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What is the function of the AIRE gene at the embryonic stage?

Aire regulates the expression of differentiation-associated genes and self-renewal of embryonic stem cells. Aire and Deaf1 help regulate the ectopic expression of diverse tissue-specific antigens to establish self-immune tolerance. Knockdown of Aire in mouse ESCs resulted in significantly decreased clone-forming efficiency as well as attenuated cell cycle, suggesting Aire plays a role in ESC self-renewal. Aire promotes the expression of the pluripotent factor Lin28 and the self-renewal of ES cells.

[19008896, 21952165, 22540148, 20226168, 19302042]

The negative selection of T cells in the thymus is necessary for the maintenance of self tolerance. Medullary thymic epithelial cells have a key function in this process as they express a large number of tissue-specific self antigens that are presented to developing T cells. Mutations in the autoimmune regulator (AIRE) protein cause a breakdown of central tolerance that is associated with decreased expression of self antigens in the thymus. In this Review, we discuss the role of AIRE in the thymus and recent advances in our understanding of how AIRE might function at the molecular level to regulate gene expression. Mesenchymal stem cells (MSCs) are emerging as a promising immunotherapeutic, based largely on their overt suppression of T lymphocytes under inflammatory and autoimmune conditions. While paracrine cross-talk between MSCs and T cells has been well-studied, an intrinsic transcriptional switch that programs MSCs for immunomodulation has remained undefined. Here we show that bone marrow-derived MSCs require the transcriptional regulator Aire to suppress T cell-mediated pathogenesis in a mouse model of chronic colitis. Surprisingly, Aire did not control MSC suppression of T cell proliferation in vitro. Instead, Aire reduced T cell mitochondrial reductase by negatively regulating a proinflammatory cytokine, early T cell activation factor (Eta)-1. Neutralization of Eta-1 enabled Aire(-/-) MSCs to ameliorate colitis, reducing the number of infiltrating effector T cells in the colon, and normalizing T cell reductase levels. We propose that Aire represents an early molecular switch imposing a suppressive MSC phenotype via regulation of Eta-1. Monitoring Aire expression in MSCs may thus be a critical parameter for clinical use. Embryonic stem cells (ESCs) are pluripotent stem cells from early embryos. It has been well recognized that ESC genomes are maintained in a globally transcriptional hyperactive state, which genetically poised ESCs to the high differentiation potential. However, the transcription factors regulating the global transcription activities in ESCs are not well defined. We show here that mouse and human ESCs express two transcription factors, Aire and Deaf1. Previously known to function in the thymus stromal cells and peripheral lymphoid organs respectively, Aire and Deaf1 help regulate the ectopic expression of diverse tissue-specific antigens to establish self-immune tolerance. Differentiation of ESCs greatly reduced Aire and Deaf1 expression, in a pattern similar to the pluripotent factors, Oct4 and Nanog. Knockdown of Aire in mouse ESCs resulted in significantly decreased clone-forming efficiency as well as attenuated cell cycle, suggesting Aire plays a role in ESC self-renewal. In addition, some differentiation-associated genes that are sporadically expressed in ESCs were reduced in expression upon Aire knockdown. These results suggest that transcription factors such as Aire and Deaf1, which exert global transcriptional regulatory functions, may play important roles in self-renewal of ESCs and maintaining ESC in a transcriptionally hyperactive state. Mutations in the transcriptional regulator, Aire, cause APECED, a polyglandular autoimmune disease with monogenic transmission. Animal models of APECED have revealed that Aire plays an important role in T cell tolerance induction in the thymus, mainly by promoting ectopic expression of a large repertoire of transcripts encoding proteins normally restricted to differentiated organs residing in the periphery. The absence of Aire results in impaired clonal deletion of self-reactive thymocytes, which escape into the periphery and attack a variety of organs. In addition, Aire is a proapoptotic factor, expressed at the final maturation stage of thymic medullary epithelial cells, a function that may promote cross-presentation of the antigens encoded by Aire-induced transcripts in these cells. Transcriptional regulation by Aire is unusual in being very broad, context-dependent, probabilistic, and noisy. Structure/function analyses and identification of its interaction partners suggest that Aire may impact transcription at several levels, including nucleosome displacement during elongation and transcript splicing or other aspects of maturation.

What is the principle of the PAR-CLIP methodology?

In particular, PAR-CLIP utilizes a photoactivatable nucleoside for more efficient crosslinking. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA.

[21559008, 23706177, 22213601, 24297251, 22152485, 22844102, 21851591, 21816340, 20371350, 21572407, 22885304, 20644507, 23368412, 22926237]

