Sea turtles (Chelonoidea) are a charismatic group of marine reptiles that occupy a range of important ecological roles. However, the diversity and evolution of their feeding anatomy remain incompletely known.Caretta caretta) and Kemp’s ridley (Lepidochelys kempii), for a better understanding of sea turtle functional anatomy and morphological variation. In both taxa the temporal region of the skull is enclosed by bone and the jaw joint structure and muscle arrangement indicate that palinal jaw movement is possible. The tongue is relatively small, and the hyoid apparatus is not as conspicuous as in some freshwater aquatic turtles. We find several similarities between the muscles of C. caretta and L. kempii, but comparison with other turtles suggests only one of these characters may be derived: connection of the m. adductor mandibulae internus into the Pars intramandibularis via the Zwischensehne. The large fleshy origin of the m. adductor mandibulae externus Pars superficialis from the jugal seems to be a characteristic feature of sea turtles.Using computed tomography and classical comparative anatomy we describe the cranial anatomy in two sea turtles, the loggerhead (C. caretta and L. kempii the ability to suction feed does not seem to be as well developed as that found in some freshwater aquatic turtles. Instead both have skulls suited to forceful biting. This is consistent with the observation that both taxa tend to feed on relatively slow moving but sometimes armoured prey. The broad fleshy origin of the m. adductor mandibulae externus Pars superficialis may be linked to thecheek region being almost fully enclosed in bone but the relationship is complex.In  Caretta caretta, Chelonia mydas, Dermochelys coriacea, Eretmochelys imbricata, Lepidochelys kempii, Lepidochelys olivacea, and Natator depressus (sensu Fritz and Havaš Sea turtles (Chelonioidea) are adapted to a marine lifestyle in possessing flippers, hydrodynamic shells, lacrimal salt glands, and a specialised physiology for diving epressus   and their phylogenetic relationship to other amniotes remains contentious e.g. . Althougids e.g. . Althougids e.g. , there aids e.g. .EunotosaurusEarly cladistic studies placed turtles within anapsids close to or within captorhinids, pareiasaurs, or procolophonids e.g. . HoweverAs stated above, the precise phylogenetic position of Chelonioidea within crown turtles (Testudines) remains uncertain Ch. mydas is the sister taxon of E. imbricata , as well as support for a grouping of C. caretta and L. kempii + L. olivaceaC. caretta and L. kempii diverged from one another during the Miocene  but this has yet to be fully surveyed. The variation makes it difficult to identify diagnostic skull shape differences between the two taxa that are size independent, especially because adult C. caretta are much larger than adult L. kempii, many published images lack scale bars or measurements  is relatively large compared to the snout region . Large specimens of C. caretta possess a prominent spine extending from the posterior edge of the squamosal . In C. caretta and L. kempii, the squamosals meet the parietals and, medial to this, the supraoccipital projects posteriorly to form the crista supraoccipitale  . However, this feature may not be present in adults The lower jaws are heavily built in both taxa and are fused at the symphysis . The denentrally . The posentrally . The dorM009/08; . This feC. caretta and L. kempii are covered by scales composed of thickened epidermis and keratin L. kempii than it is in C. caretta.The heads of both The hyoid apparatus supports the tongue, pharynx, and floor of the mouth et al. Within living Testudines, two distinct mechanisms retract and move the neck/head region: the cryptodire condition, where the neck is moved and folded in a vertical plane, and the pleurodire condition, where the neck is moved and folded in a horizontal plane L. kempii (M009/08) has a relatively wide neural arch from which two processes extend posterolaterally over the prezygapophyses of the axis. The atlas centrum and neural arch are anteroposteriorly short .The atlas is notably different from other neck vertebrae. That of M009/08; . The antM009/08; . The assM009/08; . The axiM009/08; . The fouM009/08; . These hC. carettaCh. mydasDermochelys coriaceaE. imbricataLepidochelys kempii or Natator depressus are known to the authors, and only a few aspects of this anatomy in L. olivacea have been reported  is defined according to possession of its own origin, course, and insertion site. In contrast to muscles s.s., however, muscular portions s.s. show some intercrossing fibres along their course. Muscular units may be partly subdivided into muscle heads. Muscle heads have their own origination or insertion site, but in their course they fuse with other muscle heads to form the discrete belly of a single muscular unit C. caretta or L. kempii: No. 18, 20, 22 (but see description of No. 21c), 24, 27 (but see description of No. 26), 30, 32–33, 39, 40, 44, 54, 56, 59, 61–62, 76, 79, and 84–85.Here we follow the system proposed by Werneburg C. caretta is usually described first followed by the differences found in L. kempii where present.In the following account, the anatomy of C. caretta and L. kempii this large ligament is visible between the jugal and the jaw joint  below the shallow ventrolateral emargination . Direct examination and removal of the surrounding muscles reveals the aponeurosis to be broadly trapezoidal in medial view with the anteroventral corner inserting onto the coronoid  and mandibular (V3) branches of n. trigeminus The m. adductor mandibulae externus , was previously figured by Schumacher . In acco. kempii  but in tC. caretta, Schumacher L. kempii  represents the third unit of m. adductor mandibulae externus (No. 17–21). Pars superficialis lies in the lateral area of the postorbital adductor cavity, partially covering the Pars medialis (No. 17) caudally, but previous descriptions of its origin and insertion in C. caretta and attach to the dorsal or lateral surface of the coronar aponeurosis. Other descriptions  was found to originate by three distinct heads within the lateral part of the adductor chamber (C. caretta the remainder of the third muscle head (No. 21c) inserts into a broad aponeurosis that is closely apposed to the lateral surface of the lower jaw (surangular) but wraps ventrally to insert on the ventrolateral edge of the dentary and angular  is slightly different. The medial portions  originate from the prootic and opisthotic, but not from the parietal, with the second head (No. 21b) having a relatively larger area of origin posteriorly. The lateral fold in the coronar aponeurosis (to which No. 21 a+b insert) is also less obvious  and maxillary branches (V2). In the species described here, it includes the Pars pseudotemporalis principalis (No. 23), Pars intramandibularis (No. 25), and two Partes pterygoidei  The m. adductor mandibular internus (No. 23–28) is innervated by n. trigeminus (V) and is located between its ophthalmic  and the Pars pseudotemporalis superficialis (No. 24). The former is said to arise from the ventrolateral surface of the parietal and to converge on a tendon which inserts onto the lower jaw posteromedial to the adductor chamber. Posterodorsal to the origin site of Pars superficialis principalis (No. 23), the superficial unit (No. 24) arises from the dorsolateral surface of the processus descendens parietalis. The superficial unit converges on the Zwischensehne  is divided into two portions. Most fibres of this muscular unit originate from the lateral face of the processus descendens parietalis  is similar . The same muscle arrangement is also present in L. kempii (XT043/08)   F, G.C. caretta, the m. adductor mandibulae internus Pars pterygoideus dorsalis (No. 26) originates from the dorsal surface of the palate  and from the interorbital septum ventrolaterally, and some fibres may derive from the processus descendens parietalis. The posteriormost fibres may be homologous with the m. adductor mandibulae internus Pars pterygoideus posterior (No. 27). After it passes over the posterior edge of the palatine, the muscular unit inserts on the medial face of the prearticular by a tendinous sheet that is continuous posteriorly with the tendinous sheet of the Pars pterygoideus ventralis (No. 28)  is more certain. The Pars pterygoideus dorsalis (No. 26) originates from a smaller area  (In (No. 28) . Some fi(No. 28) . In L. kietalis) , and 9A ietalis) .C. caretta, this unit is composed of two muscle heads that are separated superficially but merge at their origin and insertion sites. The larger and more medial head (No. 28a) originates from the posteroventral edge of the palatine and the complete ventral surface of the pterygoid  was described and figured by Schumacher , the Sehnenspiegel is not as clear and the fleshy insertion of the smaller head (28b) does not extend as ventrally . However, it may not be homologous with the m. adductor mandibulae posterior of some other reptiles because of apparently different developmental origins This muscular unit is innervated by n. trigeminus (V) and is located posterior to the mandibular branch  has been described as having two heads of origin in  prootic . It exte prootic . Accordientrally .This muscle is innervated by n. trigeminus (V) and connects the ventral aspects of the lower jaws C. caretta, the Pars intermandibularis (No. 42) and Pars oralis (No. 43) are separated ventrally, whereas in L. kempii they are completely continuous  in C. caretta.In contrast to m. intermandibularis (No. 31), the m. constrictor colli complex  is innervated by n. facialis (VII). The intermandibular unit of m. constrictor colli (No. 42) lies between the posteroventral edges of the lower jaws and continues anteriorly to m. intermandibularis (No. 31) in both species. A median raphe is visible. The m. constrictor colli Pars spinalis (No. 41) originates from the lateral aspects of the anterior cervical vertebrae and wraps beneath the throat where it inserts into a median raphe. The m. constrictor colli Pars oralis (No. 43) originates from a midline raphe on the dorsal edge of the neck, passes ventrad, and inserts into a ventral median raphe. In ntinuous . Below tThe m. depressor mandibulae (No. 45) is a large fleshy muscle located at the posterolateral corner of the skull and is innervated by n. facialis (VII)  also originates from the ventral and lateral faces of the squamosal (Video S3) and has a tendinous attachment in its lateralmost region.The muscle is composed of lateral (No. 45a) and medial (No. 45b) parts divided by an internal tendinous divison but both components merge prior to their insertion . The