Spinal and bulbar muscular atrophy (SBMA) is the first member identified among polyglutamine diseases characterized by slowly progressive muscle weakness and atrophy of the bulbar, facial, and limb muscles pathologically associated with motor neuron loss in the spinal cord and brainstem. Androgen receptor (AR), a disease-causing protein of SBMA, is a well-characterized ligand-activated transcription factor, and androgen binding induces nuclear translocation, conformational change and recruitment of coregulators for transactivation of AR target genes. Some therapeutic strategies for SBMA are based on these native functions of AR. Since ligand-induced nuclear translocation of mutant AR has been shown to be a critical step in motor neuron degeneration in SBMA, androgen deprivation therapies using leuprorelin and dutasteride have been developed and translated into clinical trials. Although the results of these trials are inconclusive, renewed clinical trials with more sophisticated design might prove the effectiveness of hormonal intervention in the near future. Furthermore, based on the normal function of AR, therapies targeted for conformational changes of AR including amino-terminal (N) and carboxy-terminal (C) (N/C) interaction and transcriptional coregulators might be promising. Other treatments targeted for mitochondrial function, ubiquitin-proteasome system (UPS), and autophagy could be applicable for all types of polyglutamine diseases. Spinal and bulbar muscular atrophy (SBMA) was first described in 1897 by a Japanese neurologist, Kawahara . It is c So far, nine polyglutamine diseases are known including SBMA, Huntington's disease, dentatorubral-pallidoluysian atrophy, and six forms of spinocerebellar ataxia (SCA), known as SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17 , 10. The However, the observation that the same genetic mutation in nine different proteins results in nine different diseases highlights both the significance of a specific protein context other than the polyglutamine tract and the role of normal protein function in the pathogenesis of polyglutamine diseases . Direct  In SBMA, expanded polyglutamine tracts are associated to lower levels of transcription of androgen-responsive genes , 26, whi So far, therapeutic interventions have been developed to target a number of events occurring through native AR functions upon ligand binding. Although no treatments have been established in SBMA, this review illustrates several therapeutic strategies based on the native function of AR and the common mechanisms shared by polyglutamine diseases.Due to the X-linked transmission, SBMA exclusively affects males and is transmitted by clinically unaffected or mildly manifesting female carriers. A unique gender-specific feature of SBMA is well recapitulated in both vertebrate and invertebrate animal models of the disease , 31. In  Leuprorelin is a potent luteinizing hormone-releasing hormone analog that decreases the production of testosterone and its more potent derivative, dihydrotestosterone (DHT), and has been used for the treatment of a variety of sex hormone-dependent diseases including prostate cancer, endometriosis, and central precocity . Treatmeα-reductase inhibitor, dutasteride. The observation that motor neurons degenerating in SBMA express high levels of 5-α-reductase suggests that the conversion of testosterone to DHT represents a potential therapeutic target  domain near the ligand-bound carboxyl terminus (N/C interaction) , 43. Thi Another strategy for reducing toxicity of mutant AR is based on alteration of the morphology of the oligomers. Recently, Jochum and colleagues demonstrated that the pathogenic AR mutants formed oligomeric fibrils up to 300–600 nm in length, whereas annular oligomers 120–180 nm in diameter were formed by the nonpathogenic receptors . They shDrosophila model of SBMA .–70.68–70α (RXRα) , renderiα (RXRα) . This report indicates the importance of link and equilibrium between these two degradative systems . In part Other therapeutic approaches to augment autophagy induction are the use of rapamycin, an mTOR inhibitor , and treγ-coactivator 1 (PGC-1)   and the SBMA is the first identified member of nine polyglutamine diseases , and due


Holding on to one's strategy is natural and common if the later warrants success and satisfaction. This goes against widespread simulation practices of evolutionary games, where players frequently consider changing their strategy even though their payoffs may be marginally different than those of the other players. Inspired by this observation, we introduce an aspiration-based win-stay-lose-learn strategy updating rule into the spatial prisoner's dilemma game. The rule is simple and intuitive, foreseeing strategy changes only by dissatisfied players, who then attempt to adopt the strategy of one of their nearest neighbors, while the strategies of satisfied players are not subject to change. We find that the proposed win-stay-lose-learn rule promotes the evolution of cooperation, and it does so very robustly and independently of the initial conditions. In fact, we show that even a minute initial fraction of cooperators may be sufficient to eventually secure a highly cooperative final state. In addition to extensive simulation results that support our conclusions, we also present results obtained by means of the pair approximation of the studied game. Our findings continue the success story of related win-stay strategy updating rules, and by doing so reveal new ways of resolving the prisoner's dilemma. Evolutionary game theory provides a powerful mathematical framework for studying the emergence and stability of cooperation in social, economic and biological systems Since the pioneering work of Nowak and May Regardless of the details of mechanisms that may promote the evolution of cooperation in the spatial prisoner's dilemma game, most frequently, it was assumed that individual players learn from their neighbors to update their strategies, and that they do so more or less in every round of game. But in reality, we are much less prone to changing our strategy (see In this paper, we thus introduce a so-called win-stay-lose-learn strategy updating rule as follows. When satisfied, players maintain their strategies and do not attempt to change them. When dissatisfied, however, players proceed as it is traditionally assumed, i.e., by attempting to imitate the strategy of one of their neighbors We start by presenting the results as obtained when the cooperators and defectors are distributed uniformly at random, each thus initially occupying half of the square lattice. As the main parameters, we consider the aspiration level The results obtained by means of pair approximation are presented in In order to obtain a more complete picture about the joint effects of the aspiration level and the temptation to defect, we show the simulation results as a function of both These transitions can be understood as follows. On a square lattice with nearest neighbor interactions, the payoffs of a cooperator and a defector are given by tion see .Since the strategy changes of individual players are determined by their satisfaction, we proceed with the results on the satisfaction rates in the population as a function of i.e. the population is initialized with two neighboring straight lines of cooperators, as shown in Because initial conditions are relevant for the evolutionary success of cooperators in spatial games tors see , for , bLastly, we elaborate on how cooperators can resist the invasion by defectors. For this purpose, we consider the special initial conditions depicted in efectors , when , In summary, we have studied the impact of the win-stay-lose-learn strategy updating rule on the evolution of cooperation in the spatial prisoner's dilemma game. Unlike in the majority of previous works, in our case the strategy updating is not unconditional, but rather it depends on the level of satisfaction of individual players. The latter is determined by the aspiration level, which we have considered as a free parameter. If the payoff of a player is equal or higher than its aspiration, it is assumed that this player is satisfied and that there is thus no immediate need of changing its strategy. Conversely, if the payoff is lower than the aspired amount, the player will attempt to adopt the strategy of one of its nearest neighbors in the hope that it will reach the desired success. With this setup, we have found that if all players retain their strategies when being satisfied then the evolution of cooperation is remarkably facilitated. Especially for intermediate values of the aspiration parameter, e.g., It is instructive to discuss the differences between this work and related previous works Our work also has some parallels with other models not necessarily incorporating aspirations. Namely, the model introduced recently in The spatial prisoner's dilemma game is staged on a square lattice of size Player In addition to the simulation results of the proposed spatial game, we also present the results of pair approximation


Mdr10/0 mice, we found quinacrine reached concentrations of ∼80 µM without any signs of acute toxicity. Additionally, we examined the distribution and metabolism of quinacrine in the wild-type and Mdr10/0 brains. In wild-type mice, the co-administration of cyclosporin A, a known P-gp inhibitor, resulted in a 6-fold increase in the accumulation of quinacrine in the brain. Our findings argue that the inhibition of the P-gp efflux transporter should improve the poor pharmacokinetic properties of quinacrine in the CNS.The lipophilic cationic compound quinacrine has been used as an antimalarial drug for over 75 years but its pharmacokinetic profile is limited. Here, we report on the pharmacokinetic properties of quinacrine in mice. Following an oral dose of 40 mg/kg/day for 30 days, quinacrine concentration in the brain of wild-type mice was maintained at a concentration of ∼1 µM. As a substrate of the P-glycoprotein (P-gp) efflux transporter, quinacrine is actively exported from the brain, preventing its accumulation to levels that may show efficacy in some disease models. In the brains of P-gp–deficient  Sc formation, we and others found that phenothiazines and acridines like quinacrine lowered the levels of PrPSc in scrapie-infected neuroblastoma (ScN2a) cells Sc by 50% in cell cultures were reported to be ∼1 µM and ∼7 µM extracellularly and intracellularly, respectively In the search for inhibitors of PrPin vitroin-vitro study suggested that quinacrine may be a potent compound for treating glioblastomas by inducing autophagic vacuoles leading to tumor cell death Quinacrine is an acridine-based compound that was first synthesized in 1931  and initially used for the prophylaxis and treatment of malaria. Quinacrine has also been used to treat giardiasis, systemic lupus erythematosis and rheumatoid arthritis in vivo compared to effective concentrations determined in vitro. In this paper, we report on the pharmacokinetics of quinacrine in wild-type (wt) and Mdr10/0 mice in which the mdr1a and mdr1b genes encoding the P-gp transporters were ablated Mdr10/0 mice without signs of toxicity. We also determined the half-life (t1/2) of quinacrine in the brains, spleens, kidneys, and livers of wt and Mdr10/0 mice. Using the intrinsic fluorescence of quinacrine, we were able to observe the anatomic distribution of quinacrine in the brains of quinacrine-treated mice. To increase the concentration of quinacrine in the brains of wt mice, we administered the P-gp inhibitor cyclosporine A (CyA), which produced a 6-fold increase in brain quinacrine levels.The delivery of quinacrine to the mouse brain is complicated by its rapid efflux via the P-glycoprotein (P-gp) transporter found in endothelial cells of the central nervous system (CNS) All protocols involving animal studies were approved by the University of California San Francisco Animal Care and Use Committee (AN076161-02).Quinacrine dihydrochloride, acetonitrile, methanol, quinidine, CyA, verapamil, and disulfiram were purchased from Sigma-Aldrich .Mdr10/0 mice were purchased from Charles River  and Taconic , respectively. For the orally treated group, mice were fed with 40 mg/kg/day of quinacrine in a chocolate-flavored liquid diet n = 3) were euthanized at various time points, and brain, spleen, liver, and kidney tissues were collected and flash frozen. Tissue homogenates (10% w/v) were prepared using a Precellys 24 homogenizer  in pure distilled water , aliquoted, and stored at −80°C.Approximately five-week-old male and female wt FVB and n = 4) by gavage. After 1 or 2 h of P-gp inhibitor administration, 10 mg/kg of quinacrine in PBS was given to FVB mice via gavage. As controls, Mdr10/0 mice were also treated with 10 mg/kg quinacrine in PBS via gavage. Mice were euthanized at 6 or 12 h post-quinacrine administration, and tissues were collected and flash frozen. Tissue homogenates (10% w/v) were prepared as described as above and 200-µl aliquots were stored at −80°C.P-gp inhibitor stock solutions of quinidine, CyA, verapamil, or disulfiram, were prepared in PBS or DMSO. The solutions were diluted