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs. Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP). These approaches were prone to identifying indirect or non-physiological interactions. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq). Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking. Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1). Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation. All mRNA molecules are subject to some degree of post-transcriptional gene regulation (PTGR) involving sequence-dependent modulation of splicing, cleavage and polyadenylation, editing, transport, stability, and translation. The recent introduction of deep-sequencing technologies enabled the development of new methods for broadly mapping interaction sites between RNA-binding proteins (RBPs) and their RNA target sites. In this article, we review crosslinking and immunoprecipitation (CLIP) methods adapted for large-scale identification of target RNA-binding sites and the respective RNA recognition elements. CLIP methods have the potential to detect hundreds of thousands of binding sites in single experiments although the separation of signal from noise can be challenging. As a consequence, each CLIP method has developed different strategies to distinguish true targets from background. We focus on photoactivatable ribonucleoside-enhanced CLIP, which relies on the intracellular incorporation of photoactivatable ribonucleoside analogs into nascent transcripts, and yields characteristic sequence changes upon crosslinking that facilitate the separation of signal from noise. The precise knowledge of the position and distribution of binding sites across mature and primary mRNA transcripts allows critical insights into cellular localization and regulatory function of the examined RBP. When coupled with other systems-wide approaches measuring transcript and protein abundance, the generation of high-resolution RBP-binding site maps across the transcriptome will broaden our understanding of PTGR and thereby lead to new strategies for therapeutic treatment of genetic diseases perturbing these processes. Although microRNAs (miRNAs), other non-coding RNAs (ncRNAs) (e.g. lncRNAs, pseudogenes and circRNAs) and competing endogenous RNAs (ceRNAs) have been implicated in cell-fate determination and in various human diseases, surprisingly little is known about the regulatory interaction networks among the multiple classes of RNAs. In this study, we developed starBase v2.0 (http://starbase.sysu.edu.cn/) to systematically identify the RNA-RNA and protein-RNA interaction networks from 108 CLIP-Seq (PAR-CLIP, HITS-CLIP, iCLIP, CLASH) data sets generated by 37 independent studies. By analyzing millions of RNA-binding protein binding sites, we identified ∼9000 miRNA-circRNA, 16 000 miRNA-pseudogene and 285,000 protein-RNA regulatory relationships. Moreover, starBase v2.0 has been updated to provide the most comprehensive CLIP-Seq experimentally supported miRNA-mRNA and miRNA-lncRNA interaction networks to date. We identified ∼10,000 ceRNA pairs from CLIP-supported miRNA target sites. By combining 13 functional genomic annotations, we developed miRFunction and ceRNAFunction web servers to predict the function of miRNAs and other ncRNAs from the miRNA-mediated regulatory networks. Finally, we developed interactive web implementations to provide visualization, analysis and downloading of the aforementioned large-scale data sets. This study will greatly expand our understanding of ncRNA functions and their coordinated regulatory networks. Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide binding sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible target mRNAs and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knockdown, protein but not mRNA expression of the 439 targets was specifically upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites near the start codon of target genes. These sites are functional in vitro and likely confer strong repression in vivo. We propose that GLD-1 interacts with the translation machinery near the start codon, a so-far-unknown mode of gene regulation in eukaryotes. The Photo-Activatable Ribonucleoside-enhanced CrossLinking and ImmunoPrecipitation (PAR-CLIP) method was recently developed for global identification of RNAs interacting with proteins. The strength of this versatile method results from induction of specific T to C transitions at sites of interaction. However, current analytical tools do not distinguish between non-experimentally and experimentally induced transitions. Furthermore, geometric properties at potential binding sites are not taken into account. To surmount these shortcomings, we developed a two-step algorithm consisting of a non-parametric two-component mixture model and a wavelet-based peak calling procedure. Our algorithm can reduce the number of false positives up to 24% thereby identifying high confidence interaction sites. We successfully employed this approach in conjunction with a modified PAR-CLIP protocol to study the functional role of nuclear Moloney leukemia virus 10, a putative RNA helicase interacting with Argonaute2 and Polycomb. Our method, available as the R package wavClusteR, is generally applicable to any substitution-based inference problem in genomics. The RNA-binding protein HuR, while known to stabilize cytoplasmic mRNAs, is largely nuclear. In this issue of Molecular Cell, Mukherjee et al. (2011) and Lebedeva et al. (2011) identify transcriptome-wide HuR-RNA interactions using PAR-CLIP, unveiling HuR's nuclear role in pre-mRNA processing. RNA transcripts are subject to posttranscriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases. Cross-linking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins. We developed a method for CLIP data analysis, and applied it to compare CLIP with photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) and to uncover how differences in cross-linking and ribonuclease digestion affect the identified sites. We found only small differences in accuracies of these methods in identifying binding sites of HuR, which binds low-complexity sequences, and Argonaute 2, which has a complex binding specificity. We found that cross-link-induced mutations led to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites. This bias can be substantially reduced by milder nuclease digestion conditions. Systematic analysis of the RNA-protein interactome requires robust and scalable methods. We here show the combination of two completely orthogonal, generic techniques to identify RNA-protein interactions: PAR-CLIP reveals a collection of RNAs bound to a protein whereas SILAC-based RNA pull-downs identify a group of proteins bound to an RNA. We investigated binding sites for five different proteins (IGF2BP1-3, QKI and PUM2) exhibiting different binding patterns. We report near perfect agreement between the two approaches. Nevertheless, they are non-redundant, and ideally complement each other to map the RNA-protein interaction network. RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary. A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip) defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking with immunoprecipitation followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP). CLIP was used to identify targets of a number of RBPs. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample. We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs. Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al. BACKGROUND: MicroRNAs (miRNAs) play a critical role in down-regulating gene expression. By coupling with Argonaute family proteins, miRNAs bind to target sites on mRNAs and employ translational repression. A large amount of miRNA-target interactions (MTIs) have been identified by the crosslinking and immunoprecipitation (CLIP) and the photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) along with the next-generation sequencing (NGS). PAR-CLIP shows high efficiency of RNA co-immunoprecipitation, but it also lead to T to C conversion in miRNA-RNA-protein crosslinking regions. This artificial error obviously reduces the mappability of reads. However, a specific tool to analyze CLIP and PAR-CLIP data that takes T to C conversion into account is still in need. RESULTS: We herein propose the first CLIP and PAR-CLIP sequencing analysis platform specifically for miRNA target analysis, namely miRTarCLIP. From scratch, it automatically removes adaptor sequences from raw reads, filters low quality reads, reverts C to T, aligns reads to 3'UTRs, scans for read clusters, identifies high confidence miRNA target sites, and provides annotations from external databases. With multi-threading techniques and our novel C to T reversion procedure, miRTarCLIP greatly reduces the running time comparing to conventional approaches. In addition, miRTarCLIP serves with a web-based interface to provide better user experiences in browsing and searching targets of interested miRNAs. To demonstrate the superior functionality of miRTarCLIP, we applied miRTarCLIP to two public available CLIP and PAR-CLIP sequencing datasets. miRTarCLIP not only shows comparable results to that of other existing tools in a much faster speed, but also reveals interesting features among these putative target sites. Specifically, we used miRTarCLIP to disclose that T to C conversion within position 1-7 and that within position 8-14 of miRNA target sites are significantly different (p value = 0.02), and even more significant when focusing on sites targeted by top 102 highly expressed miRNAs only (p value = 0.01). These results comply with previous findings and further suggest that combining miRNA expression and PAR-CLIP data can improve accuracy of the miRNA target prediction. CONCLUSION: To sum up, we devised a systematic approach for mining miRNA-target sites from CLIP-seq and PAR-CLIP sequencing data, and integrated the workflow with a graphical web-based browser, which provides a user friendly interface and detailed annotations of MTIs. We also showed through real-life examples that miRTarCLIP is a powerful tool for understanding miRNAs. Our integrated tool can be accessed online freely at http://miRTarCLIP.mbc.nctu.edu.tw.

Which drugs are utilized to treat amiodarone-induced thyroitoxicosis?

Amiodarone-induced thyrotoxicosis treatment includes anti-thyroid drugs and steroid therapy Radio Iodine Treatment (RIT) may be a safe and useful method of AIT therapy in patients with low RAIU, in whom other treatment methods are contraindicated. Lithium is a useful and safe medication for treatment of iodine-induced thyrotoxicosis caused by amiodarone. Thyrodectomy may be necessary in presence of unresponsiveness to standard medical treatments

[2781955, 12727944, 21135419, 9217642, 11901034, 16910349, 7946779, 16544025, 19675515]