latThis muscle is innervated by n. facialis (VII) and has a close association with the m. depressor mandibulae No. 45; . The thiL. kempii, this muscle also originates from cornu branchial-I but via less substantial connective tissue. It inserts on the lower jaw around and beneath the m. depressor mandibulae (No. 45) and is partly fused with the m. constrictor colli complex (No. 41–43).This muscle is innervated by n. glossopharyngeus (IX) C. caretta, the muscle extends rostrad and inserts laterally, via a tendon, on the atlas and directly into connective tissue around the m. depressor mandibulae (No. 45) and cornu brachial-I. In L. kempii, the lateral aspect of the muscle is partly fused with the m. constrictor colli complex (No. 41–43) along its course and the insertion onto the atlas is not tendinous.This muscle is innervated by n. accessorius (XI). As we only had access to head- and anterior neck material, the origin of this strap-like muscle could not be identified. However, in both species, the origin is caudal to the sixth cervical vertebrae and presumably on the plastron L. kempii, but in its place is a very thin tendinous structure between the cornu branchial-I and the squamosal.This muscle is innervated by n. accessorius (XI) and originates from the posterior end of the squamosal above the origin of m. depressor mandibulae (No. 45) and the insertion of m. collosquamosus (No. 57). M. squamosobranchiale inserts onto the distal edge of cornu branchial-I . The musThis muscle is innervated by n. hypoglossus (XII). In both taxa the muscles originates from the anterior edge of cornu branchial-I, ventral to the origin of m. branchiomandibularis visceralis (No. 47) and passes anteriorly to insert onto the corpus hyoidei .C. caretta . The insertion of the m. collosquamosus (No. 57) onto the back of the skull appears to be broader in L. kempii (and no spine is present).This muscle is innervated by n. hypoglossus (XII). It originates from cervical vertebrae-3 and -4, and possibly also from -5. It passes rostrolaterad and fans out at the level of the atlas to insert on the posterior surface of the squamosal, ventromedial to the insertion of m. squamosobranchiale (No. 53), dorsomedial to m. depressor mandibulae (No. 45), and lateral to m. atlantoepistropheooccipitis (No. 75) . The venThis muscle is innervated by n. hypoglossus (XII) The tongue of turtles is composed of several small muscular units including, for example, the mm. genioglossus (No. 63) et hypoglossus (No. 69) C. caretta, the insertion may also involve the posterior edge of the supraoccipital  inserts on the posterior edge of the squamosal so that its mediolateral edge overlies the lateral edge of the m. collooccipitalis (No. 80)  . The latThis muscle originates from the ventrolateral surfaces of the fifth to the seventh vertebrae and passes rostrad along the side of the neck before converging and inserting anteriorly in the region of the atlas and axis.The anterior unit (No. 86) has a mainly fleshy origin from the ventral surface of the atlas. The posterior unit (No. 87) is tendinous  and arisAs a part of the multiportioned m. retrahens collique, this unit (No. 88) originates from the ventral face of the carapace Caretta caretta and Lepidochelys kempii share a number of general features related to their musculature. However, available descriptions of other taxa suggest that many of these similarities are more widely distributed within turtles Eretmochelys imbricataC. caretta and L. kempii: insertion of the m. adductor mandibulae internus Pars pseudotemporalis principalis (No. 23) into a Zwischensehne and, in turn, the m. intramandibularis (No. 25). Whether this character is present in L. olivacea remains to be determined but it potentially provides a character for diagnosing the Carettini.C. caretta and L. kempii there are also several differences. These include the relative proportions of different muscle parts, the exact location of origins and insertions, and the relationship between muscles and particular aponeuroses. In L. kempii, for example, the second head of the m. adductor medialis externus Pars superficialis (No. 21b) has a relatively more extensive origin than in C. caretta  has a more ventrally located insertion than in L. kempii (L. kempii the m. depressor mandibulae (No. 45) originates from more lateral and ventral locations on the squamosal, than in C. caretta. In general muscle organisation tends to be more complicated in C. caretta than in L. kempii where the m. adductor medialis externus (No. 17–21) and m. constrictior colli complex (No. 41–43) are less finely divided, the m. branchiomandibularis visceralis (No. 47) is less distinct, and the m. squamosobranchiale (No. 53) appears to be entirely absent. Whether this is related to the greater adult size of C. caretta remains to be tested.Despite a general similarity in the cranial muscle anatomy of  caretta , 12B, wh. kempii . In L. kOne feature apparently common to Chelonioidea appears to be the presence of a large fleshy m. adductor mandibulae Pars superficialis (No. 21) with a broad origin inside the cheek and an insertion on the lateral side of the lower jaw Dermochelys coriacea remains highly pelagic and forages throughout the water column for jellyfish and salps Caretta caretta moves to coastal waters and feeds opportunistically on a variety of benthic prey  with seasonal variations Chelonia mydas also moves to near shore habitats but becomes herbivorous and feeds on sea grass and algae Lepidochelys kempii tends to move to coastal waters as an adult when it generally preys on crustaceans such as crabs L. olivacea is more oceanic Natator depressus generally inhabit coastal waters and shallow bays where they feed on soft corals, sea grass, sea pens, and soft-bodied invertebrates E. imbricata usually lives in nearshore reefs and eats sponges Sea turtles generally begin life as pelagic carnivores (or at least omnivores) but as adults they demonstrate a range of feeding ecologies Feeding within an aquatic medium is problematic because a predatory strike can deflect prey away from the mouth C. caretta and L. kempii is consistent with some use of suction feeding C. caretta and L. kempii possess a number of cranial features that suggest they are capable of forceful biting C. caretta and L. kempii tend to take rather slow moving but occasionally armoured prey such as sea pens, crabs, and molluscs The relatively small tongue in C. caretta and L. kempii possess a coronar aponeurosis into which jaw muscles from the back of the adductor chamber insert (e.g. m. adductor mandibulae externus Pars profundus [No. 19]). Because this aponeurosis loops over the otic area . Otherwise the orientation of the Partes pterygoidei  suggests their main function is to close the jaws and pull them forward, particularly when the jaws are wide open The orientations of muscle paths can provide a good indication of their role during jaw movement. For example, Curtis et al. The ligamentum quadratomaxillare Ch. mydas, E. imbricata, L. olivacea, and Natator depressus are probably broadly comparable to those of C. caretta and L. kempii, but this requires further examination with appropriate consideration for ontogenetic and geographic variation Dermochelys coriacea differs from other chelonioids The skull mechanics of Proganochelys quenstedti, Captorhinus sp., Sea turtle skulls are of particular interest to comparative anatomists because, unlike those of most extant turtles, which have variably developed ventrolateral and posterodorsal emarginations, their temporal region is almost entirely covered by bone . The posGopherus polyphemus, Hieremys annandalii, Heosemys spinosa, Terrapene carolina and T. ornata, Chelodina expansa, Chelodina novaeguinae, Carettochelys insculpta  and the medial wall of the temporal region in turtles warrants consideration. In chelonioids, the muscle’s fleshy origin should mean that resulting loading is evenly spread rather than locally concentrated at a tendinous origin, a condition that may promote the deposition of bone L. kempii does the m. depressor mandibulae encroach on the lateral surface of the squamosal, and this taxon has wider posterodorsal emarginations and a more dorsolateral origin of the m. adductor mandibulae externus Pars superficialis from the squamosal. Non-chelonioid turtles with a more posterior origin of m. depressor mandibulae include Chelydra serpentina, Emys orbicularis, Malaclemys terrapin litoralis, and Podocnemis expansaP. expansa are small Chely. serpentina, Em. orbicularis and M. terrapin litoralis are relatively large Terrapene carolina, Pelodiscus sinensis, Lissemys punctataEmydura macquariiThe relatively small posterodorsal emargination in chelonioids may be associated with the more posterior origin of m. depressor mandibulae from the squamosal D. coriacea arguably shows the least emargination  (Between 2007 and 2008 three Kingdom) . They weOsteological material was also examined from collections at and the Natural History Museum of Los Angeles County, USA (LACM); Grant Museum of Zoology, University College London, UK (LDUCZ); and University Museum of Zoology Cambridge, UK (UMZC) .Material was received and stored as frozen, but prior to dissection it was soaked overnight in diluted commercially available fabric softener. This made the tissues much easier to separate L. kempii (M009/08) and C. caretta (XT757/07) were were subjected to micro-computed tomography (micro-CT) at the University of Hull, UK using a X-Tek HMX 160 scanner using a Beryllium target and aperture of 75%. To reduce beam hardening, the X-rays were filtered through a 0.1 copper plate. M009/08 was scanned using 1149 projections averaging 64 frames per projection, whereas XT757/07 was scanned using 1113 projections averaging 32 frames per projection. Anatomical structures were segmented using the software Amira 4.1 (Konrad-Zuse-Zentrum für Informationstechnik Berlin). Voxel resolution was 0.121 mm3 for M009/08 and 0.081 mm3 for XT757/07. The scan of L. kempii (M009/08) was particularly successful and included details of the muscle arrangement  based on reconstructions from micro-CT data. The surface model has been simplified to 410000 faces. Also see (MPEG)Click here for additional data file.Video S2Cranium of of a young Lepidochelys kempii (M009/08) based on reconstructions from micro-CT data. The surface model has been simplified to 500000 faces. Also see (MPEG)Click here for additional data file.Video S3Skull of a young Lepidochelys kempii (M009/08) with parts of the right temporal region absent and jaw muscles shown. The model is based on reconstructions from micro-CT data. Also see i M009/0 with par(MPEG)Click here for additional data file.