to 100 mg/kg with PBS, based on 25 g of mouse body weight and the final injection volume of 200 µl per mouse. The P-gp inhibitor solution was administered to FVB mice (O-demethyl-quinacrine (M1), and mono-desethyl-quinacrine (M2) (10 µg/ml each) were prepared in 50% acetonitrile solution. Working solution was diluted with untreated, control 10% tissue homogenates to make 500 µl of 1 µg/ml of standard 1 solution for each tissue type. The standard 1 solution was then serially diluted two-fold. Nine standard solutions (200 µl each) including a blank solution (0 µg/ml) were frozen at −80°C. Extraction. A total of 400 µl of acetonitrile containing the internal standard (50 ng/ml of chlorophenamine) was added to 200 µl of 10% tissue homogenates or standard solutions. The samples were vortexed vigorously twice for 1 min and centrifuged at 16,000 g for 5 min.Working solutions containing quinacrine, m/z for quinacrine, 384.5>142.2 m/z for M1, 372.2>114.2 m/z for M2, and 277.2>142.2 m/z for internal standard (chlorphenamine). The column was a Betasil C18 column (50×4.6 mm) from Hypersil-Keystone and the mobile phase consisted of CH3OH/H2O/trifluoroacetic acid (45∶55∶0.05) with 1 mM ammonium formate. The flow rate was 0.8 ml/min.Concentrations of quinacrine and metabolites M1 and M2 were measured by LC/MS/MS as described previously Mdr10/0 mice were treated with 20 mg/kg of quinacrine via intravenous (IV) tail injection or 40 mg/kg/day of quinacrine in a chocolate-flavored liquid diet. At 1 and 24 h after quinacrine treatment, IV-injected mice were euthanized by CO2 asphyxiation and the cervical spine transected just below the skull. The brains were removed and snap frozen on powdered dry ice. Half of each brain was used for cryosectioning, and the other half for measurement of quinacrine concentrations. Frozen half-brains were mounted on a freezing stage using Cryo-gel  with the cerebellum facing out. Frozen coronal sections of 8 µm were cut at −22°C using a Microm HM 505E cryostat and mounted on Fisher Superfrost Plus slides. Serial sections were used for quinacrine and nuclear , Vector Laboratories, Burlingame, CA) imaging. For quinacrine, frozen sections stored at −80°C were air-dried at room temperature (RT) for 10 min, mounted with 100 µl glycerin , coverslipped, and sealed. When not on the microscope stage, slides were kept in a dark box on wet ice to slow disbursement of quinacrine in the tissue. All images were taken immediately after mounting using an inverted Leica DM IRB microscope through a FITC (fluorescein isothiocyanate) filter using a SpotFlex camera and Spot advanced software on the FITC setting. For DAPI, serial sections adjacent to those used for quinacrine imaging were air-dried at RT for 20 min and post-fixed in 4% formaldehyde  in PBS for 15 min at RT, rinsed in PBS, mounted with Vectashield Mounting Media with DAPI, and imaged as described above, but using the DAPI filter and settings. Images were taken from the brainstem immediately adjacent to the most posterior portion of the cerebellum (Bregma −7.70 mm in the MBL c57Bl/6J Mouse Brain Atlas), at the interface of the granule cell layer and molecular cell layer in Crus II lobuli ansiformis and of the white matter track in the lobulus simplex midway through the cerebellum (Bregma −7.32 mm), of layer IV in the cortex immediately above the dentate gyrus, the dentate gyrus, and the thalamus immediately below the dentate gyrus (Bregma −2.212 mm). Image files generated on the Spot program were later processed in Adobe Photoshop for publication images.Wild-type and Mdr10/0 mice after oral gavage or IV injections . In the brains of wt and Mdr10/0 mice, the quinacrine concentrations were 1.6 µM and 84 µM, respectively . The quinacrine concentration in the brains of wt mice was consistent with previously reported data Mdr10/0 mice showed higher quinacrine accumulation in the spleen and kidney than wt mice while the quinacrine concentration was similar in the liver for both wt and Mdr10/0 mice .In earlier studies, we compared the levels of quinacrine in the brains of wt and Mdr10/0 mice with 40 mg/kg/day of quinacrine continuously for up to 250 d. Remarkably, quinacrine concentrations reached approximately 200, 250, 300, and 500 µM in the brain, kidney, spleen, and liver, respectively, with only minimal signs of toxicity . In addition to discoloration of hair and/or skin, Mdr10/0 mice treated with quinacrine for 250 d showed some weight loss and reduced locomotor activity. Quinacrine concentrations in all four tissues rapidly increased after a few days of treatment and continued to increase gradually up to 250 d . No quinacrine-induced neuropathology was observed in comparison to age-matched untreated brains. Mild focal necrosis and macrophage clusters were found in the livers of both treated and untreated mice (data not shown).Next, we examined the quinacrine accumulation during long-term treatment. We treated O-demethylation and N-desethylation to yield metabolites, O-demethyl-quinacrine (M1) and mono-desethyl-quinacrine (M2), respectively Mdr10/0 mice. Both wt and Mdr10/0 mice were treated with 40 mg/kg/day of quinacrine for 31 days. Tissues were collected at various time points after halting treatment and concentrations of quinacrine, M1, and M2 were measured by LC/MS/MS.During the course of quinacrine exposure, the drug was metabolized by the processes of Mdr10/0 mice. The concentration of M2, the major metabolite of quinacrine, was less than 25% of the quinacrine concentration in both wt and Mdr10/0 mouse brains . We found a ∼100-fold difference in the brain quinacrine concentration between wt and Mdr10/0 mice . In wt mice, quinacrine and its metabolites (M1 and M2) were rapidly cleared from brain by 7 d after the cessation of treatment; in contrast, quinacrine remained elevated in the brains of Mdr10/0 mice for up to 21 d after ceasing treatment . Notably, ∼1 µM brain quinacrine was found at the end of treatment phase in wt mice and 21 d after treatment ceased in Mdr10/0 mice . The concentrations of M1 for wt mouse brains were under the detection limit.At the end of the treatment period, quinacrine and its metabolites (M1 and M2) achieved steady-state brain concentrations in both wt and Mdr10/0 mice, we examined its brain distribution using fluorescence microscopy. Mdr10/0 mice were treated either orally with 40 mg/kg/day of quinacrine for 25 d or by IV tail injection with 20 mg/kg of quinacrine for 1 h. The distribution of quinacrine in brain sections was visualized by imaging its intrinsic fluorescence. Quinacrine was clearly detectable in various regions of both orally treated and IV tail-injected Mdr10/0 mouse brains, but not in untreated and PBS-treated Mdr10/0 brains . The quinacrine signal was intense in nerve cell bodies of the cerebral cortex, thalamus, and brainstem .Having established that quinacrine accumulates and can persist for weeks in the CNS of mdr genes, we also examined quinacrine distribution in the brains of wt mice. Although the quinacrine fluorescence signal was not detectable in the brains of wt mice treated orally with 40 mg/kg/day of quinacrine for 25 d, it was measurable when wt mice were injected with 20 mg/kg of quinacrine by IV tail-injection. At 1 h after IV tail injection, the quinacrine fluorescence signals in wt and Mdr10/0 were similar . Notably, quinacrine signals were faint in the brains of wt mice collected at 3 h after IV injection (data not shown). By 24 h after IV injection, quinacrine signals were absent in the brains of wt mice but remained intense in Mdr10/0 mice . These observations argue that wt and Mdr10/0 mice accumulate quinacrine in similar regions of the brain, but at different concentrations.In order to rule out the possibility of artificial localization of quinacrine due to the compositional changes in membranes resulting from the deletion of  or 2 h  prior to quinacrine dosing by oral gavage. Tissues were then collected 6 or 24 h after quinacrine administration.In order to be useful clinically, quinacrine levels in the brain must remain high, which can be achieved by co-administration of a P-gp inhibitor. We attempted to increase quinacrine levels in the brains of wt mice by utilizing four known P-gp inhibitors: quinidine, CyA, verapamil, and disulfiram. Groups of wt mice were treated with 100 mg/kg of the P-gp inhibitor for 1 h  or 2 h .Wild-type mice treated with 100 mg/kg of CyA prior to quinacrine showed a 6-fold increase in brain concentrations of quinacrine compared to controls , which w, which w, which w, which w, which wP-gp is a transmembrane glycoprotein that functions as an adenosine triphosphatase energy-dependent transporter. The BBB prevents many foreign substances, such as proteins, peptides, and most drugs, from gaining access to the CNS. P-gp is expressed in the endothelial cells of the BBB and functions as an active drug efflux pump. The P-gp pump keeps the concentrations of a wide variety of foreign substances low and thereby provides an important protective barrier against many potentially toxic molecules. P-gp also removes many drugs from the CNS Sc from the brains of scrapie-infected mice In a recent study, the utility of quinacrine in combination with a γ-secretase inhibitor cleared PrP. Although we have shown that inhibition of P-gp with CyA enhanced bioavailability of quinacrine in the brain, it is likely that the administration protocol is suboptimal to achieve maximal and sustained quinacrine concentrations in the brain for prolonged periods. Quinacrine was cleared from the CNS in several hours when it was administered with a P-gp inhibitor in wt mice. This observation suggests that higher or frequent doses of quinacrine with a P-gp inhibitor may need to be given. In our study, CyA seemed to be a fast-acting inhibitor while verapamil was a slow-acting inhibitor: quinacrine concentrations were highest at 6 h with CyA and at 24 h with verapamil . In future studies, it might be interesting to examine combinations of P-gp inhibitors.In our study, P-gp inhibitors given prior to quinacrine administration increased the concentration of the drug up to 6-fold in wt mouse brain . AlthougSince its synthesis in 1931, quinacrine has been widely used for treatment and prevention of malaria and chronic inflammatory conditions


Authors of randomized trial reports seem to hold a variety of views regarding the relationship between missing outcome data (MOD) and intention to treat (ITT). The objectives of this study were to systematically investigate how authors of methodology articles define ITT in the presence of MOD, how they recommend handling MOD under ITT, and to make a proposal for potential improvement in the definition and use of ITT in relation to MOD.We systematically searched MEDLINE in February 2009 for methodological articles written in English that devoted at least one paragraph to ITT and two other paragraphs to either ITT or MOD. We excluded original trial reports, observational studies, and clinical systematic reviews. Working in teams of two, we independently extracted relevant information from each eligible article. Of 1007 titles and abstracts reviewed, 66 articles met eligibility criteria. Five (8%) did not provide a definition of ITT; 25 (38%) mentioned MOD but did not discuss its relationship to ITT; and 36 (55%) discussed the relationship of MOD with ITT. These 36 articles described one or more of three statements: complete follow-up is required for ITT (58%); ITT and MOD are separate issues (17%); and ITT requires a specific strategy for handling MOD (78%); 17 (47%) endorsed more than one relationship. The most frequently mentioned strategies for handling MOD within ITT were: using the last outcome carried forward (50%); sensitivity analysis (50%); and use of available data to impute missing data (46%).We found that there is no consensus on the definition of ITT in relation to MOD. For conceptual clarity, we suggest that both reports of randomized trials and systematic reviews separately consider and describe how they deal with participants with complete data and those with MOD. Trial methodology experts, systematic review organizations, and authorities including the Consolidated Standards of Reporting Trials (CONSORT), Trialists have widely adopted the term ITT when reporting RCTs. Surveys of RCT reports, however, suggest that simply stating ‘a study employed ITT’ is potentially misleading because of large variation in its definition and application. The primary objective of this study was to systematically investigate how authors of methodology articles define ITT when outcome data are not available in all participants and how they recommend handling MOD to conduct an analysis according to ITT. The secondary objective was to make a proposal for potential improvement in the definition and use of ITT in relation to MOD.Appendix S1).The full details of the protocol are presented in the Methods section of M.A.’s MSc thesis  to necessarily mean MOD unless the authors of methodology articles made it clear they referred to loss to follow-up or MOD. Vice versa, we use the term “complete follow-up” to indicate no missing outcome data.We considered as relevant methodology