Treatment of amiodarone-induced thyrotoxicosis (AIT) with thionamide, lithium or radioactive iodine is ineffective. This particular form of hyperthyroidism is long-lasting because of the slow elimination of amiodarone. Therefore, an alternative therapy is necessary, especially for patients who need to continue permanent administration of the drug. We report 2 cases of AIT: in one case, amiodarone was interrupted; in the other case, amiodarone was continued because of recurrent ventricular tachycardia resistant to classical antiarrhythmic drugs. Both patients were successfully treated with propylthiouracil (PTU) and dexamethasone (DXT). Amiodarone-induced thyrotoxicosis (AIT) may occur either in the presence of underlying thyroid disease (type I AIT) or in apparently normal thyroid glands (type II AIT). Type II AIT, a destructive thyroiditis, often favorably responds to glucocorticoids. Iopanoic acid (IopAc) is an iodinated cholecystographic agent that inhibits deiodinase activity and reduces the conversion of T(4) toT(3). It has recently been reported that cholecystographic agents restore euthyroidism in patients with type II AIT. We describe the results of a prospective randomized study conducted in 12 patients with type II AIT treated with either iopanoic acid (group A, n = 6) or glucocorticoids (group B, n = 6). Serum free T(3) levels normalized rapidly in both groups after 7 d, from 0.75 +/- 0.20 ng/dl (11.5 +/- 3.1 pmol/liter) to 0.46 +/- 0.10 ng/d (7.1 +/- 1.7 pmol/liter), P < 0.01, and from 0.58 +/- 0.10 ng/dl (9.0 +/- 1.2 pmol/liter) to 0.34 +/- 0.03 ng/dl (5.2 +/- 0.5 pmol/liter), P < 0.003, in groups A and B, respectively (P = NS). Serum free T(4) levels reduced at 6 months in group B [from 2.70 +/- 0.32 ng/dl (35.1 +/- 4.1 pmol/liter) to 1.0 +/- 0.04 ng/dl (13.4 +/- 0.6 pmol/liter), P < 0.0001] but not in group A (from 2.90 +/- 0.6 ng/dl (38.0 +/- 7.5 pmol/liter) to 2.30 +/- 0.4 ng/dl (35.6 +/- 6.1 pmol/liter, P = 0.39; P = 0.005 group B vs. group A). All patients in both groups became euthyroid and had their amiodarone-induced destructive thyroiditis cured as defined by normalization of both serum free T(4) and free T(3) levels, during both drugs therapy. However, patients in group B were cured more rapidly than patients in group A (43 +/- 34 d vs. 221 +/- 111 d, respectively, P < 0.002). This study shows that, albeit both drugs are effective, glucocorticoids are probably the drug of choice for more rapidly curing type II AIT. OBJECTIVE: To determine the frequency, risk factors, clinical features, and management of amiodarone-induced thyroid dysfunctions. DESIGN: Retrospective study. SETTING: A regional hospital in Hong Kong. PATIENTS: Patients who had been prescribed amiodarone for at least 6 months from 1 October 2005 to 30 September 2007. RESULTS: A total of 390 patients (mean age, 70 years; standard deviation, 9 years; 54% male) with a median follow-up of 43 (interquartile range, 25-69) months were studied. Hypothyroidism developed in 87 (22%) of the patients (mean age, 72 years; standard deviation, 7 years; 56% male) and thyrotoxicosis in 24 (6%) of the patients (65 years; 11 years; 54% male). Increased baseline thyrotropin (thyroid-stimulating hormone) level appeared to be predictive of amiodarone-induced hypothyroidism, in which a thyroid-stimulating hormone level of 4 mIU/L or above was associated with a 4.7-fold increase in the risk (95% confidence interval, 1.9-11.7; P<0.001). Compared with those who remained euthyroid on amiodarone, thyrotoxicosis developed in younger patients. In these patients, the classical symptoms of thyroid dysfunction were frequently absent, although worsening of underlying arrhythmias, their cardiac condition, weight loss, and over-warfarinisation were suggestive of amiodarone-induced thyrotoxicosis. In both amiodarone-induced thyrotoxicosis and hypothyroidism, the disease course was benign. Patients with the former showed a good response to anti-thyroid drugs and steroid therapy. CONCLUSIONS: Amiodarone-induced thyroid dysfunction is common among our population. As the clinical presentations are usually vague and atypical, regular biochemical monitoring of thyroid function is warranted, particularly in patients with elevated baseline thyroid-stimulating hormone level. The disease course of amiodarone-induced thyrotoxicosis is usually benign and remits with timely administration of anti-thyroid medications, with or without corticosteroids. PURPOSE: Amiodarone hydrochloride is an iodine-rich drug effective in the control of various tachyarrhythmias. It is known to cause refractory to thyrotoxicosis, which usually does not respond to regular antithyroid drugs. Lithium bicarbonate is a medication used to treat psychiatric disorders; it also influences thyroid production and release of hormones. We tried it in combination with propylthiouracil (PTU) for the treatment of amiodarone-induced thyrotoxicosis. PATIENTS AND METHODS: Twenty-one patients were studied. The first group (n = 5) was treated by amiodarone withdrawal only. The second group (n = 7) received PTU (300 to 600 mg), and the third (n = 9) PTU (300 mg) and lithium (900 to 1350 mg) daily. Patient selection was not randomized. The PTU + lithium group had more severe symptoms and signs of thyrotoxicosis, as well as thyroxine levels at least 50% above the upper limit of normal. They also had been on a longer course of amiodarone treatment (34.3 +/- 11.9 months) than the PTU-only (11.4 +/- 7.5) and the no-treatment (7.8 +/- 4.2) groups. RESULTS: While there was no difference between the first two groups in time until recovery (10.6 +/- 4.0 versus 11.6 +/- 0.5 weeks, respectively), the group receiving lithium normalized their thyroid function tests in only 4.3 +/- 0.5 weeks (P < 0.01 versus both other groups). T3 levels normalized even earlier-by 3 weeks of lithium treatment. No adverse effects of lithium were encountered, and the medication was stopped 4 to 6 weeks after achieving a normal clinical and biochemical state. CONCLUSIONS: We conclude that lithium is a useful and safe medication for treatment of iodine-induced thyrotoxicosis caused by amiodarone. We would reserve this treatment for severe cases only. Further studies are needed to find out whether in patients with this troublesome complication lithium therapy could permit continuation of amiodarone treatment. A female patient was admitted to our Department for total thyroidectomy in amiodarone-induced thyrotoxicosis. The drug was prescribed for ventricular arrhythmia and atrial paroxysmal fibrillation in dilated cardiomyopathy due to chronic aortic regurgitation with left ventricular dysfunction (ejection fraction 35%; Class Functional NYHA III) and moderate-severe respiratory insufficiency. The cardiologist-anesthetist team has allowed to evaluate the surgical-cardiovascular-anesthesiologic risks and the balance between the improvement by the amiodarone administration for the arrhythmia, and the discontinuation of this treatment in order to prevent aggravation of the thyrotoxicosis. These hypotheses were subsequently discharged for the two reasons listed below: - several other antiarrhytmic drugs (that didn't show equivalent efficacy as amiodarone in preventing or converting such ventricular and atrial arrhythmias) may be proposed in the place of amiodarone. However, this could expose the patient to an arrhythmia; - a clear proof that the suspension of amiodarone can allow restoring normalization of the thyroid function doesn't exist. Therefore, the patient has been successfully submitted to the surgical intervention and in the follow-up we brought her back to a state of normalized thyroid function and cardiovascular conditions. In patients that cannot safely discontinue amiodarone or when medical therapy is ineffective in controlling thyrotoxicosis, thyroidectomy is the treatment of choice. Two patients with amiodarone-induced thyrotoxicosis were treated successfully with potassium perchlorate and carbimazole while treatment with amiodarone was continued. These antithyroid drugs were stopped after the patients had became clinically and biochemically euthyroid. During follow up, when treatment with amiodarone continued, thyrotoxicosis did not recur. Amiodarone-induced thyrotoxicosis seems to be a transient condition that can be treated successfully with a short course of antithyroid drugs without stopping amiodarone treatment. Among the amiodarone-induced thyroid dysfunctions, thyrotoxicosis is the most troublesome and with the highest rate of morbidity and mortality. Treatment consists in the use of a high dose of anti-thyroid drugs and steroids in an isolated form or in combination. Association of several other drugs have been proposed for the treatment of refractory cases. In this study we report the case of a 40 y.o. patient, with a history of idiopatic dilated miocardiopathy, who developed severe amioradone-induced thyrotoxicosis after heart transplantation. Since the patient did not respond to an initial treatment consisting of a high dose of anti-thyroid drugs combined with steroids, a low dose of lithium carbonate was added for a short period of time, which resulted in normalization of the thyroid function. In this case, the addition of lithium carbonate to the two other drugs resulted in a successful and safety therapy in controlling amiodarone-induced thyrotoxicosis. INTRODUCTION: Amiodarone (AM) is frequently used in the therapy of patients with cardiac disorders. However, due to high iodine content, it has side effects on thyroid function. The use of radioiodine therapy (RIT) in amiodarone-induced thyrotoxicosis (AIT) with low radioactive iodine uptake (RAIU) is still controversial. In these patients therapeutic choices for refractory disease include surgery, antithyroid drugs, or glu ocorticosteriods. AIM: The aim of the study was to evaluate the efficacy of RIT in patients presenting AIT and low RAIU in two-year follow-up. PATIENTS AND METHODS: 40 patients (25 men and 15 women) aged from 63 to 83 years (x +/- SD: 66.2 +/- 5.0 years; median: 65 years) treated with RIT were included into the study. In these patients AM therapy was essential for the underlying heart disorder, while surgery, antithyroid drugs or glucocorticosteroids, were contraindicated. Forty seven patients with toxic multinodular goiter (TMNG) (39 women and 8 men), matched for age (67 +/- 12 yr; range 54-89 yr), were enrolled into the study as a comparative group. The diagnostic procedures included baseline thyroid function tests (thyrothropin - TSH, free triiodothyronine - fT3 and free thyroxine - fT4 levels), thyroid autoantibodies measurement (antithyroglobulin autoantibodies - TgAb, antithyroid peroxidase autoantibodies - TPOAb, anti-TSH receptor autoantibodies - TRAb), thyroid ultrasonography, thyroid scintiscan and RAIU assessment. RESULTS: Serum values of TSH, TgAb, TPOAb and TRAb were undetectable in both groups. In patients with AIT fT4 level was 18.7 to 38.7 pmol/l (mean: 27.1 +/- 5.8) and fT3 concentration was 3.9 to 5.6 pmo/l (mean: 5.7 +/- 1.4), while in TMNG patients level of fT4 was 31.5 to 22.2 pmol/l (mean: 25,3 +/- 5,8) and fT3 concentration was 3.8 to 4,2 pmo/l (mean: 4,2 +/- 0,2). Mean RAIU values after 5h and 24h in AIT patients were 2.3 +/- 0.5 and 3.1 +/- 0.9%, while in TMNG patients were 18,0 +/- 3,8 and 35,7 +/- 9,1%, respectively. A significant difference (p<0.001) between 5h and 24h RAIU in AIT compared to TMNG was noted. In all patients with AIT, a dose of 800 MBq of 131I was administered. During two-year-observation recurrence of hyperthyroidism was observed in two patients (5%) with TMNG. These patients received a second radioiodine dose 16.2 +/- 15 months later (the mean re-treatment dose was 735.93 +/- 196.1 MBq). In comparison, none of the patients with AIT required a second 131I dose and only one patient (2.5%) 6 months after ablative 131I dose needed anti-thyroid medication. Transient hypothyroidism was observed in only two patients (5%) with AIH, though was not observed in TMNG. During follow-up time, no sudden deaths in AIT patients were observed; one patient was diagnosed with prostate cancer, and in one patient acute toxic hepatitis after AM occurred. CONCLUSION: RIT may be a safe and useful method of AIT therapy in patients with low RAIU, in whom other treatment methods are contraindicated.