Blocking the androgen receptor (AR) activity is the main goal of therapies for advanced prostate cancer (PCa). However, relapse with a more aggressive, hormone refractory PCa arises, which harbors restored AR activity. One mechanism of such reactivation occurs through acquisition of AR mutations that enable its activation by various steroidal and non-steroidal structures. Thus, natural and chemical compounds that contribute to inappropriate (androgen-independent) activation of the AR become an area of intensive research. Here, we demonstrate that genistein, a soy phytoestrogen binds to both the wild and the Thr877Ala (T877A) mutant types of AR competitively with androgen, nevertheless, it exerts a pleiotropic effect on PCa cell proliferation and AR activity depending on the mutational status of the AR. Genistein inhibited, in a dose-dependent way, cell proliferation and AR nuclear localization and expression in LAPC-4 cells that have wild AR. However, in LNCaP cells that express the T877A mutant AR, genistein induced a biphasic effect where physiological doses (0.5-5 µmol/L) stimulated cell growth and increased AR expression and transcriptional activity, and higher doses induced inhibitory effects. Similar biphasic results were achieved in PC-3 cells transfected with AR mutants; T877A, W741C and H874Y. These findings suggest that genistein, at physiological concentrations, potentially act as an agonist and activate the mutant AR that can be present in advanced PCa after androgen ablation therapy. Prostate Cancer (PCa) is the most common malignancy and the second leading cause of cancer death among men in United States . In Asiain vitro studies genistein reduced expression and transcriptional activity of the androgen receptor (AR) ). However, virtually all of those patients relapse with a more aggressive, hormone refractory (castration-resistant) form of PCa which does not require circulating androgen, but still depends on functional AR for growth and progression. There are several proposed mechanisms for the molecular switch of PCa from an androgen-dependent to an androgen-independent state, including evidence to suggest that the growth of most recurrent PCa is driven by inappropriate activation of the AR –32. AR ain vitro studies genistein downregulated AR transcription and PSA protein expression in PCa cells and inhibited their growth -R1881 with or without 0.1–1,000-fold molar excess of unlabeled competitor ligands  for 90 min at 37°C. Cells were washed with phosphate buffer, and the bound 3[H]-R1881 was extracted in ethanol for 30 min at room temperature and detected by scintillation counting. This analysis was done by using the method described by Siddique et al. . Briefly-1 Å-2 restraints on the complex and followed by 500ps of constant pressure equilibration with 0.5 kcal mol-1 Å-2 restraints on the complex at 300K. After equilibration, 20 ns production run was performed to assess free energy convergence and coordinates were extracted every 2 ps. Root mean squared deviation (RMSD) analyses were performed using the ptraj module of Amber 11. MM/PBSA is a well-established method to calculate binding free energies -R1881competitive binding assay showing that genistein, starting at low physiological doses (1µmol/L), effectively competed with labeled androgen for binding to both the WT and mutant AR in LAPC-4 and LNCaP cells, respectively. Collectively, these data indicate that genistein binds to the AR, but with more affinity and stability to the T877A mutant AR than to the WT AR. In line with the ability of genistein to enhance the AR activity in PCa cells with the T877A mutation, AR, a member of the nuclear hormone receptor family, is activated by binding to its specific ligand, dihydrotestosterone, or is constitutively activated –69. We sUltimately, assessment of the status of AR activity is critical in elucidating the biological impact of this potential AR binding of genistein in both LNCaP and LAPC-4 cells. Thus, we examined the effect of a range of genistein doses on AR transcriptional activity in both cell lines, using expression of the AR-responsive gene PSA as a surrogate marker. Given that AR possesses an autoregulatory function, we employed the amount of AR expression as another indicator of AR activation. Suppression of LAPC-4 cell proliferation was tightly associated with a reduction of AR protein and mRNA levels, as well as PSA expression in response to increasing genistein from physiological to supraphysiological doses. Similarly, in LNCaP cells, the biphasic proliferation response was associated with a parallel biphasic response of AR and PSA expression. Collectively, our data provide strong evidence that the differential response of LNCaP and LAPC-4 cells to genistein is AR mediated and T877A mutation dependent. in vitro studies examining the effects of genistein on PCa cells have yielded conflicting results; some studies demonstrated that genistein downregulates AR expression and inhibits cell proliferation [Results from other feration ,26,27, wferation –47. Our in vitro studies with purified soy compounds in cancer cells may not reflect effects of dietary soy on prostate cancer in vivo, epidemiological observations suggest a protective role of soy in PCa [Although y in PCa ,5, impliy in PCa ,8. Levely in PCa , indicatCumulatively, our data add a new insight to the understanding of the bioactivities of genistein in PCa, which are affected by several factors including the dose of genistein and the mutational status of the AR. This study indicated that genistein at low, physiologically relevant exposure levels can be an endocrine disruptor and can result in altered activity of multiple tumor-derived mutant AR alleles. Therefore, genistein, especially at lower doses, can function as a potential ‘‘hormone sensitizer’’ of the mutant ARs present in advanced PCa following androgen ablation therapy, thereby possibly contributing toward therapeutic relapse in soy consuming PCa patients who have cancers carrying the T877A or similar mutations such as W741C and H874Y. The T877A AR variant is of obvious biological relevance as it has been reported in up to 12.5% of hormone-refractory PCa ,72,73 anInvestigations with the orthotopic xenograft models using human PCa cell lines with these AR-LBD mutations are warranted in order to validate our findings, given the close resemblance of these models to the actual prostate tissue conditions of stromal cells and physiological and hormonal milieu. Furthermore, it is essential to investigate whether other soy phytoestrogens such as daidzein, quercetin, and equol have similar effects. It will be also of interest to assess contributions of AR cofactors to genistein-mediated transactivation of mutant ARs. In fact, it has been reported that certain cofactors can enhance AR transactivation in the presence of non-androgenic steroids and that deregulation of these cofactors had been implicated in prostate cancer evolution to androgen independence –77.


Although androgen receptor (AR) function has been extensively studied, regulation of the AR gene itself has been much less characterized. In this study, we observed a dramatic reduction in the expression of androgen receptor mRNA and protein in hyperproliferative prostate epithelium of keratin 5 promoter driven E2F1 transgenic mice. To confirm an inhibitory function for E2F1 on AR transcription, we showed that E2F1 inhibited the transcription of endogenous AR mRNA, subsequent AR protein, and AR promoter activity in both human and mouse epithelial cells. E2F1 also inhibited androgen-stimulated activation of two AR target gene promoters. To elucidate the molecular mechanism of E2F-mediated inhibition of AR, we evaluated the effects of two functional E2F1 mutants on AR promoter activity and found that the transactivation domain appears to mediate E2F1 repression of the AR promoter. Because DNMT1 is a functional intermediate of E2F1 we examined DNMT1 function in AR repression. Repression of endogenous AR in normal human prostate epithelial cells was relieved by DNMT1 shRNA knock down. DNMT1 was shown to be physically associated within the AR minimal promoter located 22 bps from the transcription start site; however, methylation remained unchanged at the promoter regardless of DNMT1 expression. Taken together, our results suggest that DNMT1 operates either as a functional intermediary or in cooperation with E2F1 inhibiting AR gene expression in a methylation independent manner. Androgens are required for prostate gland development and for prostatic function and glandular maintenance in the adult male TATA and CAAT), but is rich in GC sequences Transcriptional regulation of AR is cell specific and age-dependent ARFThe E2F family of transcription factors control cell proliferation by regulating cell cycle progression de novo methylation during early embryogenesis The DNA methyltransferases  play an integral role in the epigenetic regulation of many genes. All DNMTs except for 3L have a catalytic domain that facilitates the transfer of methyl groups from S-adenosyl-methionine to cytosines located in CG dinucleotides. DNMT 3a and 3b are generally responsible for genome-wide In this study, we explored a regulatory mechanism that controls the endogenous expression of AR in prostate epithelium. We investigated the effects of the transcription factor E2F1 on AR mRNA and protein expression in both human and mouse prostate epithelial cells. We demonstrate how E2F-1, a classical transcriptional activator, might cooperate with DNMT1 to repress AR transcription in the prostate gland.Accumulating evidence suggests that increased E2F1 activity reactivates several aspects of benign and malignant disease including increases in cellular proliferation To determine if E2F1 directly represses AR transcription, we examined whether exogenous expression of E2F1 reduces AR mRNA levels in prostate epithelial cells. Stable E2F1 over-expressing clones were established in mouse prostate epithelial cells (PrE) and two clones, PrE2F1-1 and PrE2F1-2, were expanded and characterized. Total RNA was harvested and subjected to Northern blot analysis for the detection of AR and E2F1 mRNA. Both clones exhibited increased E2F1 mRNA and significantly reduced AR mRNA  and protThe findings from the transgenic animals indicated that E2F1 might be driving a proliferative and undifferentiated phenotype. We had previously observed the AR-regulated prostate specific antigen (PSA) gene was down regulated following E2F1 over expression suggesting a repressive activity of E2F1 on AR target genes through the repression of AR To determine if E2F1 exerts repressive activity directly on the AR promoter, we utilized a 1.5 kb (−1571 to +131 bp) mouse AR promoter construct  cloned u1–284) abrogated the inhibitory effect of E2F1 on the AR promoter  failed to significantly relieve E2F-mediated inhibition . Howeverpromoter . As expepromoter . These rWe have previously shown that the DNA methyltransferase 1 (DNMT1) gene, which typically functions to maintain the methylation and repression of specific genes, was trans-activated by E2F1 To understand how DNMT1 functions to represses AR expression, we explored the possibility for DNMT1 to physically associates with the AR gene. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis identified both DNMT1 and E2F1 associated regions across the whole hPrEC genome (data not shown). H-peak analysis 5-aza-2′-deoxycytidine (5-Aza) and observed no change in AR transcription  relieved AR promoter repression by inhibiting access of endogenous E2F1 to E2F targeted promoters. All of these observations correlated with variations in activation of the E2F-target gene promoter DHFR-Luc, and changes in expression levels of endogenous cell cycle regulatory proteins consistent with E2F1 activity. The use of E2F1 functional mutants suggests that the transcriptional regulatory domain of E2F1 comprises this repressive activity possibly through the interaction of an intermediary co-repressor. Based on studies showing that DNMT1 is both a target Sharma et al. have recently demonstrated that E2F1 transactivates the AR gene on a depleted RB1 background in an engineered model of castrate resistant prostate cancer TTTGCGG/CG/CAAA), furthermore the E2F1 DNA binding domain was not required for repression of the AR promoter suggesting that E2F1 does not bind directly to the AR promoter, but cooperates with other regulatory proteins to repress AR. Our data demonstrate that the carboxy-terminal transactivation domain was essential for E2F1 suppression of the AR promoter  re-expression at both the transcriptional and protein level, results from the targeted inhibition of DNMT1 in ER negative breast cells in vitro chromatin array methylation assays. DNMT1 is also known to directly interact with the enhancer of zeste 2 (EZH2) protein, which mediates H3K27me2/3, in the context of the Polycomb repressive complexes (PRC2/3) DNMT1 appears to function in concert with other factors to regulate gene expression. Heterochromatin protein 1 (HP1) GST fusions pull down DNMT1, 3a, and 3b Clearly, mechanisms for AR amplification and mutation play a role in prostate cancer progression, however, loss of AR has been reported in a subset of hormone-independent cancers, including a complete loss in some cases We suggest that the repression of AR may facilitate a proliferative state in normal transit amplifying basal cells. However, androgens are generally understood to promote epithelial proliferation in AR–responsive cell lines, such as LNCaP and in the regenerating prostate gland following castration. Some suggest that androgens may contribute to re-expanding cellular populations by directly activating androgen receptors expressed in the epithelium. While studies demonstrate that the proliferation of the developing epithelium is supported by the secretion of growth factors from AR activated stromal cells LNCaP and DU145 cell lines obtained from American Type Culture Collection, Rockville, MD and BPH-1 cells received from Dr. Simon Hayward, Vanderbilt University Medical Center For the development of the PrE-E2F1 stable cell lines, PrE cells were transfected with pcDNA3-E2F1 (kindly provided by W. Kaelin −9 M for 24 h after 15 h of serum starvation. All 5-aza-2′-deoxycytidine (5Aza) (Sigma) treatments were done in either complete culturing media used for DU145 or hPrEc lines. Fresh media containing 1 µM 5Aza was added every 24 h for a total of 72 h and DMSO treatments were matched as vehicle controls.All synthentic androgen (R1881) treatments were done at 1032P] dATP using the random oligonucleotide-primer labeling kit (STRATAGENE) and purified on STRATAGNE Nucleotide Push Columns following manufacturer's protocol. The [α-32P] dATP labeled probes were hybridized to a Duralon-UV membrane (STRATAGENE) at 65°C overnight in hybridization buffer  while rotating. The membrane was subsequently washed twice for 45 min each in 20 mM Na2HPO4, pH 7.2 and 5% SDS followed by two additional washes for 45 min each in 20 mM Na2HPO4, pH 7.2 and 1% SDS. The membranes were exposed to X-ray film  overnight and visualized by autoradiography.Total RNA was prepared using QIAGEN RNA Easy kit per manufacturer's protocol . Twenty micrograms of RNA was resolved by gel electrophoresis under denaturing conditions and RNA was transferred to a Duralon-UV membrane  overnight by capillary action in 20× SSC buffer (3 M NaCl and 0.3 M Na Citrate). RNA was crosslinked to the membrane by UV cross linking. A 1.6 kb human AR cDNA fragment was isolated from CMV3-hAR3.1  using a HindIII and NheI restriction enzyme sites. A 1.5 mouse AR cDNA fragment was isolated from CMV5-mAR  using HindIII restriction enzyme sites. The human and mouse AR cDNA fragments were gel purified using QIAGEN Gel Purification Kit, per manufacturer's protocol and subsequently labeled with [α-Cells were either trypsinized, centrifuged and washed one time with PBS then lysed with RIPA buffer  or lysed directly in the plate. The hPrEC lines were harevested 4 days post infection, while stably infected BPH-1 cells were collected 4 days post selection in puromycin (1 µg/ml). Protein concentrations were determined using Bradford Protein Assay Reagent , following manufacturer's protocol. For Western blot analysis, protein extract was subjected to gel electrophoresis either on a Tris-glycine polyacrylamide gel (Invitrogen) (LNCaP and mPrE) or on a Nu Page 3–8% Tris-acetate polyacrylamide gel (Invitrogen) (BPH-1 and hPrEC). The gel was transferred to Optitran nitrocellulose membrane  by electrophoresis for 1 hour at 45 V. The membrane was blocked in 10% nonfat dry milk in TBST  for 1 hour at room temperature and immunoblotted with primary antibodies for AR , E2F1 , Rb (Pharmingen), PCNA , E-Cadherin (H-108 Santa Cruz), Cyclin E , DNMT1 , β-actin , or actin . The membrane was incubated with secondary antibody conjugated to horseradish peroxidase  and the bands were detected using ECL  detection system, following manufacturer's protocol.5 cells per 6 well dish and incubated at 37°C overnight. Stably infected BPH-1s with DNMT1 shRNAs were plated at a 1 to 60 passage into a 12 well dish and incubated at 37°C overnight. Cells were co-transfected with 1 µg/ml of either of the following promoter-luciferase reporter constructs; DHFR-Luc, E2F-Luc and CRE-Luc were kindly provided by G. Denis, Boston University, Boston, MA 1–284, or Eco132)  and human GAPDH (forward: TGCACCACCAACTGCTTAGC and reverse: GGCATGGACTGTGGTCATGAG) primers in a Mastercycler ep realpex2  using SYBR green PCR Master Mix  to amplify the cDNA with the following PCR conditions; denatured at 95°C for 3 min and subjected to 40 cycles . The primers were used in a separate PCR and electrophoresed on a gel to verify the presence of a single amplicon from the cDNA. Each sample reaction in the qRTPCR was done in triplicate in a 96 well plate format. Cycle threshold units were obtained using Mastercycler ep realpex2 software. Data was analyzed using the 2−ΔΔCT method Total RNA was extracted by scraping and collecting cells in TRizol  (1 ml per 60 mm dish). The lysate was added at 1 ml to a pre-spun 2 ml heavy phase lock gel tube , incubated for 5 min at room temperature, and combined with chloroform. After the mixture was centrifuged at 12,000×g for 10 min at 4°C, the resulting aqueous mixture above the wax plug was removed and mixed together with 500 µl of isopropanol, and incubated for 10 min at room temperature. The mixture was centrifuged into a pellet at 12,000×g for 10 min at 4°C and washed 1 time in 70% ethanol. RNA was reconstituted in 35 µl of UltraPure Distilled Water  and quantitated with the NanoDrop Spectrophotometer ND-1000 , treated with DNase I (Invitrogen), then converted to cDNA using the Thermoscript RT PCR Reaction System (Invitrogen) according to the manufacture's protocol. The qRTPCR was conducted with human AR , following 2 washes in cold PBS. The harvested cells were pelleted at 5,000 rpm for 6 min at 4°C and washed once with cold PBS containing protease inhibitors. Lysates were prepared using the reagents in the Magna ChIP A kit  according to manufacture instructions, however, the lysis buffer available was substituted with 400 µl of SDS lysis buffer (Millipore) containing kit supplied protease inhibitors. The chromatin in the lysate was sheared to ≤600 bps in a 2 ml tube placed in a Covaris S2  water bath set to the following cavitation parameters: duty cycle, 20%; intensity, 5; cycles per burst, 200; cycle time, 30 sec; and cycles, 30. The sheared chromatin was processed and immunoprecipitated with 5 µg of either DNMT1  or rabbit IgG  using the reagents and instructions provided in the Magna ChIP A kit. The purified ChIP DNA was retrieved with 40 µl of elution buffer C. The DNA sample was amplified with a two step PCR program (Denaturation at 95°C for 10 min and 40 cycles of 95°C for 15 sec and 60°C for 1 min) using SYBR green PCR Master Mix in a StepOnePlus Real-Time PCR thermocycler (Applied Biosystems) employing the following primers; human E2F site A (forward: GACTCGCAAACTGTTGCATT and reverse: TACAGCACTGGAGCGGCTA), Site B (forward: CCTAGCAGGGCAGATCTTGT and reverse: TCCCCTTCTCTTGCTCAGAA), Site C (forward: GGTAGGAAGTGGCTGAATTCTGGATGA and reverse: CCCTGCCCATGCACCTGCTC), PS2 (forward: TTCCGGCCATCTCTCACTAT and reverse: CGGGGATCCTCTGAGACA), and ABCB1 (forward: TCTAGAGAGGTGCAACGGAAGCCA and reverse: CCTGCCCAGCCAATCAGCCT). An extended program  was used to create a melting curve that was analyzed with the StepOne software v2.1 package to verify that the primers only amplify a single amplicon from genomic DNA. Each sample reaction in the qPCR was done in triplicate in a 96 well plate format. Cycle threshold units were obtained using StepOne software v2.1. Data is represented as a percent of input using a derivation of the 2−ΔCT method For each ChIP 1×10GGGAGTTAGTTTGTTGGGAG and reverse: TCCTACCAAACACTTTCCTTACT), were created with Methyl Primer Express v1.0. Amplification of the bisulfite converted gDNA was accomplished using special ZymoTaq PreMix (Zymo Research) polymerase to facilitate the production of amplicons with A overhangs using the following PCR program: denature at 95°C for 10 minutes, run 35 cycles , run a final extension at72°C for 7 min, and hold at 4°C. PCR product was combined with pCR8/GW/TOPO TA cloning vector (Invitrogen) in a mixture prescribed by the manufacture to facilitate the insertion of the amplified products into the plasmids which contain sequencing primer sites that flank the insert. Plasmids were transformed and plated in One Shot Top 10 chemically competent cells (Invitrogen) per manufacturer's instructions and at least 12 bacterial colonies were individually grown in 5 ml of LB containing spectinomycin (100 µg/ml). Plasmids were harvested from the bacteria using the Wizard Plus SV Miniprep kit (Promega) and sequenced with the M13 forward and reverse primers at the University of Michigan DNA sequencing core.Genomic DNA (gDNA) was extracted from cells using the Wizard Genomic DNA Purification kit (Promega) and quantified with the NanoDrop Spectrophotometer ND-1000 . A 250 ng sample of DNA was bisulfite converted using the EZ DNA Methylation-Direct kit  according to the instructions provided by the manufacturer. The following bisulfite converted DNA specific primers, targeting a region in the AR (NM_000044) minimal promoter (forward: Data showing significance was analyzed using 2-tailed Student's t test. P<0.05 was accepted as the level of significance.