articles those that assessed how ITT was defined in other trial publications, discussed how ITT should be defined or reported, or examined methods that may be employed to address the issue of MOD when conducting an ITT analysis. These could include journal articles, reviews, editorials, or letters to the editor.The concept of ITT has two main facets: (1) how to deal with participants for whom outcome data are available, and (2) how to deal with participants for whom outcome data are not available. If all participants had outcome data, all of the three ITT definitions (see below) on our data extraction form would lead to identical terminology and identical practice: analysis in the randomization arm for all participants for whom investigators have recorded the outcome of interest, regardless of protocol deviations and participant compliance. In case of MOD under ITT, we identified three mutually exclusive characterizations of ITT  and refer to these characterizations as definitions:This definition requires complete (100%) follow-up under ITT. That is, if any outcome data was missing then ITT is violated.An ITT analysis can be conducted in the presence of MOD as long as the MOD was handled in a particular, clearly specified manner. In this definition, the author could state that you ‘must’ impute data in a particular manner under ITT. Also under this definition, authors could state that you ‘may’ allow for more than one way to handle MOD by stating several ‘desirable’ strategies of dealing with MOD.In this definition, how one deals with MOD is irrelevant to the definition of ITT. In other words, ITT is conducted simply by ‘analyzing as randomized’ irrespective to how the investigator dealt with MOD.We included publications that devoted at least one paragraph to ITT and two other paragraphs to either ITT or MOD. Eligible articles mentioned the terms “intention to treat” or “intent to treat” in the title or abstract and were published in peer-reviewed journals either as articles, editorials, or letters to the editor. Because we were interested in capturing methodological discussions and fundamental concepts of ITT and not a description of how ITT was applied, we excluded original reports of RCTs, observational studies, and clinical systematic reviews. We excluded articles written in languages other than English.Appendix S2 presents the detailed search strategy for MEDLINE using the Ovid platform to identify methodological papers from 1950 to February 2009.A research librarian (N.B.) trained in health research methodology developed an initial pilot search strategy for MEDLINE. The librarian and an investigator (M.A.) subsequently used relevant articles identified through a pilot search to refine the search strategy. Working in teams of two, investigators  independently screened titles and abstracts in duplicate. When one investigator deemed an abstract as potentially eligible, the full text document was retrieved. Next, pairs of investigators independently assessed eligibility of full text publications based on eligibility criteria. Disagreements were resolved by discussion and consensus or if necessary by third party arbitration (G.G.).Appendix S3). Data extractors chose from the following options to describe an author’s stance for each of the three definitions of ITT in relation to MOD above: ‘sole definition,’ ‘definition desirable,’ ‘definition possible but undesirable,’ ‘definition mentioned but preference unclear,’ ‘definition specifically excluded,’ and ‘definition not mentioned’  working in teams of two independently extracted the different descriptions of ITT in relation to MOD from each eligible article using standardized, pre-piloted forms  to determine the degree of agreement between pairs of reviewers and interpreted it according to Landis and Koch . The MEDLINE search identified 1007 articles Figure 1There was substantial agreement among investigators for title and abstract screening  and almost perfect agreement for the full text screening . For duplicate extraction on the definition of ITT, extractors reached almost perfect agreement  when averaging the kappas for all response categories.The included articles discussed ITT and MOD on at least half a page and up to 15 pages and were published in journal types distributed among sub-specialty (46%), statistics (24%), methods (21%), and general medical journals (9%). Of the 66 articles, 5 (8%) did not provide a specific definition of ITT, 25 (38%) defined ITT and mentioned MOD but did not discuss its relationship with ITT, and 36 (55%) defined ITT and discussed the relationship of MOD to ITT.Of the 36 articles that addressed MOD in relation to ITT, 19 (53%) mentioned a sole definition for ITT and 17 (47%) provided multiple possible definitions for ITT. Of the 19 that provided only one definition for ITT, 7 argued that complete follow-up is required under ITT, one that ITT and MOD are separate issues, and 11 that ITT involves specific strategy for MOD. Of the 17 that held more than one definition for ITT as possible, 15 (42%) articles provided two definitions, and two (6%) articles considered all three definitions as possible for ITT Table 1.In total, 21 articles considered ‘complete follow-up required’ as a possible definition of ITT in relation to MOD: Seven (33%) concluded that this is the sole definition for ITT, 10 (48%) thought it was a desirable option, none thought it was undesirable, and 4 (19%) had an unclear preference. Seven articles specifically excluded this definition of ITT and 8 did not mention it. Soares and Carneiro, for instance, advocated the ‘complete follow-up required’ definition:“Other methods used to solve this problem include… carry forward of last observation response, explicit allocation of poor outcome, implicit assumption of good or poor outcome, and use of the group average… However, no imputation method can provide an unbiased assessment of the treatment effect unless the assumptions about the missing data are valid… Full application of intention-to-treat is possible only when complete outcome data are available for all randomized subjects”.In the 28 articles that suggested ITT involves a specific strategy for MOD, complete case analysis (16 articles), last outcome carried forward (14 articles), sensitivity analysis (14 articles), and use of available data to impute missing data (13 articles) were the most frequently mentioned strategies. Less frequently mentioned were the worst case scenario (9 articles), the best case scenario (6 articles), ‘all had outcome event’ (8 articles), ‘all had no outcome event’ (5 articles), and multiple imputation (3 articles); 11 articles mentioned strategies other than these. Of the 16 articles that mentioned complete case analysis, 13 (81%) specifically excluded this strategy under ITT Table 2.“We examined a total of 14 ad hoc strategies for dealing with missing values. These can be roughly classified into numerical imputational strategies (last observation carried forward (LOCF), mean and regression based methods) and non-parametric strategies (rank and dichotomization based methods). We included CCA [complete case analysis] as the non-ITT strategy for reference purposes only.”Unnebrink and Windeler, for example, list several methods of dealing with missing data that are valid strategies under ITT, but specifically excluded complete case analysis as a valid option: “Intention-to-treat analysis cannot minimize bias introduced by loss to follow-up, that is, patients whose outcome status is unknown… To improve the applicability of study results to individual patients, investigators should improve study design to ensure protocol adherence with minimal loss to follow-up. Finally, loss to follow-up can result in exactly the same sort of bias as a per protocol analysis. Therefore, if there is significant loss to follow-up, statements that investigators conducted an “intention-to-treat analysis” generally provide little reassurance.”Six articles included, as a possible definition of ITT, our third characterization, i.e. ITT and MOD are separate issues. One argued that this was the sole definition for ITT. This systematic review of methodology articles found great variation in the proposed definitions of ITT in relation to MOD and in how MOD should be handled in RCTs under the ITT principle. Given the high proportion of RCTs with MOD for the primary outcome (60–75%) and the increasing use of the term ITT in RCT reports (up to 80%), this constitutes a serious problem. Although others have shown that the concept of ITT is inconsistently applied in clinical trials, A potential limitation of our systematic review is that we focused our search exclusively on MEDLINE, articles in English, and articles that mentioned the terms “intention to treat” or “intent to treat” in the title or abstract potentially leading to less representative findings. Searching multiple databases and broadening our eligibility criteria might have increased the generalizability of our results. However, we would likely have found even more variability in definitions of ITT, suggesting that our findings in this respect are if anything conservative. Strengths of our review include our success in capturing a variety of journal types . We modified the data extraction form throughout the study to best reflect the data and to accommodate new insights from articles as they were acquired. We sought a criterion for a substantive discussion of the concepts of ITT and its relation to MOD and chose a criterion of three paragraphs. The criterion we chose is arbitrary, and one could argue that a criterion based on word rather than paragraph count would have been more appropriate. We found, however, that eligible articles did provide substantive discussion, and we were able to classify these articles according to their suggestions regarding ITT and MOD. Whether a less stringent criterion could have achieved the same goal with more eligible articles remains open to question.Of those who considered ITT and MOD to be related issues, the authors endorsed either imputing data and/or requiring complete follow-up. Of 16 articles that mentioned it, 13 (81%) excluded complete case analysis as a method of dealing with MOD under ITT; the others expressed the view that complete case analysis was a possibility under ITT, although it was an undesirable strategy. The position that MOD should be treated as a separate issue from ITT may implicitly accept complete case analysis as an acceptable alternative. Among clinical trialists, complete case analysis is the most popular approach to handle MOD: a recent review showed that complete case analysis was used in about half of RCTs reporting ITT. “replaced mention of ‘Intention to treat’ analysis, a widely misused term, by a more explicit request for information about retaining participants in their original assigned groups.”Several institutions have acknowledged that varying meanings of the term ITT is problematic. In its updated 2010 statement, CONSORT “1. Keep participants in the intervention groups to which they were randomized, regardless of the intervention they actually received. 2. Measure outcome data on all participants. 3. Include all randomized participants in the analysis.”“there is no clear consensus on whether all criteria should be applied.  While the first is widely agreed, the second is often impossible and the third is contentious.” If there are MOD the Cochrane handbook mentions two options, complete case analysis and data imputation, recommending a sensitivity analysis in either case. Both options involve assumptions about missing data and may be problematic, but using imputation achieves the ‘ITT quality label’ and the – in our view equally justified – complete case analysis does not. It acknowledges that We propose a solution to the problem: First, we must separate the issue for which there is consensus, the analysis of those for whom outcome data is available, from the issue in which no consensus exists, how to conduct an analysis according to the ITT principle in the presence of MOD. Both issues are important items for assessing the risk of bias in RCTs. Substantial MOD as well as ‘per protocol’ or ‘as treated’ analyses may introduce bias. To date, while authors have suggested reasonable alternatives for handling MOD, We found a large variation in the definition of ITT in relation to MOD among methodology articles and how MOD should be handled in RCTs under ITT. We therefore propose to separate the ITT principle from handling of MOD in order to increase clarity and transparency of reporting trials and systematic reviews. We define four distinct sub-categories of ITT depending on the presence of patients with MOD and, if present, how investigators handle such patients in their analysis Table 3.Appendix S1MSc thesis of MA including study protocol.(PDF)Click here for additional data file.Appendix S2MEDLINE search strategy.(DOC)Click here for additional data file.Appendix S3Data extraction forms.(DOC)Click here for additional data file.Appendix S4Detailed results and list of included studies(DOC)Click here for additional data file.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.