How is spastic diplegia diagnosed?

Diagnosis of spastic diplegia is mainly carried out with through clinical gait analysis (CGA), with variations such as 1-minute walk, LSU, and 10-meter walk tests, or Gross Motor Function Measure-88 (GMFM-88). Other methods used for evaluation of patients include brain magnetic resonance imaging (MRI) and motor function, presence of epileptic episodes, and IQ or developmental quotient.

[24901761, 21310339, 25276040, 24239880, 17826455, 20829081, 10427678, 1743414, 23085499, 25700542, 24409030, 24182356, 16344032, 2816867, 15157997, 23121133, 2600179, 7270517, 12549749, 17509240]

Argininemia is caused by a deficiency of arginase 1, which catalyzes the final step in the urea cycle, i.e., the cytosolic hydrolysis of arginine to ornithine and urea. In contrast to other urea cycle disorders, hyperammonemic encephalopathy is rarely observed in patients with argininemia. Rather, most exhibit an insidious onset and progression of neurologic manifestations, including spastic diplegia. We describe the first Korean patient with argininemia, manifesting as slowly progressive spastic diplegia. Our patient carries c.[32T>C]+[913G>A] (p.[Ile11Thr]+[Gly305Arg]) mutations in the ARG1 gene. The latter mutation was not previously reported. Although argininemia is a very rare disease, it is recognized as a pan-ethnic disorder. We conclude that argininemia should be considered more frequently in the differential diagnosis of a patient with slowly progressive neurologic manifestations, especially progressive spastic diplegia, even in a population where argininemia was previously unknown. OBJECTIVE: To determine the relationships between spasticity, strength, and the functional measures of gait and gross motor function in persons with spastic diplegia cerebral palsy (CP). DESIGN: Retrospective, cross-sectional study. SETTING: Hospital clinic. PARTICIPANTS: Ninety-seven participants (49 boys, 48 girls; mean age+/-standard deviation, 9.11+/-4.8 y) with spastic diplegia CP were tested once. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: A KinCom dynamometer was used to objectively measure spasticity (ankle plantarflexors, knee flexors, hip adductors) and maximum strength (ankle dorsiflexors and plantarflexors, knee flexors and extensors, hip abductors and adductors). A gait analysis was conducted to evaluate linear variables (gait speed, stride length, cadence) and kinematic variables (ankle dorsiflexion, foot progression, knee and hip flexion, pelvic tilt at initial contact and ankle dorsiflexion, knee and hip flexion, pelvic tilt, trunk rotation range of motion) during gait. Gross motor function was measured using the Gross Motor Function Measure (GMFM-66) and separately, the GMFM walking, running & jumping dimension. Multiple linear regression analysis was used to determine the relationships between spasticity, strength, gait, and the GMFM (P<.05). RESULTS: Spasticity did not account for a substantial amount of explained variance in gait and gross motor function (up to 8% for the GMFM walking, running & jumping dimension). Moderate to high correlations existed between strength and gait linear data and function, accounting for up to 69% of the explained variance (strength and GMFM-66, r2=.69). CONCLUSIONS: For this cohort of participants with spastic diplegia CP who ambulated with or without an assistive device, strength was highly related to function and explained far more of the variance than spasticity. The results may not be generalized to those with more severe forms of CP. OBJECTIVE: This study examined the construct validity of the In-Hand Manipulation Test (IMT) by assessing the test's ability to discriminate between samples of children with and without known fine motor problems. METHOD: The IMT was administered to 55 children without known fine motor problems and 24 children with spastic diplegia who had mild to moderate fine motor problems. Construct validity was estimated by evaluating how accurately the IMT classified the children as having or not having fine motor problems on the basis of total score. RESULTS: A discriminant analysis indicated that IMT total score correctly classified 83.33% of the participants as having or not having fine motor problems. CONCLUSION: The IMT has adequate construct validity to classify the participants of this study and for continued use as a research instrument to assess children's in-hand manipulation skills. Additional validity studies of the IMT are needed with other samples of children before its use for clinical purposes. Leg movements in the supine position of 49 infants with spastic diplegia (three to 11 months corrected age) were examined. Only simultaneous flexion and extension of the hips and knees were seen, with exceptional isolated hip movements; the simultaneous movements had synergic features. When the knees were flexed, the hips were flexed, abducted and externally rotated, and the ankles were dorsiflexed. When the knees were extended, the hips were extended, adducted and internally rotated and the ankles were plantar-flexed. Hip flexion combined with knee extension (leg elevation) and isolated knee movements were not seen in diplegic infants, but were seen in all control preterm infants with a good prognosis, after five and six months corrected age, respectively. The absence of these movements is a useful diagnostic item for spastic diplegia. Hereditary spastic paraplegia (HSP) and spastic diplegia (SD) patients share a strong clinical resemblance. Thus, HSP patients are frequently misdiagnosed with a mild form of SD. Clinical gait analysis (CGA) has been highlighted as a possible tool to support the differential diagnosis of HSP and SD. Previous analysis has focused on the lower-body but not the upper-body, where numerous compensations during walking occur. The aim of this study was to compare the full-body movements of HSP and SD groups and, in particular, the movement of the upper limbs. Ten HSP and 12 SD patients were evaluated through a CGA (VICON 460 and Mx3+; ViconPeak(®), Oxford, UK) between 2008 and 2012. The kinematic parameters were computed using the ViconPeak(®) software (Plug-In-Gait). In addition, the mean amplitude of normalised (by the patient's height) arm swing was calculated. All patients were asked to walk at a self-selected speed along a 10-m walkway. The mean kinematic parameters for the two populations were analysed with Mann-Whitney comparison tests, with a significant P-value set at 0.05. The results demonstrated that HSP patients used more spine movement to compensate for lower limb movement alterations, whereas SD patients used their arms for compensation. SD patients had increased shoulder movements in the sagittal plane (Flexion/extension angle) and frontal plane (elevation angle) compared to HSP patients. These arm postures are similar to the description of the guard position that toddlers exhibit during the first weeks of walking. To increase speed, SD patients have larger arm swings in the sagittal, frontal and transversal planes. Upper-body kinematics, and more specifically arm movements and spine movements, may support the differential diagnosis of HSP and SD. Author information: (1)Division of Pediatric Neurology, Department of Pediatrics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. (2)Division of Medical Genetics, Department of Pediatrics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. (3)Division of Neurosurgery, Department of Surgery, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. (4)Division of Pediatric Orthopedics, Department of Surgery, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. (5)Division of Developmental Pediatrics, Department of Pediatrics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. [Purpose] This study aimed to investigate the effects of Vojta therapy on spatiotemporal gait parameters in children with spastic diplegia. [Methods] The study population consisted of 3 children diagnosed with spastic diplegia. The subjects were treated with Vojta therapy for 8 weeks and followed up for 8 weeks after completion of the therapy. Vicon motion analysis was used to determine the subjects' spatiotemporal gait parameters. [Results] The following results were noted in the changes of each joint angle in the sagittal plane after Vojta therapy. Subject 1 remained in phase throughout the entire gait cycle and did not show any noticeable improvement, even demonstrating a negative range of motion when compared to the baseline. Subject 2 showed a normal anti-phase in heel strike, and the mid-stance, and swing phases. Subject 3 showed a normal anti-phase in heel strike and mid-stance, but the anti-phase during the swing phase was not significantly different from the baseline. For subjects 2 and 3, compared to the baseline, the range of motion of the hip and knee increased but the range of motion of the ankle decreased. [Conclusion] The findings of this study indicate that Vojta therapy can do a good role in improve the spatiotemporal gait parameters of children with spastic diplegia. AIM: The aim of this study was to explore the physical