FOXO – which regulates growth arrest under nutrient stress – mediates a nutritional polyphenism in the horned beetle, Onthophagus nigriventris. Male beetles in the genus Onthophagus vary their mating strategy with body size: large males express horns and fight for access to females while small males invest heavily in genitalia and sneak copulations with females. Given that body size and larval nutrition are linked, we predicted that 1) FOXO expression would differentially scale with body size  between males and females, and 2) manipulation of FOXO expression would affect the nutritional polyphenism in horns and genitalia. First, we found that FOXO expression varied with body size in a tissue- and sex-specific manner, being more highly expressed in the abdominal tissue of large (horned) males, in particular in regions associated with genitalia development. Second, we found that knockdown of FOXO through RNA-interference resulted in the growth of relatively larger copulatory organs compared to control-injected individuals and significant, albeit modest, increases in relative horn length. Our results support the hypothesis that FOXO expression in the abdominal tissue limits genitalia growth, and provides limited support for the hypothesis that FOXO regulates relative horn length through direct suppression of horn growth. Both results support the idea that tissue-specific FOXO expression may play a general role in regulating scaling relationships in nutritional polyphenisms by signaling traits to be relatively smaller.We investigated whether insulin signaling, known to mediate physiological plasticity in response to changes in nutrition, also facilitates discrete phenotypic responses such as polyphenisms. We test the hypothesis that the gene  FOXO, which subsequently inhibits growth Variation in the quality and quantity of nutrition is a ubiquitous challenge during development both within and across generations. Organisms have evolved a wide range of behavioral, developmental, and physiological mechanisms to cope with such variation. Central among the developmental and physiological mechanisms is the insulin signaling pathway, which permits major adjustments in growth, development, body size, lifespan and behavior in response to nutrition  that support the entire larval development of individual offspring. The body size of an adult is largely influenced by the size and nutritional quality of their brood ball We sought to test the role of insulin signaling in nutritionally induced phenotypic plasticity using the sneaker-fighter polyphenism in horned beetles. Beetles in the genus FOXO, because it has been linked to variation in scaling relationships in a range of systems FOXO expression levels in developing horn tissue, relative to expression in the abdomen, of small, hornless males compared to large, horned males. Furthermore, recent work in Drosophila suggests that FOXO expression may regulate tissue-specific responses to nutritional variation We tested the hypothesis that insulin signaling plays a role in the development of this nutritional polyphenism in mating tactics in horned beetles, using a combination of observation and manipulation of patterns of gene expression. We chose to focus on one important player in the insulin signaling pathway, FOXO in this nutritional polyphenism. First, we investigated the hypothesis that FOXO regulates horn size relative to body size. Mechanistically, in this case, the activation of FOXO in the developing horn tissue of small, nutritionally stressed males is expected to repress horn growth. This hypothesis predicts that FOXO expression would be elevated in the developing horn rudiments of small males, and that knockdown of FOXO would result in larger horn length for a given body size. Second, we address the hypothesis that FOXO regulates genitalia investment relative to body size. Past research has suggested that costly horns FOXO expression in other developing regions, such as the genitalia, resulting in the repression of their growth. This hypothesis predicts that FOXO expression will be elevated in developing abdominal and genitalia tissues of large, horned males and that knockdown of FOXO would result in larger genitalia size for a given body size. Our results suggest that FOXO plays an important role in nutritional polyphenisms, negatively regulating relative trait size for both horns and genitalia.We sought to test two hypotheses for the potential role of FOXO transcript (912 bp of coding sequence) in Onthophagus nigriventris. Using BLASTp, this region significantly matched FOXO from Tribolium castaneum  with 79% matching amino acid identity. Additionally, we sequenced FOXO in several species of Onthophagus and aligned the protein sequences. We found that Onthophagus beetles, relative to Tribolium, have a large insertion (5–9 amino acids) in the coding region of the gene  was not correlated with body size in the developing horn region (the thoracic epidermis) of either males or females sacrificed as first day pupae  FOXO expression in the developing horns or prothorax of any of these samples  of large, horned males, but much less abundant in the corresponding tissue regions in small, hornless males. Contrary to our initial expectations, FOXO expression is indistinguishable in developing pupal horn tissues of large and small males.Taken together, our expression results suggest that FOXO RNAi had significant effects on body size and development time in males, but not females. Relative to control-injected individuals, RNAi males had significantly larger pupal thorax widths and pupal weights, extended length of their third instar, and marginally significantly larger adult thorax widths  or genitalia scaling relationships and thus was not included in subsequent analyses.x widths , Table 2cted see . However1,52F = 5.75, P = 0.02). Residual horn length did not differ between control and treatment males in the pupal stage . In females, which express a small horn as pupae and a ridge as adults, we observed no differences in horn-body size relationships between control-injected and RNAi individuals for either the pupal  or adult stage .We also detected a moderate effect of RNAi on horn expression. Relative to control-injected individuals, RNAi male adults had significantly longer horns relative to their body size , control: 1.58 (0.009), FOXO: 1.61 (0.009), see also F1,50 = 3.23, P = 0.07; size effect: F1,50 = 1,28, P = 0.26; see also FOXO regulates not only body size, but also the relative growth of traits important in a nutritional polyphenism – horns and genitalia.FOXO-injected  individuals. We tested for differences in relative FOXO expression in an ANOVA that included expression of the control gene actin , and a measure of body size . We found a significant effect of dsRNA injection on FOXO expression . Using the methods of Pfaffl FOXO expression, relative to control-injected individuals, for the abdomen and thorax, respectively. Because our validation was on individuals injected with 1.0 ug FOXO dsRNA, all analyses focused on individuals injected with at least 1.0 ug dsRNA.We used quantitative real-time PCR to validate the RNAi knockdown. We harvested thoracic and abdominal tissue from 20 male and female first-day pupae (within 24 hours of pupation) of control-injected (N = 11) and FOXO in the direct regulation of relative horn size in males. However, our data provide support for the hypothesis that FOXO regulates the relative size of male genitalia, thus playing an important role in trait integration across polyphenic morphs. These results are consistent with recent observations that FOXO expression regulates tissue-specific responses to nutritional variation in DrosophilaFOXO and insulin signaling, and future directions for investigating FOXO as a player in nutritional plasticity.We present evidence that suggests insulin signaling plays a role in nutrition-induced phenotypic plasticity in horned beetles. We found only modest support for a role of FOXO in particular. We found that FOXO knockdown affected both development time and body size . Variation in nutrient availability or timing of knockdown may result in variation among studies in the effect of FOXO knockdown on body size.We found that ody size , Fig. 4.ur study , Fig. 4.FOXO on body size in the present study was specific to males   of horned males .In situ hybridizations suggested that ed males , 3, althFOXO in the thoracic tissue, resulting in the repression of horn growth FOXO expression in other body regions which signals organs within those tissues to grow proportionally smaller. We sought to test this hypothesis further via knockdown of FOXO expression.Taken together, the results of the qPCR and in situ hybridization run counter to the initial hypothesis that small, nutritionally stressed males would express FOXO affected horn and genitalia size, relative to body size. We found that FOXO knockdown had significant effects on genitalia size relative to body size  such that relative differences in genitalia size were most pronounced in smaller sized individuals. It is possible that growth of horns in large males prevented genitalia growth by means of nutrient limitation despite the signal for continued growth stimulated through depressed FOXO expression, although future research is necessary to test this mechanism. FOXO RNAi also significantly affected the size of thoracic horns relative to body size, although the effect was modest overall  was weak in the prothorax of small, hornless males at the first day pupae stage , show significant differences in expression between polyphenic morphs in a range of systems FOXO expression in one body region is directly triggered by the growth of tissue in another body region (such as horns) or alternatively, is regulated by overall body size. Either scenario suggests different mechanisms by which FOXO results in trait integration, which could be addressed experimentally by manipulating horn size (via ablation) and nutrition (nutrient supplement or starvation) independent of body size. An additional interesting line of inquiry would be to integrate insulin signaling with endocrine signaling, a known mediator of polyphenisms The results of this work suggest several promising areas of future research. A key question for future studies is to investigate whether Onthophagus nigriventris were collected from populations in Waimea, Hawaii. No specific permits were required for the described field work  and we had owner permission for collecting on private land. Beetles were maintained in laboratory colonies using established methods Onthophagus taurus had identified a 704 base pair fragment that matched to FOXO . PCR fragments were cloned into a pSC-A vector with the Strataclone PCR Cloning kit . Clones were sequenced using M13 primers and BigDye PCR reactions , under RNase-free conditions: all dissecting tools were treated with RNase-Zap . Immediately after removal, tissue was placed in 350 µl Buffer RLT 1% v/v BME . Tissue was ground using a sterile, RNase-free pestle fit to the 1.5 µl microcentrifuge tube  and immediately frozen in liquid nitrogen before being stored at −70 C until RNA extraction.RNA was extracted from individual tissue samples using the RNeasy Mini Kit , following standard kit protocols. The optional on-column DNA digest was performed (using the Qiagen RNase-free DNase set) to reduce any genomic DNA contamination during the qPCR analyses. Total RNA was eluted in 25 µl RNase-free water , and quantified using a Nanodrop . Extracted RNA had an average yield of 7.07 µg, and an average purity of 260/230 = 1.94, 260/280 = 2.14. Total RNA was stored at −70°C until further processing for qPCR analyses.Total RNA was converted to cDNA using the Quantitect Reverse Transcription Kit  and standard kit protocols. Briefly, 250 ng total RNA was incubated with 2 ul gDNA wipeout buffer at 42°C for 2 minutes before being combined with Quantiscript reverse transcriptase, 5× buffer, and primer mix and incubated at 42°C for 15 minutes, followed by 85°C for 3 minutes. cDNA was quantified using a Nanodrop 1000 Spectrophotometer  and stored at −20 until the qPCR was performed. cDNA had an average yield of 1022 ng/ul, 260/280 ratio of 1.81 and 260/230 ratio of 2.24.FOXO  was performed using SYBR green PCR kit  and standard kit protocols. Prior to running our samples, we executed a minimum of five preliminary qPCRs to test a range of primers, control genes and test conditions. We designed primers for qPCR using Oligoanalyzer and PrimerQuest/Primer3 . Primers were tested at two concentrations over a four-fold dilution series of cDNA . Based on the Ct data for these standard curves