In the rodent brain the hemodynamic response to a brief external stimulus changes significantly during development. Analogous changes in human infants would complicate the determination and use of the hemodynamic response function (HRF) for functional magnetic resonance imaging (fMRI) in developing populations. We aimed to characterize HRF in human infants before and after the normal time of birth using rapid sampling of the Blood Oxygen Level Dependent (BOLD) signal. A somatosensory stimulus and an event related experimental design were used to collect data from 10 healthy adults, 15 sedated infants at term corrected post menstrual age (PMA) (median 41 + 1 weeks), and 10 preterm infants (median PMA 34 + 4 weeks). A positive amplitude HRF waveform was identified across all subject groups, with a systematic maturational trend in terms of decreasing time-to-peak and increasing positive peak amplitude associated with increasing age. Application of the age-appropriate HRF models to fMRI data significantly improved the precision of the fMRI analysis. These findings support the notion of a structured development in the brain's response to stimuli across the last trimester of gestation and beyond. ► First systematic characterization of the BOLD signal HRF in human neonates. ► A maturational trend in HRF morphology was identified. ► Application of empirical HRF models significantly improved neonatal fMRI analysis. In the third trimester of gestation and the first months of postnatal life, the human brain undergoes a dramatic but structured sequence of maturation resulting in the establishment of the cortex and its associated framework of structural and functional connectivity . The impBlood Oxygen Level Dependent (BOLD) contrast functional Magnetic Resonance Imaging (fMRI) is non-invasive and can identify patterns of activation across the whole brain . DevelopfMRI has not been widely applied to the human neonatal population due to a history of methodological challenges and inconsistent results . Central2), show marked developmental changes during the perinatal period  has been well characterized and found to be reproducible and consistent across different populations , but evel period .In this study we systematically characterized the ontogeny and morphology of the hemodynamic response to neural stimulation before and after the normal time of birth using an event-related experimental design and a somatosensory stimulus, and investigated the possible confounding effects of oral sedative medication by measuring global CBF. We hypothesized that the neonatal HRF differs from the canonical adult waveform; that there is a systematic maturational trend; and that application of an age-appropriate HRF in the analysis of fMRI data would significantly improve the identification of functional responses.The work was approved by the NHS research ethics committee, and written subject  consent was obtained prior to all sessions of data acquisition.All neonatal subjects were recruited from the Neonatal Intensive Care Unit and Postnatal wards at the Queen Charlotte and Chelsea Hospital, London, UK during a period of 18 months between 2010 and 2011. A total of 19 preterm infants and 22 infants at term equivalent Post-Menstrual Age (PMA) were scanned. Data sets were excluded from the analysis if the sequence was not able to be completed (due to the subject waking), or due to excessive motion. The final study group see  thereforMR imaging was performed on a Philips Achieva 3-Tesla system  with an eight channel phased array head coil. All infants were assessed by a pediatrician prior to the scan, and the infants' temperatures, oxygen saturations and heart rates were monitored throughout the scan . Ear profMRI data was acquired with a single shot echo-planar imaging (EPI) sequence lasting 8 min and 37 s (parameters: (TR) 500 ms; (TE) 45 ms; (flip angle) 90°; (matrix) 64 ∗ 64; (resolution(x ∗ y ∗ z)) 3.125 ∗ 3.125 ∗ 4 mm, total 1000 volumes). A relatively short TR was chosen  as our goal was to characterize the BOLD signal HRF, and a faster sampling rate has been shown to be important when characterizing the HRF waveform particularly with respect to identifying the time to onset . To alloAn event-related experimental design was used to acquire a sampled BOLD HRF following a brief (1 s) stimulus during which the subject's right hand was moved passively. It has previously been shown that robust changes in BOLD contrast can be identified and used to characterize the HRF using stimuli as brief as 0.1–0.3 ms . To ensuwww.fmrib.ox.ac.uk/fsl)  . Each fu.uk/fsl) . In partData was first processed using FEAT  and standard pre-statistics processing steps were applied: motion correction , slice-timing correction, non-brain tissue removal, spatial smoothing (FWHM 5 mm), global intensity normalization and highpass temporal filtering (cut-off 50 s)) . Head moThe BOLD signal time-series was extracted and averaged from a region of interest (ROI); defined as voxels above the 90th centile in z-score within the cluster of activation in the contralateral primary somatosensory cortex identified with the GLM analysis and the complete fit of the data derived from an F-test of the parameter estimates from the individual basis functions . The timAnimal and adult fMRI work have demonstrated that changes in the HRF peak amplitude and time to peak can be artificially induced by the experimental manipulation of baseline CBF . To inveo; (resolution(x ∗ y ∗ z)) 0.6 ∗ 0.6 ∗ 4.0 mm; ) 120 cm/s) (www.doc.ic.ac.uk/~dr/software/)   sequence . A multi20 cm/s) . Instant20 cm/s) ). The coftware/) . The CSFeonates) .To test the value of the HRF waveforms derived in the main study, they were applied into the GLM analysis of 6 preterm (median age 34 + 0 weeks PMA (32 + 2–34 + 5) and 6 term equivalent (median age 40 + 5 weeks PMA (39 + 0–43 + 3) infant data sets from a previously collected study group . A blockFollowing passive motor stimulation of the right hand lasting 1 second, clusters of functional activation were identified in the contralateral (left) primary somatosensory cortex in all 3 subject groups . As obseA clear developmental trend in the shape parameters of the HRF was identified, characterized by a reduction in the time to positive peak and an increase in positive peak amplitude with increasing age . In the Global CBF data was acquired from a total of 14 term born infants who were then subdivided into two paired groups (those who were sedated for scanning and those who were not) by postmenstrual age at the time of scan. There were no significant differences between the two groups in the age of the infants at scan (Mann–Whitney–Wilcoxon test: p = 0.5198); the weight (Wilcoxon signed rank test: p = 0.3750); occipito-frontal head circumference (p = 0.8438); or brain volume (p = 0.1562). No significant difference was identified in the global CBF between the paired sedated (median: 22.40 ml/100 g/min) and unsedated groups (median: 20.78 ml/100 g/min) (Wilcoxon paired signed rank test: p = 0.4688). These CBF values are in good agreement with those previously described using diverse measurement techniques .To test the value of the derived HRF models the empirical waveforms were then convolved into the GLM analyses of data collected from 12 further infants, using a block paradigm of somatosensory stimulation . In 6 prThis process was repeated in a group of 6 infants at term corrected PMA on whom data had been collected using an identical experimental paradigm. In an exemplar study b convoluUsing a combination of optimized fMRI scanning parameters, an appropriate and precise somatosensory stimulus, and an event-related experimental design, we have been able to characterize the morphology of the BOLD contrast HRF waveform in the developing human brain. As described in the rat brain, a systematic maturational change in the morphology and parameters of the HRF was seen , both inIn comparison to the canonical form seen in the mature adult brain, the amplitude of the HRF positive peak was found to be significantly less in the developing neonatal brain. In addition, the time taken to attain the positive peak amplitude of the HRF was found to decrease significantly with increasing age. The physiological reasons underlying these differences are likely multi-factorial, and involve many stages of the neurovascular coupling cascade which ultimately culminates in the hemodynamic changes responsible for the BOLD contrast response . Due to Of note, robust electrophysiological responses to simple somatosensory stimuli can be elicited at a significantly younger age than reliable BOLD signal responses have been described in both animal and human subjects . AlthougA localized increase in CBF is known to be the key to the positive peak of the BOLD response through the change in signal which results from an increase in local oxygenated hemoglobin (Hb) . ArteriaGlobal CBF is known to increase dramatically during early human development, with preterm infant brain values approximately half that of a full term infant, with a further twofold increase in adult life . Given tNear-Infrared Spectroscopy (NIRS) studies have shown that quantitative measures of cerebral blood volume (CBV) remain unchanged both throughout the preterm period and during the first weeks after full term gestation . This wo2) results in deoxygenated Hb accumulation and therefore a decrease in BOLD signal  . It has ed CMRO2 . This efAlthough an event-related fMRI design with a widely-spaced constant inter-stimulus interval is relatively inefficient at both detecting activity and HRF estimation , it was The term equivalent age study group consisted mostly of infants who had been born prematurely (12/15), and although previous work has suggested that the functional activity is well localized regardless of the gestational age at birth , furtherThe benefits of an age-appropriate HRF for convolution into the GLM have been demonstrated here in two example preterm and term infant groups. This effect was most marked in the preterm infant group, where a cluster of positive activation in the primary somatosensory cortex was only identified when an age-appropriate HRF was used in the GLM design model, incorporating the significantly longer time taken to achieve the positive peak. In the term infant group, a large area of positive functional activation was identified irrespective of the HRF model used, and a significant difference was not seen when comparing the age-specific and adult canonical HRF models. This is explained by the relative similarity in the time to the positive peak for the term infant and adult groups, which will lead to a similar positive overshoot time regardless of the proportionately deeper undershoot period. However, at shorter inter-stimulus intervals than used in this work, the rise rate and amplitude of BOLD signal change would be significantly reduced should the next period of stimulation occur during the undershoot . It is nIn summary, we provide characterization of the HRF in the healthy human brain before and around the normal time of birth, and demonstrate a developmental trend in early human HRF morphology similar to that seen in the rodent brain. Moreover, the data provide evidence that the marked changes in brain structure known to occur in the third trimester of human development are also accompanied by a sequence of maturation in the brain's hemodynamic responses to stimulation. These maturational changes are likely to be due to both probable developmental alterations in the underlying neurovascular coupling and known changes in cerebrovascular physiology. These findings demonstrate that BOLD fMRI responses can be reliably identified in neonatal subjects, and offer the potential to improve the accuracy of analysis in studies involving this population. Although the effects of sedation cannot be completely excluded, the described HRF parameters still remain relevant, and can be applied to much needed future fMRI studies in this vulnerable population.TA, NT, NM, SJC collected the data; TA, MV, SJC optimized the imaging sequences; AM, AA, EB designed and manufactured the stimulus; TA, GF, CFB analyzed the data; TA and ADE planned the study and wrote the manuscript.The authors have no conflicts of interest to disclose.