status and gait patterns of children with spastic diplegia secondary to human immunodeficiency virus encephalopathy (HIVE). METHOD: A cross-sectional study was conducted on children diagnosed with HIVE and spastic diplegia. Sociodemographic and clinical background information was obtained, followed by three-dimensional gait analysis (3DGA) and a physical examination including assessments of muscle tone, strength, motor control, contractures, and bony deformities of the lower extremities. RESULTS: Fourteen children (eight males, six females; mean age 5 y 8 mo [SD 9 mo], range 4 y 4 mo-6 y 10 mo) were studied. The cohort was divided into two groups based on distinctive gait patterns. Nine participants in group I showed only limited abnormalities. Group II displayed a more pathological gait pattern including stiff knee and equinus ankle abnormalities. Results of 3DGA, as with the physical examination outcomes, showed increased impairments from proximal to distal (except for hip extension). INTERPRETATION: This study provides a first description of distinctive gait patterns and related physical characteristics of children with HIVE and spastic diplegia. Further research is necessary. OBJECTIVE: To assess the reliability and validity of a newly described classification of sagittal plane alignment in spastic diplegic gait. DESIGN: Twenty split-screen videos of children with spastic diplegia, Gross Motor Function Classification System levels I to III, were viewed on 2 occasions, 6 weeks apart, by 5 raters. The sagittal plane alignments of the right and left lower limbs in gait were classified separately as true equinus, jump knee, apparent equinus, or crouch, based on the published classification. A fifth category, nonclassifiable, was used if classification was not possible. We then used sagittal plane kinematic data to confirm the classification for each subject and these were compared with rater classification scores, which used the video information only. SETTING: Tertiary-level children's hospital. PARTICIPANTS: Three pediatric orthopedic surgeons and 2 pediatric orthopedic residents. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Gait classification scores derived from visual observation were compared among and within raters. The gait classification scores derived from visual observation were compared with the scores derived from sagittal plane kinematic data to assess validity. RESULTS: A moderate correlation was found among the 5 raters within each session, with an interrater weighted kappa score of .45 in session 1 and .49 in session 2. The intrarater, weighted kappa scores showed a moderate to substantial level of agreement between sessions, ranging from .50 to .68. The classification scores of individual raters had moderate validity when compared with classifications derived from the sagittal plane kinematic data. However, there was a substantial level of agreement between the consensus opinion and the classification obtained using the kinematic data as well as the video recordings (weighted kappa=0.8). CONCLUSIONS: This classification has only moderate reliability and validity when a single experienced rater views the 2-dimensional gait videos. However, the consensus opinion derived from the scores of 5 raters considerably improves the validity of the assessment. A retrospective study examined early neurodevelopmental behaviors of children with spastic diplegia, spastic hemiplegia, and quadriplegia (spastic, athetoid, or mixed) who had been followed up longitudinally in a high-risk infant follow-up clinic. Compared with peers with normal outcomes, children with all three types of cerebral palsy had significantly lower scores on the Bayley Mental Scale at 4 months of age; children with hemiplegia and quadriplegia also scored significantly lower on the Bayley Motor Scale. On the Movement Assessment of Infants at 4 months of age, the children with hemiplegia and quadriplegia showed significantly higher risk scores than the nonhandicapped group. The Movement Assessment of Infants was more than three times as sensitive as the Bayley Motor Scale in detecting motor abnormalities in 4-month-old infants with diplegia and more than twice as sensitive in detecting early abnormalities of hemiplegia. At 1 year of age, however, the Bayley Motor Scale was extremely sensitive in picking up motor deficits in children with all three types of cerebral palsy. The objective of this prospective study was the application of proton magnetic resonance spectroscopy in children with spastic diplegia (SD) to determine the metabolite profile of SD children in the left basal ganglia, and to assess the relationship of this profile with motor and mental development. Patients with SD showed reduced ratios of N-acetylaspartate (NAA)/creatine (Cr), NAA/choline (Cho), NAA/myo-inositol (mI), Cho/NAA, Cho/Cr and Cho/mI in the basal ganglia compared to a well-matched control group. On the other hand, we noted increased Cr/NAA, Cr/Cho and mI/NAA ratios in the SD patients as compared with controls. NAA/mI ratios were positively correlated with the severity scale of cerebral palsy in SD children. There was also a significant correlation between Cr/NAA and mental retardation. Increased Cr/NAA, Cr/Cho and mI/NAA ratios in SD children may suggest the existence of the compensatory mechanisms in these patients. The NAA/mI ratio could be used as an additional marker of SD severity and Cr/NAA as a marker of the mental retardation. AIM: To investigate the association between magnetic resonance imaging (MRI) patterns and motor function, epileptic episodes, and IQ or developmental quotient in patients born at term with spastic diplegia. METHOD: Eighty-six patients born at term with cerebral palsy (CP) and spastic diplegia (54 males, 32 females; median age 20 y, range 7-42 y) among 829 patients with CP underwent brain MRI between 1990 and 2008. The MRI and clinical findings were analysed retrospectively. Intellectual disability was classified according to the Enjoji developmental test or the Wechsler Intelligence Scale for Children (3rd edition). RESULTS: The median ages at diagnosis of CP, assignment of Gross Motor Function Classification System (GMFCS) level, cognitive assessment, and MRI were 2 years (range 5 mo-8 y), 6 years (2 y 8 mo-19 y), 6 years (1 y 4 mo-19 y), and 7 years (10 mo-30 y) respectively. MRI included normal findings (41.9%), periventricular leukomalacia, hypomyelination, and porencephaly/periventricular venous infarction. The frequency of patients in GMFCS levels III to V and intellectual disability did not differ between those with normal and abnormal MRI findings. Patients with normal MRI findings had significantly fewer epileptic episodes than those with abnormal ones (p=0.001). INTERPRETATION: Varied MRI findings, as well as the presence of severe motor dysfunction and intellectual disability (despite normal MRI), suggest that patients born at term with spastic diplegia had heterogeneous and unidentified pathophysiology. To investigate the etiology of spastic diplegia (SD) of prematurity, we compared the prenatal, perinatal, and neonatal course of 18 preterm infants with SD to that of a control group of preterm infants without SD. No significant differences between the group with SD and the control group were found in most of the perinatal and neonatal factors analyzed. Significant differences were found in birth weight, birth head circumference, and the one-minute Apgar score. Controlling for gestational age, infants with SD weighed less at birth, had smaller heads, and were more often briefly neurologically depressed. Intracranial hemorrhage and neonatal seizures occurred significantly more often in infants with SD. Fifteen infants with SD were believed to be neurologically normal at the time of nursery discharge. These findings suggest the importance of prenatal factors in optimally treated preterm infants in whom SD develops. Patients with hereditary spastic paraplegia (HSP) often resemble patients with mild spastic diplegia (SD), although their motor limitations differ. The aim of this study was to analyse quantitatively the gait of HSP and SD subjects in order to define the gait pattern in HSP and the differences between the two conditions. Fifteen subjects with HSP, 40 patients with SD and 20 healthy subjects underwent gait analysis (GA). The spatio-temporal and kinematic parameters at the proximal joints were found to be similar in HSP and SD, whereas the most significant differences were found at the knee and ankle joints. Both groups displayed a tendency for knee hyperextension in the midstance phase, but the duration of this hyperextension was longer in the HSP patients. This study shows that GA complements traditional clinical evaluations, making it possible to distinguish, clearly, between motor ability in HSP and in SD patients; the duration of the knee hyperextension during midstance was found to discriminate between the two gait patterns.

Which is the genetic defect causing Neurofibromatosis type 1?

Neurofibromatosis type 1 (NF1) is due to all types of mutations in the neurofibromin (NF1) gene.