and the dissociation curves from these runs, we chose one primer pair for 2 of standard curve generally 0.99) and consistent dissociation curves. Final primers for actin were based off of O. nigriventris actin sequences . The appropriateness of actin as a control gene was further verified by microarrays that directly hybridized tissues of male Onthophagus taurus of varying size FOXO expression across individuals of different sizes.We used published microarray data FOXO primers were run with 250 ng of cDNA. Standard curves (over a 4-fold dilution), no-template controls and no-reverse transcription controls were included for each primer pair for each qPCR run. Quantitative real time PCR was performed with a Stratagene MX3000P system ; SYBR green and ROX (the reference dye) fluorescent measurements were taken every cycle for 45 cycles. PCR settings were as follows: 95°C for 15 min; 45 cycles of 94°C for 15 second, 57°C for 30 second and 72°C for 30 seconds; followed by a 55°C–95°C dissociation curve. A small subset of samples  failed to amplify and were dropped from the final analysis.All samples of a particular tissue from a particular sex were run simultaneously  over four qPCR runs. Based on previous primer optimizations, we ran 1.2 ul of 10 uM actin primers with 250 ng amount of cDNA while 0.6 ul of 10 uM FOXO to vary with body size and nutrition, we included body size in all analyses. We controlled for the amount of tissue harvested by using a control housekeeping gene (actin) in all analyses FOXO expression. This represents an extension of the multiple regression methods developed by Yuan et al We focused on the crossing threshold (referred to as Ct from here-on), for all qPCR analyses. Because we hypothesized In situ-hybridizations were performed as described previously FOXO fragment cloned for RNAinterference, described below . M13 primers  were used to amplify the relevant fragment, which was gel-extracted, using the Qiaquick Gel Extraction kit . We used the MEGAscript kit  to synthesize RNA. Briefly, an in-vitro transcription reaction was run overnight at 37°C using 1 ug of PCR product, NTP mix, reaction buffer and either the T7 or T3 enzyme mix. Control dsRNA was generated using the same methodology applied to vectors with no inserted PCR product. Template DNA was removed by adding 1 ul Turbo DNase (37°C for 15 minutes); RNA was recovered by precipitating with 30 ul LiCl (and 30 ul water and chilling at −20°C for 4 hours). Concentration of product was verified using a Nanodrop (average = 3.57 ug.ul). Complementary RNA strands were annealed by mixing equal amounts of RNA (from with T7 and T3 reactions), heating to 80°C for 5 minutes and then slowly cooling to room temperature over 4 hours in an insulated beaker. dsRNA was brought to 3 ul with injection buffer  and stored at −70°C until injection.Double-stranded RNA was produced by first using the RNAi FOXO RNA, suspended in 3 ul of injection buffer. Control individuals were injected with 1.0 ug of double-stranded vector RNA suspended in 3 ul of injection buffer. Beetles were injected between day 6 and 10 of the third instar (mean (SD) = 8.31 (1.47)); variation in time of injection did not affect body size or horn-body size scaling. Injections occurred under RNase-free conditions using a gas-tight 1801 Hamilton syringe with a 32-gauge needle. Larvae were injected in the anterior, dorsal abdomen, just under the cuticle.Treatment individuals were injected with either 0.5, 1.0, 1.75, or 2.5 ug of double stranded 1,39F = 0.02, P = 0.88).We measured several phenotypic traits of larvae, pupae and adults. We used pupal thorax width, pupal mass and adult thorax width as measures of body size. The length of the third ( = last) instar, measured in days, was used as an estimate of development time. The third instar constitutes the dominant feeding stage of onthophagine larvae. We measured thoracic horn length (in pupae and adults) as described in FOXO-knockdown individuals . We used an ANOVA to test for significant differences between control and knock-down individuals, which allowed us to control for differences in body size among individuals. We also estimated the degree of knockdown (fold change) using the methods of Pfaffl actinE = 0.91; FOXOE = 1.16).We validated our RNAi knockdown using real-time quantitative PCR. Dissection and qPCR methods were identical to those described above for untreated individuals. We compared gene expression in the prothoracic epidermis and dorsal abdominal epidermis of 11 control-injected and 9 We fit a sigmoidal curve based on a 4-parameter Hill equation to male body size-horn scaling data using Sigma Plot 2001. This equation was used to calculate expected pupal or adult horn length for a given body size (thorax width). Residual horn length was calculated as the difference between observed and expected horn length for a given body size. For body size-genitalia scaling relationships we used standard ANOVAs instead of reduced major axis methods (errors in variable statistics) sometimes used in allometries Figure S1FOXO protein sequences of five Onthophagus species compared to Tribolium castaneum. Shown are the first 300 amino acids of the gene. The conserved forkhead domain lies approximately between amino acids 80 and 170 (highlighted with the light dotted line). The gene region that made up the RNAi and in situ probe follows the conserved domain (highlighted in heavy dotted line). Darker grays indicate more conserved amino acids.(TIF)Click here for additional data file.Figure S2Validation of FOXO RNAi knockdown. Shown are least square means from an ANOVA that included expression of the control gene (actin) and body size  of control-injected and knockdown individuals harvested as first-day pupae (N = 20). Gene expression was measured in the prothoracic epidermis and the abdominal epidermis.(TIF)Click here for additional data file.Table S1Effect of degree of FOXO knockdown on body size. Shown are results of linear regressions considering the effect of FOXO knockdown  on body size. In this analysis, control individuals were treated as “0.”(DOC)Click here for additional data file.Table S2Onthophagus; primers used in qPCR analyses; and primers used to clone a non-homeodomain fragment of FOXO from O. nigriventris to be used in RNAi and in situs. Base pair regions are with reference to this original FOXO sequence  and actin sequence (GenBank accession FG541406.1). A series of primers (sequences available upon request) were used to clone larger fragments of FOXO in the original species (taurus) and the focal species (nigriventris) before designing the primers reported below, some of which  worked in a range of species to clone out fragments of FOXO.Primers. Shown are primers used to clone multiple fragments of FOXO from several species of (DOC)Click here for additional data file.


The strengthening of international criminal law through an increased focus on the right to reparation and rehabilitation for victims of crimes against humanity represents an important challenge to health professionals, particularly to those in the field of trauma research and treatment. A brief outline of some developments in the field of international law and justice for victims of gross human rights violations is presented, with a focus on the right to reparation including the means for rehabilitation. The fulfillment of this right is a complex endeavor which raises many questions. The road to justice and reparation for those whose rights have been brutally violated is long and burdensome. The active presence of trauma-informed health professionals in this process is a priority. Some of the issues raised within the context of states’ obligations to provide and ensure redress and rehabilitation to those subjected to torture and gross human rights violations are discussed, and in particular how rehabilitation can be understood and responded to by health professionals. Strengthening of the international system to ensure accountability for crimes against humanity and justice for victims involves a stronger focus on the right to reparation, including the means for rehabilitation after torture and other gross human rights violations.A major challenge today is how the international community can ensure that the justice it seeks in fact represents a “just” justice to those who have experienced gross violations of human rights. Necessary attention must be paid to the rights and needs of those involved in the process as victims, witnesses, or other parties. What contributions can the field of traumatology make to secure justice and reparation for those affected in a way that takes into account what they have endured? There is a need to discuss what rehabilitation as a form of reparation means and how this can be dealt with in practice. These questions are discussed below with a focus on how the idea of rehabilitation as a form of reparation or redress has developed within international human rights treaties. How may it be understood and dealt with from the point of view of health professionals? What are the challenges involved in fulfilling this right to those who have survived serious human rights crimes?Demand for the rights of victims and survivors of crimes against humanity to justice, to truth and to various forms of reparation has in recent years become an issue of high priority. In particular the right to reparation for harm suffered as part of international justice has been established as a way in which persons exposed to gross human rights violations can be compensated for what happened to them . Attempts at bringing former or present dictators to court, for instance the late Augusto Pinochet from Chile, Charles Taylor from Sierra Leone who have been tried at the International Criminal Court (ICC), or Omar Bashir from Sudan, the first head of state to be charged by this same court, are usually followed with great public interest, in particular by the survivors of the human rights abuses committed . The recently adopted General Comment nr. 3 to article 14 of the Convention Against Torture, argues that rehabilitation “should be holistic and include medical and psychological care as well as legal and social services”. Furthermore rehabilitation “refers to the restoration of function or the acquisition of new skills required as a result of the changed circumstances of a victim in the aftermath of torture or ill-treatment. It seeks to enable the maximum possible self-sufficiency and function for the individual concerned, and may involve adjustments to the person's physical and social environment. Rehabilitation for victims should aim to restore, as far as possible, their independence, physical, mental, social and vocational ability; and full inclusion and participation in society” . Based on this definition, the obligations of states to provide redress, and in particular, the means for rehabilitation, should be based on these principles and include the elements described in the GC3.009. The www.irct.com, www.ishhr.com, www.hhri.org).When discussing how rehabilitation fits into the larger scope of reparation under international law, it is necessary to look into the comprehensive work of rehabilitation with victims of torture and other gross human rights violations that have been carried out for years in the real world, by non-governmental centers, organizations and networks, and to a lesser extent by the states themselves. Health professionals working at such centers and the networks with which these centers are affiliated, like the International Rehabilitation Council for Torture Victims (IRCT) or the International Society for Health and Human Rights (ISHHR) have closely followed the developments in international human rights law. They have been strong voices for the right to redress and the need for rehabilitation services for all victims of torture IRCT, , irrespeReparative measures are intended to acknowledge harm, as well as repair or compensate whether state or non-state actors acting under the color of law commit the violations. One understanding of the term reparation denotes a process in which a person tries to come to terms with what has happened and enter a process of healing. It can refer to something that takes place in the individual, a complex psychological process that may be endangered or supported, but never directed or managed by others. Conditions can be favorable and beneficial or they can be detrimental and destructive to the process, but the process as such goes on in the hands, mind, and heart of the person Hamber, . Even whhuman rights law. That is the way in which states compensate loss and suffering, including through such actions, such