Polycystic ovary syndrome (PCOS), one of the most common and complex endocrine disorders affecting up to 15 % of reproductive age women, is considered a predominantly hyperandrogenic syndrome according to the Androgen Excess Society. It is generally accepted that androgens determine the characteristic features of PCOS; in this context, a hyperactive androgen receptor (AR) at the levels of the GnRH pulse generator in the hypothalamus and at the granulosa cells in the ovary, skeletal muscle or adipocytes senses initially normal testosterone and dihydrotestosterone as biochemical hyperandrogenism and might be a crucial connection between the vicious circles of the PCOS pathogenesis.Polymorphism of the AR gene has been associated with different androgen pattern diseases. Several studies have demonstrated an association between AR with increased activity encoded by shorter CAG repeat polymorphism in the exon 1 of the AR gene and PCOS, although there are conflicting results in this field. The phenomenon is more complex because the AR activity is determined by the epigenetic effect of X chromosome inactivation (XCI). Moreover, we must evaluate the AR as a dynamic heterocomplex, with a large number of coactivators and corepressors that are essential to its function, thus mediating tissue-specific effects. In theory, any of these factors could modify the activity of AR, which likely explains the inconsistent results obtained when this activity was quantified by only the CAG polymorphism in PCOS. Although the aetiology remains unknown, some investigators consider that androgen excess is the sine qua non of PCOS [5], which is consistent with the theory that androgens determine the characteristic features of the syndrome, for example, oligo-anovulation and infertility, inhibition of follicle development with polyfollicular ovarian morphology, hirsutism, acne or androgenic alopecia [10]. Polycystic ovary syndrome (PCOS) is a complex endocrine disorder [17]. These data provide evidence that androgen excess is a key factor in the PCOS etiopathogeny.  Experiments conducted in animal models as well as equivalent natural situations in humans, i.e., pregnancy in PCOS or congenital adrenal hyperplasia, demonstrated that exposure to androgen excess during the foetal life and infancy determines the PCOS features in adult life, with LH hypersecretion, oligomenorrhoea, polyfollicular enlarged ovaries, insulin resistance, abdominal obesity, impaired insulin response to glucose and dyslipidemia [20]. In these adult women with PCOS, the blockade of androgen action by flutamide administration restored the sensitivity of GnRH to estradiol and progesterone, thus supporting the role of an increased androgenic activity mediated by the androgen receptor (AR) to reduce the progesterone negative feedback at the level of the GnRH pulse generator [21]. More specifically, experiments conducted in animal models showed that a special KNDy subpopulation of cells in the arcuate nucleus, which colocalise three important neuropeptides, kisspeptin, neurokinin b and dynorphin, express steroid receptors that include AR and connect with GnRH neurons in a network, are responsible for transporting the testosterone influence on GnRH neurons in the hypothalamic GnRH pulse generator [22].  In women with PCOS, the abnormality in the regulation of hypothalamic GnRH secretion, with persistently rapid GnRH pulsatility, pituitary synthesis of LH over that of FSH, and increased LH concentrations and LH/FSH ratios, has been explained, at least in part, by a decreased sensitivity of the GnRH pulse generator to the progesterone suppression [24], an increased androgenic activity could amplify insulin resistance as soon as testosterone in physiological concentrations enhanced the insulin-induced IRS-1 serine 636/639 phosphorylation in differentiated rat skeletal muscle myotubes [25]. AR is present in human addipose tissue, at the level of preadypocites and adipocytes [27] and Corbould demonstrated that testosterone induces a selective insulin resistance in cultured subcutaneous adipocytes of women, with action mediated by the classical AR, given that AR antagonists, cyproterone acetate and flutamide attenuated the effect of testosterone on the glucose uptake [28].  Although the insulin resistance of PCOS partially has a genetic substrate . In this condition, compensatory hyperinsulinaemia in synergistic action with LH will amplify all steroidogenesis and specifically testosterone synthesis at the level of responsive ovarian theca cells. The androgens, either circulatory or produced locally, impair the ability of primary follicles to grow normally, with a marked increase in the number of growing preantral and antral follicles, particularly in the population of growing and classic primary follicles, leading to the typical polycystic ovarian morphology [30]. Several studies have demonstrated that high-dose androgen treatment of transsexual women induced enlarged ovaries with increased numbers of cystic follicles and theca-interstitial hyperplasia, which meet the morphological criteria for PCOS [33]. Although the gonadotropins were suppressed in these subjects, their ovaries were not suppressed. These observations suggested that androgens per se might induce ovarian follicular and theca interstitial growth [7]. These data, correlated with experiments in non-human primates [7] and the evidence for expression of AR in the human ovary, at germinal epithelium, granulosa cells of antral follicles, corpus luteum, and thecal and stromal cells [34], indicated that the characteristic polycystic ovarian morphology, with enlarged ovaries and numerous small follicular cysts, might be the result of direct, receptor-mediated androgen activity.  Whereas the classic target tissues such as muscle and fat manifest insulin resistance in PCOS, the ovaries of these patients remain sensitive to insulin or perhaps hypersensitive to it  .  The AR, encoded by the AR gene located at Xq11-q12, is a nuclear transcription factor and member of the steroid receptor superfamily [42]. After ligand binding, AR adopts an active conformation that facilitates the dissociation of heat shock proteins, dimerisation, and binding to response elements in the promoters of responsive genes in the nucleus. The N-terminal activation domain accounts for more than 60% of the AR protein and has a potent transcriptional activator function (AF-1) at the level of androgen response elements [43]. The unbound AR is inactive in the cytoplasm as a large dynamic heterocomplex, together with heat shock proteins (Hsp70 and Hsp90) and their co-chaperones [44], partial androgen insensitivity syndrome (Reifenstein syndrome) [45] or prostate cancer [44].  So far, have been described different point mutations, deletions or insertions at the level of the AR gene that influence the activity of AR in different diseases, such as complete androgen insensitivity syndrome (CAIS) [49]; however, there is no uniform consensus [50].  Moreover, has been shown that the activity of AR is influenced by a genetic polymorphism in exon 1 of the AR gene. There are two polymorphic trinucleotide repeat fragments, CAGn and GGNn, in the first exon of the AR gene, encoding two polyglutamine and polyglycine tracts in the N-terminal activation domain. Although the functional consequences of variation in the length of the GGNn fragment are unclear, several in vitro studies have evidenced an inverse relationship between the length of the CAG polymorphic repeat  and the AR transcriptional activity [47], whereas the expansion of the CAG repeat length of over 40 is related to SBMA, a rare neuromuscular disorder characterised by spinal and bulbar muscular atrophy and is associated with androgen insensitivity, decreased virilisation, testicular atrophy, reduced sperm production, and infertility [51]. Expanded CAGn repeat in the AR gene modulates other male-specific phenotypes, such as prostate growth under androgen substitution in hypogonadal men [52] or different phenotypic features in untreated patients with Klinefelter syndrome [54].  In vivo studies support the majority of functional data, which demonstrate a negative correlation between the CAG repeat number and AR activity. Healthy men whose AR has a CAG repeat length of greater than 28 have an increased incidence of impaired spermatogenesis and infertility [59] or premature pubarche [60].  Conversely, shorter AR polyglutamine tracts and thus a more transcriptionally active AR have been associated with prostate cancer [CAG polymorphism of AR gene in PCOS69].  According to functional and in vivo data, CAG repeat polymorphism, encoding an AR with increased activity, might play a role in PCOS pathogenesis. However, several studies centred on the association between the CAG repeat polymorphism of the AR gene and PCOS have conflicting results [36]; this finding is consistent with that of another study that described all of the subjects with 15 or fewer CAG repeats as part of the PCOS group [37]. Azziz et al and Schuring et al reported significantly lower CAG repeats in patients with PCOS than in controls [69], a finding consistent with our preliminary data that demonstrated significantly shorter biallelic means of CAG repeats in patients with PCOS than in normal women [67].  Some authors observed that short androgen receptor CAGn alleles tended to appear more frequently in women with PCOS than in controls [70].  Mohlig et al demonstrated that in women with PCOS, the AR CAG repeat polymorphism has metabolic consequences by modifying the impact of testosterone on insulin resistance, which is consistent with prior known functional data. In a statistical model that described 42.5% of the HOMA-IR variation, the elevated testosterone increased the insulin resistance in carriers of short CAG lengths; this effect attenuated with increasing biallelic CAG length until it turned into the opposite at a CAG length of longer than 23 [68]; however, when compared PCOS groups constructed by androgen levels, one of these studies showed a trend for lower CAG biallelic means in the subgroup of women with PCOS and lower testosterone levels, which was consistent with a role for androgen hyperactivity in the development of the syndrome [61].  Other studies showed no difference in CAG repeat length between patients with PCOS and controls, regardless of the fact that both alleles were considered together or separately [62].  Contrary to the theory of an inverse relation between CAG repeat number and receptor activity, the study published by Hickey et al has shown that infertile women with PCOS exhibited a greater frequency of CAG alleles or biallelic means greater than 22 repeats compared with both the fertile controls and the general population [68] nor acne [68] was significantly associated with the CAG repeat polymorphism, whereas other studies found that short CAG repeat was associated with hirsutism and acne [71].  