[14722917, 2129297, 16323217, 21567923, 16835897]

One of the main features of neurofibromatosis type 1 (NF1) is benign neurofibromas, 10-20% of which become transformed into malignant peripheral nerve sheath tumors (MPNSTs). The molecular basis of NF1 tumorigenesis is, however, still unclear. Ninety-one tumors from 31 NF1 patients were screened for gross changes in the NF1 gene using microsatellite/restriction fragment length polymorphism (RFLP) markers; loss of heterozygosity (LOH) was found in 17 out of 91 (19%) tumors (including two out of seven MPNSTs). Denaturing high performance liquid chromatography (DHPLC) was then used to screen 43 LOH-negative and 10 LOH-positive tumors for NF1 microlesions at both RNA and DNA levels. Thirteen germline and 12 somatic mutations were identified, of which three germline (IVS7-2A>G, 3731delT, 6117delG) and eight somatic (1888delG, 4374-4375delCC, R2129S, 2088delG, 2341del18, IVS27b-5C>T, 4083insT, Q519P) were novel. A mosaic mutation (R2429X) was also identified in a neurofibroma by DHPLC analysis and cloning/sequencing. The observed somatic and germline mutational spectra were similar in terms of mutation type, relative frequency of occurrence, and putative underlying mechanisms of mutagenesis. Tumors lacking mutations were screened for NF1 gene promoter hypermethylation but none were found. Microsatellite instability (MSI) analysis revealed MSI in five out of 11 MPNSTs as compared to none out of 70 neurofibromas (p=1.8 x 10(-5)). The screening of seven MPNSTs for subtle mutations in the CDKN2A and TP53 genes proved negative, although the screening of 11 MPNSTs detected LOH involving either the TP53 or the CDKN2A gene in a total of four tumors. These findings are consistent with the view that NF1 tumorigenesis is a complex multistep process involving a variety of different types of genetic defect at multiple loci. The locus for the gene causing neurofibromatosis type 1 (NF1) was bracketed to a region on the long arm of chromosome 17 by means of genetic linkage analysis. When the limits of resolution for genetic mapping were reached physical mapping methods were used to map the NF1 gene precisely, with reference to translocation breakpoints in NF1 affected individuals who harboured constitutional chromosomal translocations on chromosome 17. The region of DNA located between two translocation breakpoints has been cloned and a DNA sequence encoding a 11-13 kb mRNA identified. That this sequence shows deletions and point mutations in NF1 affected individuals and not in normal controls provides strong evidence that it is indeed the NF1 gene. The genetic defect in NF2 has been mapped to chromosome 22 by studies of chromosomal loss in tumours associated with this disease. Subsequent linkage analysis of NF2 pedigrees has confirmed this location. DNA markers that bracket the NF2 locus to a region of 5-10 Mb have been identified. Neurofibromatosis type 1 (NF1) is a common tumor predisposition syndrome affecting approximately 1 in 4,000 persons. It is an autosomal-dominant disorder with half of the cases resulting from spontaneous mutations. This genetic defect leads to the formation of benign tumors or neurofibromas of the peripheral nervous system. Dermal neurofibromas may cause local discomfort and itching but are rarely associated with neurological deficit and do not undergo malignant change. The more extensive plexiform neurofibromas produce neurological complications in 27%-43% of patients with NF1 and may undergo malignant degeneration in 5% of cases. Patients with NF1 who develop pain or new neurological symptoms should have a rapid and thorough assessment for malignancy. In this report, we illustrate this point by presenting a patient who developed acute shoulder pain and weakness due to malignant degeneration of a plexiform neurofibroma involving the left brachial plexus, and review the literature on this subject. Neurofibromatosis type 1 and Noonan syndrome are both common genetic disorders with autosomal dominant inheritance. Similarities between neurofibromatosis type 1 and Noonan syndrome have been noted for over 20 years and patients who share symptoms of both conditions are often given the diagnosis of neurofibromatosis-Noonan syndrome (NFNS). The molecular basis of these combined phenotypes was poorly understood and controversially discussed over several decades until the discovery that the syndromes are related through disturbances of the Ras pathway. We present an infant male with coarse facial features, severe supravalvar pulmonic stenosis, automated atrial tachycardia, hypertrophic cardiomyopathy, airway compression, severe neurological involvement, and multiple complications that lead to death during early infancy. The severity of clinical presentation and significant dysmorphic features suggested the possibility of a double genetic disorder in the Ras pathway instead of NFNS. Molecular analysis showed a missense mutation in exon 25 of the NF1 gene (4288A>G, p.N1430D) and a pathogenic mutation on exon 8 (922A>G, p.N308D) of the PTPN11 gene. Cardiovascular disease has been well described in patients with Noonan syndrome with PTPN11 mutations but the role of haploinsufficiency for neurofibromin in the heart development and function is not yet well understood. Our case suggests that a double genetic defect resulting in the hypersignaling of the Ras pathway may lead to complex cardiovascular abnormalities, cardiomyopathy, refractory arrhythmia, severe neurological phenotype, and early death. Neurofibromatosis type 1 (NF1), characterized by skin neurofibromas and an excess of café-au-lait spots, is due to mutations in the neurofibromin (NF1) gene. Identifying the genetic defect in individuals with the disease represents a significant challenge because the gene is extremely large with a high incidence of sporadic mutations across the entire gene ranging from single nucleotide substitutes to large deletions. In the present study, we have used a combination of techniques (heteroduplex analysis, sequencing, loss of heterozygosity and quantification of gene dosage) to define the genetic defect in 68 individuals from a cohort of 107 NF1 Taiwanese patients of Chinese origin. Fifty-eight were initially identified using heteroduplex analytical techniques and confirmed by sequence analysis. A further five were identified by direct sequence analysis alone. The reminders were shown to carry large deletions in the NF1 gene by demonstrating loss of heterozygosity that was confirmed by gene dosage measurements using quantitative-PCR techniques. Mis-sense, non-sense, frame-shift or splice-site mutations were identified across the entire gene of which the majority (45/68) were novel in nature. The detection rate with the various analytical techniques and the types of mutation detected are consistent with published data involving both individuals and large cohort studies from other ethnic backgrounds.

Which is the human selenoprotein that contains several Se-Cys residues?

Selenoprotein P, that contains 10 selenocysteines.

[19345254, 11122377, 10692426, 7637580, 20417644, 15104205, 9288402, 17000762, 15777501]