as public apologies and rehabilitation. The term reparation is about what states do to right the wrongs, either because they see the need to do this, or because they are required to do so as a result of judicial proceedings brought forth by those who have suffered damages. Examples are the payments provided by the government of Chile to people tortured under the Pinochet regime, and by the government of Argentina to persons affected by disappearances and torture during the dirty war. Post-WWII Germany has given compensation to Jews following the Holocaust, to workers in the factories during the Second World War and issued programs of reparation for Jews returning to Germany after the war. An example of claims for compensation which have never been met includes the long struggle for redress from the government of Japan to the victims of their military's sexual slavery during WWII, or “comfort women” mostly from Korea. These examples deal with acts of reparation in response to events that took place long back.Nevertheless, the focus here is on forms of reparation and redress as the term used in rehabilitation has been referred to in the Convention on the Rights of the Child (CRC), the Convention on Economic, Social and Cultural Rights (CESC), and not least in the Convention on the Rights of Persons with Disabilities (CRPD). The duty of states to provide some form of rehabilitation to defined groups or to persons with certain characteristics or experiences is referred to and this obligation goes beyond the general concept of right to health.The right to and in need of rehabilitation services. Should this right be limited to the person's own national state, or should it be considered a right that could be exercised everywhere? Should victims of torture be entitled to rehabilitation, regardless of where they are and who tortured them  has defined rehabilitation as a form of reparation  provides that “The Court shall establish principles relating to reparations to, or in respect of, victims, including restitution, compensation and rehabilitation”  referred directly to the need for developing rehabilitation services, stressing that victims of torture “obtain redress and are awarded fair and adequate compensation and receive appropriate socio-medical rehabilitation” and encouraged “the development of rehabilitation centers for victims of torture”. In his report to the General Assembly in August 2010 (A/65/273) in his capacity as UN Special Rapporteur on Torture, Professor Manfred Nowak devoted a whole section to the role of rehabilitation centers for victims of torture, stating that it follows from the obligation of the Convention Against Torture that torture rehabilitation centers are established and that such centers must provide holistic treatment for survivors. He also makes an important point about the role rehabilitation centers have in providing evidence to hold perpetrators accountable. The active use of the Manual on Effective Investigation and Documentation of torture, the so-called Istanbul Protocol Philippines, May 2009, CAT/C/SR.868: 56). Similarly, asking the state party whether it “makes physical, psychological, and social rehabilitation services available to victims of torture or cruel, inhuman, or degrading treatment” .States may also be informed thatAzerbaijan, May 2010, CAT/C/SR.909: 35).Reparation within the meaning of Article 14 has three dimensions: moral, financial, and medical. The payment of compensation alone is not enough; it is equally necessary to ensure victims the means necessary for their rehabilitation. It would be interesting to know in how many cases of torture the courts have ordered the payment of compensation to victims and whether there are rehabilitation programs in place  Offer full reparation, including fair and adequate compensation for the victims of such acts, and provide them with medical, psychological and social rehabilitation .The committee is deeply concerned about: (a) Persistent and consistent reports of torture and ill-treatment allegedly carried out by the State party's security forces and services, especially in district police stations, gendarmeries and remand centers, and the apparent impunity enjoyed by the perpetrators of such acts; … (d) Reports that torture and ill-treatment are commonly used on prisoners of war and political opponents. In the conclusions for Chad, it was pointed out thatAs can be seen, states are asked both about their legislation and about the actual existence of such services even in countries where most of the persons subjected to torture have arrived as refugees and where the state is not necessarily responsible for the torture. The Committee Against Torture is probably the treaty body that most frequently, most directly, and even most critically raises issues related to redress and rehabilitation. Despite most of the questions coming under the umbrella of rehabilitation as part of redress or compensation there are, as in the examples, also frequent references to services provided to torture victims. States can be asked about what kind of rehabilitative services they provide to traumatized refugees arriving in the country, or to victims of trafficking or others subjected to torture and ill-treatment, either within or outside the state. Are these services a form of reparation or a provision of necessary services to persons at risk? Again, it has been argued under the purview of universality both in civil and criminal jurisdiction, that one could actually be redressing violations committed by another country. Should there be established procedures permitting victims to recover reparation in countries other than where the actual torture took place Hall, ? This reRehabilitation to victims of torture depends on the clarification of some questions. First of all, the question of who has the right to redress after human rights violations must be addressed, as well as how to ensure that rehabilitation as part of redress is actually being carried out. Then, there is the question as to when and where rehabilitation can take place, by whom and what services will be available. These are questions about principles and definitions, but the questions are also closely linked to practical challenges on the ground and a political will to implement is what required.It seems fair to say that all persons subjected to torture and ill-treatment and their dependents, in particular in cases of death or the disappearance of a person, should be entitled to redress, and with a possibility to access rehabilitation. This is a twofold obligation. At the procedural level, states must enact legislation and establish a complaint mechanism, investigation bodies, and institutions capable of determining the right to and awarding redress for a victim of torture and ill-treatment. At the substantive level, states must ensure that a victim of torture or ill-treatment obtains full and effective redress and reparative measures, including compensation and the means for as full rehabilitation as possible. Rehabilitation is a substantive right. Health professionals must be active in the process of ensuring that it is realized. Effective rehabilitation also rests on formal decisions that must be taken before rehabilitation as a form of redress can be provided. Must compensation be based on decisions taken by a court, and as such be delayed until liability has been established, or are other venues to redress possible. Must a perpetrator be identified, found guilty, and even convicted in court for the crime of torture before redress, including rehabilitation, can take place?Some states will argue that the right to compensation can only be realized through a court order if a court recognizes the right of a victim of torture to compensation from the State, the victim will received compensation, including restitution if rights, adequate and equitable financial remedies, medical care and rehabilitation , and that the state, through financing such services, actually fulfill their obligation to provide redress. This would mean that NGOs in Chile, Argentina, or the Philippines could use their expertise and provide rehabilitative services mandated and financed by the state. The state would then implement its obligations to provide means to rehabilitation and redress. This discussion is far from unproblematic, but it should be encouraged and possible ways in which to move in relation to this should be looked for.For those receiving rehabilitation this is highly relevant, because for the survivor, it is possible that not general comment on this article, as referred to above. The working document was reviewed by state parties, by NGOs, and by others engaged in victims’ rights. The general comment refers to many of the important conditions surrounding reparation in the form of rehabilitation and focuses on the importance of victim participation . It is my hope that the adoption of general comment no. 3, explaining and clarifying the obligations of state parties under article 14 of the UN Convention Against Torture and Other Cruel, Inhuman or Degrading Treatment or Punishment, will be one more step toward justice and reparation for all victims of torture. It does not happen by itself, and health professionals also have important contributions to make here.There is a need for clarification on a number of issues related to the right to redress and in particular, in relation to the substantiation and implementation of these rights. One way to clarify, specify, and strengthen provisions and state obligations under article 14 of the Convention against Torture has been to develop and adopt a no to impunity” and yes to accountability for perpetrators, must not preclude or overshadow the many hardships involved in obtaining justice for the survivors and their familes. This progress toward justice does not necessarily seem beneficial or meaningful for healing, but the solution does not lie in less justice but in “better justice”. It lies in closer participation with survivors, stronger advocacy for sound and secure ways of implementing the rights referred to and active involvement of the knowledge and understanding gained within traumatology. After all, this is about severe trauma and life-long consequences. It is about lack of protection and insecurity, about possible aggravating and retraumatizing factors, but it is also about ways out of the trauma. Ways of regaining dignity and reconstruction.This article has been motivated by many challenges involved in the international aspirations for justice for crimes against humanity, in particular in the challenges that the victims and survivors are facing and the possible contributions and participations of health professionals in this field. Reparation has been discussed as an important element of justice, with a special focus on how the right to rehabilitation can be understood in the context of justice and reparation. Rehabilitation to survivors of torture regarded as a free-standing right to all persons who have been subjected to torture and crimes against humanity and as a basic form of reparation or redress have been outlined and some implications discussed. The torture convention and the rights and obligations laid down in this, are crucial but there is a need for further elaboration and to implementation. Active participation of trauma-informed personnel could contribute to the actual fulfillment in practice of the right to redress and to as full rehabilitation as possible. Their involvement should also imply that this is carried out in a way that ensures participation by and respect for those who have been severely affected by violence. Finally, the road to justice is long, and the many battles that have been won in relation to “