Regarding clinical hyperandrogenism of PCOS, several studies found that neither hirsutism  [69]. DNA methylation is an important epigenetic mechanism in X chromosome inactivation [72], which normally occurs in early embryogenesis as a random event. A nonrandom X inactivation, either due to environmental exposures or allelic differences, may contribute significantly to the expression of PCOS [38]. Experimentally induced animal models, in monkeys or sheep, showed that in utero hyperandrogenism exposure may lead to abnormalities of epigenetic reprogramming in foetal reproductive and metabolically active tissues and may produce an impact on the neuroendocrine development, gonadal differentiation and pancreatic organogenesis [16], which ultimately results in a PCOS phenotype in the adult life [16]. Prenatally androgenised female rhesus monkeys developed a PCOS-like phenotype as adults with increased visceral adipose tissue and basal serum insulin levels [73], conditions that correlated with different DNA methylation patterns in both infant and adult visceral adipose tissue [74]. An equivalent, naturally occurring situation with foetal exposure to the androgen excess occurs in pregnant women with PCOS [77].  In addition to the conflicting data on the association between genetically encoded CAG repeats and PCOS, we should consider the X chromosome inactivation (XCI) as a significant phenomenon present in females because the AR gene is located on the X chromosome; therefore, the AR activity is determined not only by the AR genotype but also by the epigenetic effect of XCI .  We hypothesise that a hyperandrogenemic environment could modify the process of methylation, including the AR expression in the active X chromosome, even when it appears in adult life, based on the data that demonstrated a loss of methylation of the LH receptor in the ovary of a mouse model with PCOS induced by the administration of dehydroepiandrosterone .  In this context, supplementary information can be derived by investigating the methylation pattern of AR’s entire sites near the polymorphic CAG repeats because all of those CpG sites may be involved in regulating AR expression in the active X chromosome [69]. One important study demonstrated that total methylation of peripheral blood leukocyte DNA is unaltered in patients with PCOS vs. matched controls [84].  b. We should consider that XCI can vary in different tissues. In all of the previous studies, XCI was generally tested by using blood samples, which does not appear to reflect the condition in androgen target tissues [85]. Future genetic and physiologic studies are required to identify the functional interactions between the CAG repeats and these AR coregulators [38]. 3. Finally, a large number of AR coactivators and corepressors have been identified as critical to the AR function. When they mediate tissue-specific androgen effects, a variation in the expression or function of these coregulators might contribute to the development of PCOS [Genetic data that demonstrate a hyperactive androgen receptor in PCOS are limited to the evaluation of the CAG repeat polymorphism of the AR gene and are inconsistent, thereby requiring more in vitro studies to identify functional AR polymorphisms that affect AR transactivity and different PCOS phenotypes. The important role that AR with increased activity plays in the PCOS pathogenesis is interesting in light of continuous efforts to understand this complex disease, which might represent an important connection between the multiple characteristic pathways, including the androgens, neuroendocrine and insulin axis.


A differential mass spectrometry analysis of secreted proteins from juvenile and senescent Podospora anserina cultures revealed age-related differences in protein profiles. Among other proteins with decreased abundance in the secretome of senescent cultures a catalase, termed PaCATB, was identified. Genetic modulation of the abundance of PaCATB identified differential effects on the phenotype of the corresponding strains. Deletion of PaCatB resulted in decreased resistance, over-expression in increased resistance against hydrogen peroxide. While the lifespan of the genetically modified strains was found to be unaffected under standard growth conditions, increased exogenous hydrogen peroxide stress in the growth medium markedly reduced the lifespan of the PaCatB deletion strain but extended the lifespan of PaCatB over-expressors. Overall our data identify a component of the secretome of P. anserina as a new effective factor to cope with environmental stress, stress that under natural conditions is constantly applied on organisms and influences aging processes. Podospora anserina represents a well-studied model organism for aging [P. anserina has a small genome [P. anserina has been demonstrated to be associated with various pathways including mitochondrial DNA (mtDNA) instability [P. anserina, as in other organisms, is not mono-factorial, but depends on many factors and a network of different, interacting molecular pathways [The filamentous fungus or aging -4. P. anl genome  that is l genome , is tracl genome , and is l genome . During l genome . Aging itability -13, resptability -17, ROS tability , 18-22, tability , and apotability -26. It tpathways . In aginpathways , 29, is pathways -38. It ipathways -41.cat-3 gene in Neurospora crassa as well as deletion of catB in Aspergillus nidulans increases the sensitivity against hydrogen peroxide. Furthermore, an induction of the catalase activity caused by treatment with exogenous hydrogen peroxide, identified these catalases as part of the oxidative stress response [While the FRTA is basically addressing the impact of ROS generated by normal metabolism within a cell, biological systems are also challenged by ROS originating from the environment. For instance, food taken up as energy supply can be such a source. Moreover, the generation and secretion of ROS by organisms is a well known strategy to attack competitors in the same environmental niche or to weaken potential hosts prior to infection , 43. In response -46.P. anserina, we identified a ROS scavenging protein, PaCATB, to increase in abundance in the secretome of senescent cultures. The deletion and over-expression of the gene coding for this catalase provided evidence for a role in protecting growing P. anserina cultures against environmental oxidative stress and as such has an impact on the lifespan of this aging model.The aim of our study was to explore a potential impact of exogenous oxidative stress and it's relation to extracellular ROS defense and to aging. Towards this goal we performed a comparative secretome analysis. In comparison to secreted proteins from juvenile cultures of PaCatB over-expressing strains (PaCatB_OEx1-3) was performed by amplifying the open reading frame of PaCatB (plus ~ 500bps terminator region) by PCR using the oligonucleotides PaCatBEx1-1 (5‘-GGGGATCCAT GAAAAGGCTG CTAACG-3’) and PaCatBEx1-2 (5‘-CCAAGCTTAA AAGCTCACCG GCCAAC-3’), introducing Bam HI and Hin dIII restriction sites (underlined). The amplicon was cloned into the pExMthph backbone (BamHI / HindIII digested) resulting in the plasmid pPaCatBEx1. In this the PaCatB reading frame is under control of a strong constitutive metallothionein promoter [P. anserina wild-type spheroplasts. Transformants were selected for hygromycin-resistance and verified by Southern blot analysis. Three different strains containing one additional copy of PaCatB were subsequently further analyzed (PaCatB_OEx1-3).The generation of promoter . The plaPaCatB in wild-type strain s was performed according to a previously described method [PaCatB were amplified using the 5‘-flank specific oligonucleotides PaCatBKO1-1 (5‘-CCGGTACCCC TTTGCCGGGG GGCGTG-3’) and PaCatBKO1-2 (5‘-CCCTGCAGCT GCTGCCGCTG CCGTGC-3’) introducing KpnI and Pst I restriction sites and the 3'-flank specific oligonucleotides PaCatBKO1-3 (5‘-GGACTAGTGG AAAAGGGAAT GGGTTC-3’) and PaCatBKO1-4 (5‘-GGGCGGCCGC ACTAATATAT ATACCG-3’) with Bcu I and Not I restriction sites. The fragments were cloned into the plasmid pKO4 [Escherichia coli and a phleomycin resistance cassette for selection in P. anserina. The resistance cassette with the flanking regions was excised by degistion with NotI and KpnI and transformed into the E. coli KS272 strain which contains plasmid pKOBEG [PaCatB gene containing cosmid 19B11 [P. anserina wild-type spheroplasts. Transformants were selected by growth on phleomycin-containing medium. Successful deletion of PaCatB was indicated by phleomycin resistance and hygromycin sensitivity. The correct replacement of the PaCatB gene was verified by Southern blot analysis. The selected strain, lacking the PaCatB gene and harbouring the phleomycin gene instead, was termed ΔPaCatB.Deletion of d method . Brieflymid pKO4 , 49 nextd pKOBEG  and the id 19B11 . HomologP. anserina spheroplasts was performed as described [Production, regeneration, and integrative transformation of escribed , 53.P. anserina wild-type strain s [PaCatB over-expressing strains (PaCatB_OEx1-3) and the deletion strain (ΔPaCatB) were used. All transgenic strains are in the genetic background of wild-type strain s. Strains were grown on standard cornmeal agar (BMM) at 27 °C under constant light [P. anserina synthetic medium) [In this study the strain s , the new medium) .PaCatB and PaCatB_OEx1-3: n=8) were grown on P. anserina synthetic medium (PASM) [For incubation of strains with hydrogen peroxide, mycelia of monokaryotic isolates   containiPaCatB: n=20, PaCatB_OEx1: n=29, PaCatB_OEx2: n=11, PaCatB_OEx3: n=14). Mycelial pieces from cultures of freshly germinated ascospores were placed on race tubes containing PASM [Lifespan determination was performed with cultures originating mono-nucleate ascospores  or 3 mM hydrogen peroxide (wild-type and PaCatB_OEx1-3: n=40).Lifespan determination under oxidative stress was performed as described above. The different isolates were placed on Petri dishes with PASM containing 0.75 mM hydrogen peroxide  and were incubated for 4 days at 27 °C under constant light without shaking to prevent cell damages and consequential resultant contamination with total cell extract. The filtered liquid medium was enriched by filter tubes . Supernatants were mixed with 1:100 PIC (Protease Inhibitor Cocktail). The secreted proteins were precipitated by adding 2 volumes of ethanol and 2 volumes of acetone for 2 days at -20 °C. After centrifugation  1 ml desalinisation solution (2:1:1 water: ethanol: acetone) was added to the pellet. After 1 min of vortexing, the secreted proteins were incubated over night at -20 °C. This desalinisation procedure was repeated twice. Pellets were dried and resolved with 50 μl protein extraction puffer at room temperature.Mycelia from wild-type strain s, P. anserina strains was allowed to overgrow a cellophane foil covered PASM surface for 4 days in the dark. Subsequently, harvested mycelia were pulverized in liquid nitrogen. The protein was isolated from the powder as described [For extraction of whole cell protein, mycelia from different escribed .Secreted protein probes were purified  and eluted in IEF buffer . For the isoelectric focusing, protein (400 μg) was applied to IEF strips . The electro-focusing program was limited to 50 μA per strip and started with 250V for 15 min. Voltage rose rapidly up at 10,000 V for 3 h. This was maintained till 60,000 Vh