Selenoprotein P (Sepp1) is a secreted protein that is made up of 2 domains. The larger N-terminal domain contains 1 selenocysteine residue in a redox motif and the smaller C-terminal domain contains the other 9 selenocysteines. Sepp1 isoforms of varying lengths occur but quantitation of them has not been achieved. Hepatic synthesis of Sepp1 affects whole-body selenium content and the liver is the source of most plasma Sepp1. ApoER2, a member of the lipoprotein receptor family, binds Sepp1 and facilitates its uptake into the testis and retention of its selenium by the brain. Megalin, another lipoprotein receptor, facilitates uptake of filtered Sepp1 into proximal tubule cells of the kidney. Thus, Sepp1 serves in homeostasis and distribution of selenium. Mice with deletion of Sepp1 suffer greater morbidity and mortality from infection with Trypanosoma congolense than do wild-type mice. Mice that express only the N-terminal domain of Sepp1 have the same severity of illness as wild-type mice, indicating that the protective function of Sepp1 against the infection resides in the N-terminal (redox) domain. Thus, Sepp1 has several functions. In addition, plasma Sepp1 concentration falls in selenium deficiency and, therefore, it can be used as an index of selenium nutritional status. Most selenoproteins contain a single selenocysteine residue per polypeptide chain, encoded by an in-frame UGA codon. Selenoprotein P is unique in that its mRNA encodes 10-12 selenocysteine residues, depending on species. In addition to the high number of selenocysteines, the protein is cysteine- and histidine-rich. The function of selenoprotein P has remained elusive, in part due to the inability to express the recombinant protein. This has been attributed to presumed inefficient translation through the selenocysteine/stop codons. Herein, we report for the first time the expression of recombinant rat selenoprotein P in a transiently transfected human epithelial kidney cell line, as well as the endogenously expressed protein from HepG2 and Chinese hamster ovary cells. The majority of the expressed protein migrates with the predicted 57-kDa size of full-length glycosylated selenoprotein P. Based on the histidine-rich nature of selenoprotein P, we have purified the recombinant and endogenously expressed proteins using nickel-agarose affinity chromatography. We show that the recombinant rat and endogenous human proteins react in Western blotting and immunoprecipitation assays with commercial anti-histidine antibodies. The ability to express, purify, and immunochemically detect the recombinant protein provides a foundation for investigating the functions and efficiency of expression of this intriguing protein. When cDNA containing proteins enriched in the bovine cerebellar cortex were cloned, a clone which seemed to encode a selenoprotein P-like protein was isolated. The coding nucleotide sequence of its cDNA insert displayed high homology to rat and human selenoprotein P cDNA but contained 12 rather than 10 TGAs (12 rather than 10 selenocysteines in deduced amino acids), a tandem repeat of one CACTCC (His-Ser) and seven CATCCCs (His-Pro), and a 3' untranslated region approximately 890 bases shorter than that of rat liver selenoprotein P. RT-PCR using a set of primers flanking to the repeat displayed the existence of mRNA without the repeat. The tandem repeat and its adjacent region consisted of a similar motif of CAC/TCC/AC/T. Thus, these proteins included a (His-Pro) rich domain with a slightly negative free energy change irrespective of having the tandem repeat or not. Such His-Pro repeats reportedly exist in the segmentation gene paired or homeobox protein Om(1D) of Drosophila. Moreover, both this selenoprotein P-like protein mRNA and selenoprotein P mRNA were expressed in all the areas of the brain but most prominently in the cerebellar cortex, hippocampus, and olfactory bulb. These findings suggest the possibility that these selenoproteins are major selenium carriers in the brain and play a role in the morphological response of nerve or glial cells. Selenoprotein P (SeP) is an extracellular glycoprotein with 8-10 selenocysteines per molecule, containing approximately 50% of total selenium in human serum. An antioxidant function of SeP has been postulated. In the present study, we show that SeP protects low-density lipoproteins (LDL) against oxidation in a cell-free in-vitro system. LDL were isolated from human blood plasma and oxidized with CuCl2, 2,2'-azobis(2-amidinopropane) (AAPH) or peroxynitrite in the presence or absence of SeP, using the formation of conjugated dienes as parameter for lipid peroxidation. SeP delayed the CuCl2- and AAPH-induced LDL oxidation significantly and more efficiently than bovine serum albumin used as control. In contrast, SeP was not capable of inhibiting peroxynitrite-induced LDL oxidation. The protection of LDL against CuCl2- and AAPH-induced oxidation provides evidence for the antioxidant capacity of SeP. Because SeP associates with endothelial membranes, it may act in vivo as a protective factor inhibiting the oxidation of LDL by reactive oxygen species. OBJECTIVE: Human selenoprotein P (HSelP) is unique protein that contains 10 selenocysteines encoded by 10 inframe UGA, which typically function as stop codon. The function of HSelP remains unclear, in part due to the inability to express it by gene recombinant technique. This study is to investigate expression and purification of recombinant HSelP in prokaryotic expression system, and its activity to induce apoptosis in vitro. METHODS: The shorter HSelP isoform was cloned. After the selenocysteine (SeCys) at 40th position from N terminus of the HSelP shorter isoform was mutated into cysteine by PCR, it was expressed in E. coli. The expressed product was purified with DEAE column and identified by Western blot. Subsequently, its function on induction of mitochondrial apoptotic activity was studied. RESULTS: The mutant HSelP shorter isoform expressed in prokaryotic system was purified by DEAE column to 90% homogeneity. The purified product, HSelP280m, induced the opening of mitochondrial permeability transition pore (PTP) and decreased the transmembrane potential in a dose-dependent manner. These events could be abolished by PTP specific inhibitors. CONCLUSION: HSelP280m can induce the opening of mitochondrial PTP, which provides a basis for investigating the structure and function of recombinant HSelP.

Which package is available for analysing genomic interactions in R/Bioconductor?

r3Cseq is an R/Bioconductor package designed to perform 3C-seq data analysis in a number of different experimental designs. The package reads a common aligned read input format, provides data normalization, allows the visualization of candidate interaction regions and detects statistically significant chromatin interactions, thus greatly facilitating hypothesis generation and the interpretation of experimental results.

[23671339]

The coupling of chromosome conformation capture (3C) with next-generation sequencing technologies enables the high-throughput detection of long-range genomic interactions, via the generation of ligation products between DNA sequences, which are closely juxtaposed in vivo. These interactions involve promoter regions, enhancers and other regulatory and structural elements of chromosomes and can reveal key details of the regulation of gene expression. 3C-seq is a variant of the method for the detection of interactions between one chosen genomic element (viewpoint) and the rest of the genome. We present r3Cseq, an R/Bioconductor package designed to perform 3C-seq data analysis in a number of different experimental designs. The package reads a common aligned read input format, provides data normalization, allows the visualization of candidate interaction regions and detects statistically significant chromatin interactions, thus greatly facilitating hypothesis generation and the interpretation of experimental results. We further demonstrate its use on a series of real-world applications.

Are stress granules involved in the pathogenesis of Amyotrophic Lateral Sclerosis?

Stress granules are cytoplasmic inclusions that repress translation of a subset of RNAs in times of cellular stress, and several proteins implicated in neurodegeneration (i.e. Ataxin-2 and SMN) interact with stress granules. Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules. ALS-linked mutations in profilin 1 alter stress granule dynamics, providing further evidence for the potential role of stress granules in ALS pathogenesis. ALS mutations in FUS NLS can impair FUS nuclear localization, induce cytoplasmic inclusions and stress granules, and potentially perturb RNA metabolism.

[26557057, 23474818, 23629963, 25888396, 25429138, 24297750, 24090136, 20606625, 24920614, 21173160, 24336168, 20674093, 20699327, 19765185, 23257289, 25216585, 24312274, 22405725, 24013423, 23152885]