Rehabilitation services are less-studied aspects of the management following total knee arthroplasty (TKA) despite long-term suboptimal physical functioning and chronic deficits in muscle function. This paper describes the preliminary findings of a six-week (12 session) eccentrically-biased rehabilitation program targeted at deficits in physical function and muscle function, initiated one month following surgery. A quasiexperimental, one group, pretest-posttest study with thirteen individuals  examined the effectiveness of an eccentrically-biased rehabilitation program. The program resulted in improvements in the primary physical function endpoints  with increases of 59% and 47%, respectively. Muscle function endpoints (knee extension strength and power) also increased 107% and 93%, respectively. Eccentrically-biased exercise used as an addition to rehabilitation may help amplify and accelerate physical function following TKA surgery. In the US, over 500,000 total knee arthroplasty (TKA) surgeries are performed each year for severe knee osteoarthritis and that number is expected to increase sevenfold over the next two decades . Most TKImpairments in muscle function  parallel these physical function deficiencies. Following TKA surgery, muscle weakness is ubiquitous and directly associated with impaired physical functioning , 13–15. PCS) [Many institutions, centers, and clinics provide outpatient therapy to patients following TKA surgery. Despite this, a best-practice outpatient rehabilitation approach has not been published. This void in the peer-reviewed literature, highlighted by an NIH-sponsored expert panel  as one oPCS) . It is our contention that high muscle force producing resistance exercise can amplify muscle conditioning and optimize the level of physical function to that of healthy normative groups. Our laboratory has specifically studied the effects of resistance exercise via negative, eccentrically-induced work on muscle and mobility function in older adults with a variety of comorbid conditions. Significant improvements following eccentric exercise have been noted in muscle strength and size and in the hallmark activities underlying physical function  –27. ImpoIn this paper, we present a descriptive report of the use of an eccentrically-biased postoperative rehabilitation intervention targeted at deficits in physical function and muscle function following TKA surgery. This preliminary study examines the efficacy of a six week (12 sessions) eccentrically-biased rehabilitation program initiated one month following TKA surgery.This study was approved by the Institutional Review Board at the University of Utah and was in accordance with the Declaration of Helsinki. All participants provided written informed consent prior to participation. A quasiexperimental, one group, pretest-posttest design was used. The same investigator (YY) collected pre and posttest measures. Potential participants were recruited sequentially from one orthopaedic surgeon's two-week postoperative TKA clinic from June 2008 through June 2009. Measurements of physical function and muscle function were assessed prior to outpatient rehabilitation (third postoperative week) and one week following the rehabilitation period (eleventh postoperative week). The six-week outpatient rehabilitation began the fourth postoperative week. Thirteen individuals  see  met the All TKA procedures were performed through a minimedial parapatellar arthrotomy with minimal patella eversion , 29. A cOutpatient rehabilitation was supervised by a physical therapist and performed two times per week over a six-week period. Each of the 12 rehabilitation sessions lasted 60 minutes and included a warmup, stretching, and a progressive suite of lower extremity joint range of motion exercises, resistance exercises of the lower extremities, functional task-oriented exercises, endurance exercise and a short session of cyrotherapy. All rehabilitation components were initiated at the start of rehabilitation though the intensity, frequency and duration were adjusted based on the response to treatment. Any signs of adverse knee joint responses  resulted in a lowering of the intensity, frequency and duration of the exercises or elimination of a rehabilitation component. The resistance exercise component of each rehabilitation session utilized resistance exercise via negative, eccentrically-induced work as the major mode of lower extremity strengthening .Resistance exercise via negative, eccentrically-induced work (RENEW) of both the operative and nonoperative lower extremities was performed on a recumbent ergometer that appears like a normal stepper ergometer  . RENEW iPhysical Component Summary of the Short Form-36 (SF-36PCS) is a composite measure of physical function derived from the overall Short Form-36 [PCS composite measure provides a simplified metric of physical function that accounts for all 8 subscales of the original Short Form-36 [PCS is highly reliable (ICC = 0.90) and can readily detect changes in physical function with 5 points being the minimum clinically important difference [PCS was assessed as the primary self-report endpoint for physical function. The  Form-36 . The SF- Form-36  and prov Form-36 . The SF-fference . Partici6-Minute Walk Test (6MW) [the ability to move around” [The st (6MW) , 35 is a around” ) in adul around” . The 6MW around” . The 6MWlower extremity function scale (LEFS) is a self-report, region-specific questionnaire [The ionnaire . The LEFionnaire . ParticiStair Climbing Test (SCT) is one of the most demanding tasks for individuals after TKA [The fter TKA . The SCTfter TKA . The parGait Speed (GS) assessment at a comfortable, self-selected speed was performed over a 10 m path and timed with a stopwatch. This test is highly reliable (ICC = 0.91) [A  = 0.91) , and a c = 0.91) . The aveknee extension deficits and knee Flexion ROM measures of flexion and extension were recorded to the nearest degree using a goniometer.In order to track active range of motion (ROM) Knee Extension Maximal Voluntary Isometric Contraction (StrengthMVIC) torque was assessed in both the operative and nonoperative legs. StrengthMVIC of the quadriceps femoris muscle was evaluated at 60° of knee flexion on a dynamometer . Participants were tested three times with a two-minute rest between trials to avoid muscle fatigue. The average maximum torque value from the three trials was analyzed. The StrengthMVIC techniques, which were corrected for the influence of gravity, have demonstrated high reliability (ICC = 0.99) with important change scores being 25 Nm [leg extensor maximal voluntary power output (PowerMVPO) is a reliable (ICC = 0.91) and important variable related to measures of physical function [MVPO in both the operative and nonoperative legs using the Leg Extension (Nottingham) Power Rig . Following 4-5 practice trials, the participant performed 5 leg extension maximal efforts. The average maximal effort leg extension power was analyzed. The leg extension power rig is a feasible means of assessing muscle power across the lifespan [A test of ng 25 Nm . A measufunction  followinfunction , 43. We lifespan .t-test was used to test for differences between the prerehabilitation values and the postrehabilitation values for the dependent variables. The level of statistical significance for all tests for differences was set at P < .05. Next, to better understand the effects of the eccentrically-biased rehabilitation program, the magnitude of changes within the groups was calculated. The interval estimators of pre- to postrehabilitation changes  as well as calculation of a within-group effect size (ES) were also analyzed.Descriptive statistics were calculated for demographic variables and the primary and secondary dependent measures. The RPE, work, and pain measurement results were examined graphically. The assumptions of parametric statistical tests for the primary and secondary outcomes were assessed via tests of normality and homogeneity of variance. In all cases, the assumptions were met; therefore parametric tests were performed. First, a paired Of the total of 14 individuals who consented to participate, 13 individuals completed the full dose of eccentrically-biased rehabilitation (12 sessions over 6 weeks) and all study measures see . The oneThe total amount of work during RENEW increased approximately 5-fold over the course of the 6 weeks of rehabilitation despite a perceived level of exertion never exceeding “somewhat hard” . During P < .01) improved on all physical function outcomes and muscle function outcomes related to the operative leg  eccentrically-biased rehabilitation program induced the outcomes in this small sample of patients. Furthermore, any comparisons to existing literature should be made with caution since criteria for inclusion in previous studies are not identical and participant characteristics are likely different. The effects on the primary physical function endpoints, however, seem impressive. The normative levels of physical functioning, 50 on the SF-36PCS , were re session PCS , 45–62. utilized –60 and mutilized , 49, 63.utilized , 23, 64,utilized , 66.PCS. In our small descriptive study, the effect size (ES) of an eccentrically-biased rehabilitation program on physical and muscle function was large (ranging between ES = 1.5 and ES = 3.2). Every participant's SF-36PCS change score and over one half of the 6MW change scores exceeded the minimal clinically important difference for those measures of physical function. Overall, the level of physical function at the third postoperative month following an eccentrically-biased rehabilitation program could be considered equivalent to or better than the levels achieved in the previous RCT at the one year. Moreover, the secondary physical function endpoints in our study, that included assessments of perceived (LEFS) and performance-based abilities (SCT and GS), demonstrated large effects (ranging from ES = 1.2 to ES = 1.7) and 54–100% of the participants' change scores exceeded clinically important levels. These improvements in the secondary endpoints are important since the LEFS better represents functional activities as compared to the WOMAC [When the focus of postoperative rehabilitation is based on functional activities, short-term quality of life improvements are noted when compared to home exercise and education with small to moderate weighted means differences . Studieshe WOMAC  and the he WOMAC .MVPO), an important variable related to measures of physical function [The effect on strength over the first three postoperative months also appears greater with an eccentrically-biased program, though direct comparisons to earlier studies are challenging. Leg extension power (powerfunction  followinfunction , 43, alsfunction . Furtherfunction , was notThis is the first report of the use of eccentrically-biased rehabilitation program for the knee and hip extensors early after TKA surgery. There were no adverse events associated with this rehabilitation approach. Eccentric exercise of the knee flexors has been tested previously as a rehabilitation intervention designed to reduce terminal knee extension deficits , but notThe limitations of a quasiexperimental design with a nonblinded assessor and the inability to assign the positive findings strictly to RENEW warrant caution when extrapolating these results. Our TKA recipients underwent a multifaceted program of which RENEW was only one component. The enrolled participants were also a select group with initial high levels of function. This precludes a direct comparison to other treatment studies that incorporate a broader range on participants with less restrictive inclusion/exclusion criteria. Additionally, there was no control group to help decipher whether the positive findings were simply a result of a natural return of muscle and physical function. It may be that this cohort of participants was bound for a good result independent of whether they engaged in formal outpatient rehabilitation. Also, since the participants were only engaged in outpatient rehabilitation for 3 months, this prevents an assessment of long-term implications of this rehabilitation mode. Since it has been demonstrated that the greatest rehabilitation effects on muscle conditioning occur during the first three months following TKA surgery, and that is the typical timing and duration of postoperative rehabilitation, this limitation, however, may be one of this study's greatest strengths. That is, the findings after 12 outpatient rehabilitation sessions over three months result in normal or near-normal physical function. While we are not able to comment on whether the muscle function changes were a result of improved activation or hypertrophy of the leg muscles, we do know the effect was large. In the future, we hope to perform a randomized, controlled study with a mediation analysis that will isolate the “active muscle ingredient” following TKA surgery that constitutes the causal pathway toward normalized physical function.Utilizing RENEW as the strengthening mode of an eccentrically-biased rehabilitation program early after TKA contributed to changes in physical function to norm-based levels. This result alone suggests that the high muscle force production potential of eccentric exercise, at relatively low levels of exertion via RENEW, may contribute to an amplified level of physical function. The potential impact on best-practice rehabilitation following TKA is far reaching as greater focus on quadriceps strengthening is feasible and may be capable of optimizing outcomes.