were obtained. The strips were equilibrated in Tris base  for 10 min and then equilibrated for 10 min in the same buffer with 2.5% iodoacetamide instead of dithiothreitol. The strips were fixed on top of SDS separating polyacrylamide gels (10%) with a 0.5% agarose solution in SDS separation buffer with 0.00067% bromphenol blue. The gels were run at 6 mA/ gel for 3 h followed by 18 mA/ gel for 4 h. The current was set to 20 mA/ gel until the bromphenol blue flew out. The gels were silver stained as previously described .10 μg secreted protein were fractionated by two-phase SDS-PAGE (6-18 % separating gels) according to standard protocol . After e2O2 solution. Subsequently, the gel was transferred into 30 ml freshly prepared solution containing 2 % potassium ferricyanide and 2 % ferric chloride. The gel tray was gently agitated over a light box until appearance of a green color in the gel. After rinsing the gel with water it was scanned for further analysis.The ‘in-gel’ catalase stain was performed as described  with nat2O2 degradation was performed in quartz glass cuvette  by measuring the absorption at 240 nm in intervals of 10 sec for a total of 10 min. The cuvette was loaded with 300 μl of a 0.01 M potassium phosphate buffer (pH 7.0) and 100μl of a 300 mM H2O2 solution dissolved in 0.01 M potassium phosphate buffer (pH 7.0) was added. After reaching a constant absorption, 100 μl of a 0.02 μg/μl protein solution, dissolved in 0.01 M potassium phosphate buffer (pH 7.0), was added. The percentage absorption decrease at 240 nm per time, representing the catalase activity, was calculated based on the amount of the linear slope after addition of the protein solution. All samples were measured as triplets in three independent experiments.The photometric quantification of HProtein spots of interest were manually excised from gels and digested with trypsin . The resulting peptide mixture was desalted and analyzed using a 4800 Plus MALDI TOF / TOF Analyzer  as described before . From thP. anserina cultures have been achieved by iTRAQ labelling and nanoLC-MS/MS in combination with sample pre-fractionation by SCX (strong cathion exchange) following previously described protocols [Identification and quantitation of the secretome of juvenile and senescent rotocols . BrieflyPodospora anserina sequence database (version 55). Trypsin was used as a cleavage enzyme with a maximum of 2 missed cleavages was allowed. Carbamidomethyl (C) was chosen as a fixed modification. As variable modifications, iTRAQ 4plex (K), iTRAQ 4plex (N-term) and Oxidation (M) were chosen. The data were searched with a peptide mass tolerance of 10 ppm and a fragment mass tolerance of 0.8 Da (CID) and 0.1 Da (HCD). Only proteins identified by at least 2 peptides and mascot peptide ion scores of at least 20 were considered. The quantitative results were normalized on protein median.The data were processed described before with minor changes . The rawP. anserina, secreted proteins from juvenile and senescent wild-type cultures were isolated and analyzed by 2D SDS-PAGE and mass spectrometry . Two of them are small subunit catalases within the peroxisome (PaCATP1) or the cytosol (PaCATP2). In addition, a catalase-peroxidase (PaCAT2) is localized in the cytosol. Furthermore, two large putative catalases, PaCATA and PaCATB, are encoded by the P. anserina genome. PaCATA is predicted to be bound to the plasma membrane whereas PaCATB contains a putative secretion sequence. Homologues of PaCATB are known in various ascomycetes such as Neurospora crassa (Cat-3), Aspergillus nidulans (CatB) and Blumeria graminis (CatB) with a high similarity   and three independent PaCatB over-expression strains (PaCatB_OEx1-3) containing one extra copy of PaCatB under the control of the strong constitutive metallothionein promoter [P. anserina were selected and subsequently analyzed.In order to investigate the role of the putative secreted and age-regulated catalase PaCATB in lifespan control and ROS scavenging, we set out to generate strains in which promoter  integratPaCatB over-expression strains (PaCatB_OEx1-3) and the PaCatB deletion strain (ΔPaCatB) were verified  completely failed to decompose hydrogen peroxide while the three over-expressors (PaCatB_OEx1-3) decomposed hydrogen peroxide with a 2- to 6-fold higher rate compared to the wild-type  displayed significant increased growth rates  was characterized by an increased sensitivity against low levels of exogenous hydrogen peroxide  compared to the wild-type   Figure .To determine the induction of PaCATB activity and thereby an active mechanism of protection against hydrogen peroxide, catalase activity of whole protein extracts from different strains were analyzed Figure .P. anserina . When grown on media with added hydrogen peroxide, the wild-type strain exhibits an increased catalase B activity compared to the control without oxidative stress. While the wild-type shows an induction of PaCATB activity by hydrogen peroxide stress, the deletion strain proofs that the remaining catalases of a Figure : PaCATX P. anserina is a filamentous fungus that is an extensively studied aging model. Studies with this system were generally taking advantage of the possibility to keep the organism under well defined and controlled conditions in the laboratory and to carry out specifically designed manipulations. In this way the successful identification of a variety of individual components and molecular pathways involved in the control of aging and lifespan was possible P. anserina, the impact of the natural conditions and the interactions with many other microorganisms is only initially investigated [P. anserina cultures. The rationale behind this strategy was to identify factors secreted and effective in protecting the growing individual against environmental stress.In stigated  but may Comparing the secretome of juvenile and senescent cultures by 2D SDS-PAGE and mass spectrometry identified a number of proteins which change in abundance. Among others, PaCATB abundance decreased in the secretome of senescent cultures. Since catalases are ROS scavenging proteins and oxidative stress is of particular relevance for aging, PaCATB was selected for further studies.PaCatB over-expressors and a deletion strain were compared to the wild-type, there was a clear effect when strains were challenged with increased amounts of hydrogen peroxide in the medium. Strains with increased PaCATB were long-lived and showed an increased tolerance against hydrogen peroxide while those missing PaCATB were short-lived and highly sensitive against this ROS. The sensitivity of the PaCatB deletion strain against hydrogen peroxide is consistent with the findings in A. nidulans and N. crassa catalase deletion strains [The analyses of strains with altered PaCATB abundance revealed an age-related function of this enzyme. While no effect on lifespan was observed under standard growth conditions when  strains -46.P. anserina is growing on and may be due to the competition for the same natural resources with the many microorganisms living in this ecological niche. The secreted catalase may provide a selective advantage and help to protect against the attack by microorganisms in the immediate environment. Parasitic fungi use ROS as chemical weapons to weaken a potential host prior to successful infection [P. anserina the secreted catalase may indeed provide an efficient protection shield and to allow extraction of enough nutrients from the growth substrate and thus to go successfully through a full reproduction cycle.To the best of our knowledge this is the first report of an effect on tolerance against oxidative stress, aging and lifespan of a secreted isoform of the ROS scavenging system. Such a system may have evolved as a consequence of increased oxidative stress in the natural substrate, herbivorous dung, nfection , 61. ForP. anserina it is known that in senescent cultures hydrogen peroxide secretion is increased compared to juvenile and middle-aged cultures [P. anserina culture [In contrast to the role of an ROS scavenging systems outside the cell, there is a huge literature about the impact of cellular ROS scavenging on aging and disease. Since hydrogen peroxide generated inside the cell can cross membranes and age-related increase of intracellular ROS generation also affects extracellular ROS levels a secreted catalase may be important for protection of growing hyphae. In cultures . Togethe culture .P. anserina we generated strains in which a gene coding for the mitochondrial superoxide dismutase (PaSOD3) was over-expressed. Surprisingly, the corresponding strains were not long- but short-lived and showed different kinds of impairments. A more detailed characterization of these mutants could solve part of the counter-intuitive results. PaSod3 over-expressors were found to be impaired in components of the mitochondrial protein quality control system, like the mitochondrial matrix protease PaCLPP or the heat shock protein PaHSP60, and in mitochondrial peroxiredoxin, a protein converting hydrogen peroxide into water [Many of the different studies investigating the role of oxidative stress and of ROS scavenging systems are contradictory and the results are taken to question the FRTA , 64. Howto water . It appe


Arthrogryposis multiplex congenita is an etiopathogenetically heterogeneous disorder characterised by non-progressive multiple intra-articular contractures, which can be recognised at birth. The frequency is estimated at 1 in 3,000 newborns. Etiopathogenesis of arthrogryposis is multifactorial.We report first 26 weeks of life of a boy with severe arthrogryposis. Owing to the integrated rehabilitation approach and orthopaedic treatment a visible improvement in the range of motion as well as the functionality of the child was achieved. This article proposes a cooperation of various specialists: paediatrician, orthopaedist, specialist of medical rehabilitation and physiotherapist.Rehabilitation of a child with arthrogryposis should be early, comprehensive and multidisciplinary. Corrective treatment of knee and hip joints in infants with arthrogryposis should be preceded by the ultrasound control. There are no reports in the literature on the ultrasound imaging techniques which can be used prior to the planned orthopaedic and rehabilitative treatment in infants with arthrogryposis. The experience of our team indicates that such an approach allows to minimise the diagnostic errors and to maintain an effective treatment without the risk of joint destabilisation. Arthrogryposis multiplex congenita (AMC) is an etiopathogenetically heterogeneous disorder, characterised by non-progressive multiple intra-articular contractures, which can be recognised at birth . The freSymptoms of some forms of arthrogryposis can be found in selected monogenic diseases , chromosomal aberrations and syndromes of multi-organ congenital malformations.Arthrogryposis can also result from environmental factors, affecting the mother and the foetus, such as: infections, medications, traumas, chronic illnesses, oligohydramnios or abnormal structure of the uterus . These aOnly few reports can be found in the literature on