possibility is to use techniques similar to the procedures proposed by Aumann et al. Another obstacle that stands inon the way of a potential implementation of the mechanism is the acceptance test. We have assumed that we have a perfect GoF test function. This is somewhat similar to assuming that we have a set of samples whose number is very large  for detecting lies with the usual tests. In a real system, this solution is impractical since nodes would require to store all the historical values of the rest of players, and initially the number of samples is necessarily limited. As mentioned before, we propose that players simply generate costs using their particular distribution and apply the PIT using the successive generated samples. The CDF used for the PIT is synthesized from the existing samples Definition 5  The empirical cumulative distribution function (ECDF) Glivenko-Cantelli theoremObviously, this process has an error which tends to zero when the number of samples (rounds) increases as, by the it is proved by Regarding the GoF used, a tremendous number of GoF tests have been proposed in the scientific literature. Some of them may be applied over discrete distributions and others require continuous distributions. The Kolmogorov-Smirnov (KS) test p-value). Note that, in statistical significance testing, the p-value is the probability of obtaining a particular test statistic on the model at least as extreme as the one that was actually observed. Now, the value is accepted by the test when that p-value is over a particular acceptance threshold, We propose to use the KS test as the GoF test of QPQ. Hence, whenever a new normalized cost is issued, we check the KS test of it, together with the historical sequence of that player, so that we obtain the corresponding  and one dishonest, for a set of Finally, for the same simulation scenario, in Throughout this paper, we have presented QPQ, an algorithm for optimal allocation and execution of tasks in a distributed environment with selfish behavior. Unlike many of the preexisting works, this algorithm proposes a mechanism that does not use payment or prior information on the behavior of the players. We have demonstrated that the algorithm is tolerant to dishonest, non rational or rationally limited behaviors without punishing fair users and rewarding players proportionally to their degree of truthfulness. The proposed algorithm may be adapted using reasonable approximations so that it can be implemented in real networks with affordable computational and communication complexity. For all these reasons, we claim that this algorithm opens new horizons for the creation of novel computing frameworks where users can openly and effectively cooperate to achieve a common goal, based on the collaborative execution of simple atomic independent tasks.Despite this, the authors consider necessary to carry out further research to make QPQ robust to more sophisticated selfishness scenarios. For example, it would be necessary to consider cases in which players are not independent, and associate in groups trying to break the system's fairness. QPQ can be also made more robust by tolerating the unreliability of players or the communication between them. Observe that this can be modeled as a random process or as a possible strategy of the players. Another aspect that should be extended is related to the notion of task utility. We have assumed that all nodes have an interest in having all tasks done. However, in a real environment, it is possible that only a subset of tasks are relevant for a given node. Hence, further work should be developed to relax some of the QPQ hypotheses, and deal with this aspect. To conclude, another aspect that may be improved is investigating GoF tests other than the KS to analyze if they can provide advantages for real implementations of the algorithm .