the rehabilitation programmes for infants with AMC. Paediatricians are usually the primary care doctors for children with AMC, later, often at the time the children start to walk, they are usually referred to orthopaedists .This study presents a multi-disciplinary rehabilitation approach in a case of a child with full-blown form of arthrogryposis, complicated by perinatal hypoxia. We propose a cooperation of specialists of various disciplines, initiated as early as the neonatal period.th week of gestation. It was not until the 5th week that the pregnancy was detected. Progesteron was recommended vaginally because of the spotting. Furthermore, starting from the 8th week, clotrimazole and vitamins (Femibion) were used throughout the pregnancy. No signs of deformation within the joints were reported in the routine clinical ultrasound evaluation but the oligohydramnios observed in 33th week of pregnancy was linked with the signs of foetus poor movement described by the mother as “very calm baby” during the last weeks of the pregnancy. A boy was delivered after spontaneous labour at term (40th week of gestation) in a general poor condition  with the birth weight of 4150 g, length 56 cm and head circumference 36 cm. The child required ventilation for 4 days. Antibiotics were administered, as an intrauterine infection was suspected based on elevated C-reactive protein levels. Seizures were observed in the first days of life, most likely resulting from the perinatal hypoxia. Subsequently some abnormal EEG readings were evident, requiring a short anticonvulsant therapy with phenobarbital.The boy was born, after the first pregnancy, to young, healthy, unrelated parents. In the first weeks of the pregnancy X-rays of the head and palm were taken in the mother because of the injury. The mother had been taking NSAIDs until the 5Examination after birth revealed the presence of micrognathia, retrognathia and contractures of the joints typical for AMC: i.e. affecting the wrists, elbows and shoulders as well as legs in the form of clubfeet - scoring 4 on Dimeglio scale, accompanied by knee-joint subluxation and dislocation of the right hip joint Figures  and 2. TAn x-ray image of the lower limbs showed visible callus around the femur fractures, the occurrence of perinatal fracture and deformation typical for a clubfeet Figure . An ultrCranial and abdominal ultrasound, as well as ophthalmic examination, revealed no pathology. Examination of the induced auditory potentials showed correct results from the brain stem and bilateral hearing loss at the level of 70 dB. Echocardiography revealed haemodynamically insignificant defect of the atrial septum (ASD). Due to the massive deformation of the occipital area, brain MRI was performed, detecting no abnormalities except for the 9 mm arachnoid cyst, adhering to the left temporal pole. The congenital infections - cytomegaly, toxoplasmosis, parvovirus infection, hepatitis type B and rubeola- were ruled out. Cytogenetic investigations presented normal male karyotype - 46, XY. Submicroscopic aberrations were excluded using microarray-based comparative genomic hybridisation (array-CGH) (array(1–22)×2,×1). Osteogenesis inperfecta was ruled out in further tests. EEG performed some months later showed no pathology.th week of gestation.More than one type of joints was affected. A very specific position with symmetric limb involvement was noted: arm-wrists, elbows and shoulders, fixed knees, equinovarus feet. Typical AMC inducing factors and symptoms could be identified in this case: X-rays were taken in the mother and NSAIDs were used until the 5These could be the potential teratogenic agents in this case, but no signs of other major congenital disorders where found, cytogenetic investigations revealed no abnormalities. The clinical picture was not compatibile with any known monogenic disorder with AMC. The cause of oligohydramnios and the pathological reduction of the foetal movements had not been pinpointed. Bone fractures could only be explained by the difficulties during the labour. Perinatal asphyxia resulted in the seizures and perinatal hypotonia.Rehabilitation was initiated at the age of three weeks, the infant was retained in the hospital for eight weeks in order to stabilise the epileptic activity, to monitor nutrition (due to the lack of sucking reflex and dysphagia), and for the orthopaedic treatment.The aim of the rehabilitation was to increase the range of motion in the joints and enhance the neuro-developmental stimulation.The ultrasound was used to position the structures of the limbs. In particular, ultrasound imaging was helpful in positioning of the patella and the knee axis. The information obtained from ultrasound assessments allowed marking the structures on the skin. This simple procedure allowed for the progress of training, limiting, at the same time, the risk of the joint dislocation. Photographs of the training manouvers, with the marks on the skin, were taken over the therapy to instruct parents and other therapists.Techniques used to increase the mobility in the joints:– relaxing massage and stretching within the muscle attachments,– articular mobilization techniques in strict accordance with the limits of mobility audited under the control of the ultrasound,– corrective kinesiotaping on the dorsal side of the hand, stimulating straightening of the joints in the wrist and fingers,– mobilization of the feet according to the Ponseti method.Techniques of development stimulation– the positional therapy,– stimulation of the sensory systems, proprioceptive stimulation of the hand opening, techniques using the setting reflexes to stimulate supporting functions of the upper limbs, holding the head, etc.,– sucking reflex stimulation.Instruction for parents was an integral part of the rehabilitation: training in the laying position, placing, changing of positions, carrying, feeding, etc.Careful and repeated training of the optimal axes in which the exercises, increasing the motion range, should be performed. Recording and photographing of the presented techniques was encouraged.Orthopaedic treatment was initiated at the age of 3 months . It started with a series of manipulative plasters using a modified Ponseti's protocol (treatment of clubfeet) . The condition before the plaster manipulation is presented at the Figure Through the use of plaster casts a correction of knee joint flexion to 45° was achieved, then plasters on the lower limbs were combined with an aluminium crossbar, in order to achieve a correction of the rotation in the hip joints Figures .Thanks to the integrated rehabilitation approach and orthopaedic treatment a visible improvement in the range of motion, as well as in the functionality of the child, was achieved. The child continues orthopaedic treatment and rehabilitation. Functional assessment and joint deformation assessment had been performed four times since the child’s birth Table .Arthrogryposis in infancy is an interdisciplinary problem. Children affected by this disease require the care of paediatricians, rehabilitation professionals as well as orthopaedists.Children with arthrogryposis can be subjected to orthopaedic treatment with satisfactory results. The current technological advancements allow for an orthopaedic interventions to minimise the limitations in everyday activities . In the The ultrasound revealed features of the medial instability of the knee joints resulting from the improper earlier rehabilitation by relatives during a short stay at home.At the moment it is not possible to determine what level of motor and intellectual development the described child can reach . ParalleEarly rehabilitation of a child with arthrogryposis requires the involvement of the parents/guardians. Unfortunately, in the face of serious illness of the child very often there is a relationship crisis between the parents and even the disintegration of a relationship. Parents of a child burdened with severe congenital disease often experience disappointment, frustration and a sense of losing the control. The care for a sick child usually imposes changes of the everyday life, changes of the long-term life plans, which may result in depression and even in a post traumatic stress disorder .1. Rehabilitation of a child with arthrogryposis should be early, comprehensive and multidisciplinary.2. Corrective treatment of knee and hip joints in the case of infants with arthrogryposis should be preceded by the ultrasound control.Written informed consent was obtained from the patient’s parents for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.The authors declare that they have neither financial nor non-financial competing interests.ABG – originator of the project in its final form, exercised rehabilitation consultations, commissioned rehabilitation activities, coordinated multidisciplinary team activities, obtained funds for the publication, translated the text from Polish and was responsible for drafting a cover letter. ASR – performed all rehabilitation activities, assessed the patient in terms of functionality and was responsible for collecting data and drafting the article. SB – substantial supervision. JW – was responsible for conducting the paediatric proceedings and edited the introduction to the article. KD – performed all consultations and orthopaedic actions, was consulted on the orthopaedic aspect of the article. IMK – made the final adjustments to the text and literature. KZS – introduced final modifications to the article and table. All authors approved the final version of the article.ABG - MD, specialist of medical rehabilitation, member of Polish Rehabilitation Society, assistant in the Department of Rehabilitation.ASR- Physiotherapist, PhD, assistant in the Department of Rehabilitation, member of Polish Physiotherapy Association.SB - MD, associate professor in Medical University of Gdansk, specialist of internal medicine and medical rehabilitation, head of the Department of Rehabilitation, member of Polish Society of internal Medicine, member of Polish Rehabilitation Society, member of Rehabilitation, Physical Culture, Social Integration Committee of Polish Academy of Science.JW - MD, PhD, specialist of paediatrics and clinical genetics, head of the Department of Infant Pathology, member of European and Polish Human Genetics Society, member of Polish Registry of Congenital Malformations Working Group, member of Advisory Council – Cornelia de Lange Syndrome Association.KD - MD, PhD, specialist Of Orthopaedics, assistant in Department of Orthopaedics and Traumatology, member of Polish Orthopaedics and Traumatology Society, Polish Foot and Ankle Society and European Foot and Ankle Society.IMK - MD, associate professor in University of Warmia and Mazury, specialist of paediatrics and medical rehabilitation, director of Rehabilitation Department, member of Polish Rehabilitation Society, voivodeship regional consultant of medical rehabilitation.KZS - MD, specialist of medical rehabilitation, assistant in Department of Rehabilitation, member of Polish Rehabilitation Society and Polish Medical Writers Association.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/13/184/prepub