Stress granules (SGs) are RNA-containing cytoplasmic foci formed in response to stress exposure. Since their discovery in 1999, over 120 proteins have been described to be localized to these structures (in 154 publications). Most of these components are RNA binding proteins (RBPs) or are involved in RNA metabolism and translation. SGs have been linked to several pathologies including inflammatory diseases, cancer, viral infection, and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In ALS and FTD, the majority of cases have no known etiology and exposure to external stress is frequently proposed as a contributor to either disease initiation or the rate of disease progression. Of note, both ALS and FTD are characterized by pathological inclusions, where some well-known SG markers localize with the ALS related proteins TDP-43 and FUS. We propose that TDP-43 and FUS serve as an interface between genetic susceptibility and environmental stress exposure in disease pathogenesis. Here, we will discuss the role of TDP-43 and FUS in SG dynamics and how disease-linked mutations affect this process. Mutations in the gene encoding Fused in Sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predominantly nuclear DNA- and RNA-binding protein that is involved in RNA processing. Large FUS-immunoreactive inclusions fill the perikaryon of surviving motor neurons of ALS patients carrying mutations at post-mortem. This sequestration of FUS is predicted to disrupt RNA processing and initiate neurodegeneration. Here, we demonstrate that C-terminal ALS mutations disrupt the nuclear localizing signal (NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and patient fibroblasts. FUS mislocalization is rescued by the addition of the wild-type FUS NLS to mutant proteins. We also show that oxidative stress recruits mutant FUS to cytoplasmic stress granules where it is able to bind and sequester wild-type FUS. While FUS interacts with itself directly by protein-protein interaction, the recruitment of FUS to stress granules and interaction with PABP are RNA dependent. These findings support a two-hit hypothesis, whereby cytoplasmic mislocalization of FUS protein, followed by cellular stress, contributes to the formation of cytoplasmic aggregates that may sequester FUS, disrupt RNA processing and initiate motor neuron degeneration. Amyotrophic lateral sclerosis (ALS) is a fatal human neurodegenerative disease affecting primarily motor neurons. Two RNA-binding proteins, TDP-43 and FUS, aggregate in the degenerating motor neurons of ALS patients, and mutations in the genes encoding these proteins cause some forms of ALS. TDP-43 and FUS and several related RNA-binding proteins harbor aggregation-promoting prion-like domains that allow them to rapidly self-associate. This property is critical for the formation and dynamics of cellular ribonucleoprotein granules, the crucibles of RNA metabolism and homeostasis. Recent work connecting TDP-43 and FUS to stress granules has suggested how this cellular pathway, which involves protein aggregation as part of its normal function, might be coopted during disease pathogenesis. BACKGROUND: Mutations in calcium-responsive transactivator (CREST) encoding gene have been recently linked to ALS. Similar to several proteins implicated in ALS, CREST contains a prion-like domain and was reported to be a component of paraspeckles. RESULTS: We demonstrate that CREST is prone to aggregation and co-aggregates with FUS but not with other two ALS-linked proteins, TDP-43 and TAF15, in cultured cells. Aggregation of CREST affects paraspeckle integrity, probably by trapping other paraspeckle proteins within aggregates. Like several other ALS-associated proteins, CREST is recruited to induced stress granules. Neither of the CREST mutations described in ALS alters its subcellular localization, stress granule recruitment or detergent solubility; however Q388stop mutation results in elevated steady-state levels and more frequent nuclear aggregation of the protein. Both wild-type protein and its mutants negatively affect neurite network complexity of unstimulated cultured neurons when overexpressed, with Q388stop mutation being the most deleterious. When overexpressed in the fly eye, wild-type CREST or its mutants lead to severe retinal degeneration without obvious differences between the variants. CONCLUSIONS: Our data indicate that CREST and certain other ALS-linked proteins share several features implicated in ALS pathogenesis, namely the ability to aggregate, be recruited to stress granules and alter paraspeckle integrity. A change in CREST levels in neurons which might occur under pathological conditions would have a profound negative effect on neuronal homeostasis. TDP-43 is an RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) that is known to regulate the splicing, transport, and storage of specific mRNAs into stress granules. Although TDP-43 has been shown to interact with translation factors, its role in protein synthesis remains unclear, and no in vivo translation targets have been reported to date. Here we provide evidence that TDP-43 associates with futsch mRNA in a complex and regulates its expression at the neuromuscular junction (NMJ) in Drosophila. In the context of TDP-43-induced proteinopathy, there is a significant reduction of futsch mRNA at the NMJ compared with motor neuron cell bodies where we find higher levels of transcript compared with controls. TDP-43 also leads to a significant reduction in Futsch protein expression at the NMJ. Polysome fractionations coupled with quantitative PCR experiments indicate that TDP-43 leads to a futsch mRNA shift from actively translating polysomes to nontranslating ribonuclear protein particles, suggesting that in addition to its effect on localization, TDP-43 also regulates the translation of futsch mRNA. We also show that futsch overexpression is neuroprotective by extending life span, reducing TDP-43 aggregation, and suppressing ALS-like locomotor dysfunction as well as NMJ abnormalities linked to microtubule and synaptic stabilization. Furthermore, the localization of MAP1B, the mammalian homolog of Futsch, is altered in ALS spinal cords in a manner similar to our observations in Drosophila motor neurons. Together, our results suggest a microtubule-dependent mechanism in motor neuron disease caused by TDP-43-dependent alterations in futsch mRNA localization and translation in vivo. RNA localization pathways direct numerous mRNAs to distinct subcellular regions and affect many physiological processes. In one such pathway the tumor-suppressor protein adenomatous polyposis coli (APC) targets RNAs to cell protrusions, forming APC-containing ribonucleoprotein complexes (APC-RNPs). Here, we show that APC-RNPs associate with the RNA-binding protein Fus/TLS (fused in sarcoma/translocated in liposarcoma). Fus is not required for APC-RNP localization but is required for efficient translation of associated transcripts. Labeling of newly synthesized proteins revealed that Fus promotes translation preferentially within protrusions. Mutations in Fus cause amyotrophic lateral sclerosis (ALS) and the mutant protein forms inclusions that appear to correspond to stress granules. We show that overexpression or mutation of Fus results in formation of granules, which preferentially recruit APC-RNPs. Remarkably, these granules are not translationally silent. Instead, APC-RNP transcripts are translated within cytoplasmic Fus granules. These results unexpectedly show that translation can occur within stress-like granules. Importantly, they identify a new local function for cytoplasmic Fus with implications for ALS pathology. Mutations in fused in sarcoma (FUS) are a cause of familial amyotrophic lateral sclerosis (fALS). Patients carrying point mutations in the C-terminus of FUS show neuronal cytoplasmic FUS-positive inclusions, whereas in healthy controls, FUS is predominantly nuclear. Cytoplasmic FUS inclusions have also been identified in a subset of frontotemporal lobar degeneration (FTLD-FUS). We show that a non-classical PY nuclear localization signal (NLS) in the C-terminus of FUS is necessary for nuclear import. The majority of fALS-associated mutations occur within the NLS and impair nuclear import to a degree that correlates with the age of disease onset. This presents the first case of disease-causing mutations within a PY-NLS. Nuclear import of FUS is dependent on Transportin, and interference with this transport pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules. Moreover, proteins known to be stress granule markers co-deposit with inclusions in fALS and FTLD-FUS patients, implicating stress granule formation in the pathogenesis of these diseases. We propose that two pathological hits, namely nuclear import defects and cellular stress, are involved in the pathogenesis of FUS-opathies. Mutations in the PFN1 gene encoding profilin 1 are a rare cause of familial amyotrophic lateral sclerosis (ALS). Profilin 1 is a well studied actin-binding protein but how PFN1 mutations cause ALS is unknown. The budding yeast, Saccharomyces cerevisiae, has one PFN1 ortholog. We expressed the ALS-linked profilin 1 mutant proteins in yeast, demonstrating a loss of protein stability and failure to restore growth to profilin mutant cells, without exhibiting gain-of-function toxicity. This model provides for simple and rapid screening of novel ALS-linked PFN1 variants. To gain insight into potential novel roles for profilin 1, we performed an unbiased, genome-wide synthetic lethal screen with yeast cells lacking profilin (pfy1Δ). Unexpectedly, deletion of several stress granule and processing body genes, including pbp1Δ, were found to be synthetic lethal with pfy1Δ. Mutations in ATXN2, the human ortholog of PBP1, are a known ALS genetic risk factor and ataxin 2 is a stress granule component in mammalian cells. Given this genetic interaction and recent evidence linking stress granule dynamics to ALS pathogenesis, we hypothesized that profilin 1 might also associate with stress granules. Here we report that profilin 1 and related protein profilin 2 are novel stress granule-associated proteins in mouse primary cortical neurons and in human cell lines and that ALS-linked mutations in profilin 1 alter stress granule dynamics, providing further evidence for the potential role of stress granules in ALS pathogenesis. TDP-43, or TAR DNA-binding protein 43, is a pathological marker of a spectrum of neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. TDP-43 is an RNA/DNA-binding protein implicated in transcriptional and posttranscriptional regulation. Recent work also suggests that TDP-43 associates with cytoplasmic stress granules, which are transient structures that form in response to stress. In this study, we establish sorbitol as a novel physiological stressor that directs TDP-43 to stress granules in Hek293T cells and primary cultured glia. We quantify the association of TDP-43 with stress granules over time and show that stress granule association and size are dependent on the glycine-rich region of TDP-43, which harbors the majority of pathogenic mutations. Moreover, we establish that cells harboring wild-type and mutant TDP-43 have distinct stress responses: mutant TDP-43 forms significantly larger stress granules, and is incorporated into stress granules earlier, than wild-type TDP-43; in striking contrast, wild-type TDP-43 forms more stress granules over time, but the granule size remains relatively unchanged. We propose that mutant TDP-43 alters stress granule dynamics, which may contribute to the progression of TDP-43 proteinopathies. Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2, is that they localize to stress granules. Unexpectedly, we found that genes that modulate stress granules are strong modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster. eIF2α phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component, polyA-binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. We investigated the efficacy of a small molecule inhibitor of eIF2α phosphorylation in ALS models. Treatment with this inhibitor mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach. Amyotrophic lateral sclerosis (ALS) is an uncommon neurodegenerative disease caused by degeneration of upper and lower motor neurons. Several genes, including SOD1, TDP-43, FUS, Ubiquilin 2, C9orf72 and Profilin 1, have been linked with the sporadic and familiar forms of ALS. FUS is a DNA/RNA-binding protein (RBP) that forms cytoplasmic inclusions in ALS and frontotemporal lobular degeneration (FTLD) patients' brains and spinal cords. However, it is unknown whether the RNA-binding ability of FUS is required for causing ALS pathogenesis. Here, we exploited a Drosophila model of ALS and neuronal cell lines to elucidate the role of the RNA-binding ability of FUS in regulating FUS-mediated toxicity, cytoplasmic mislocalization and incorporation into stress granules (SGs). To determine the role of the RNA-binding ability of FUS in ALS, we mutated FUS RNA-binding sites (F305L, F341L, F359L, F368L) and generated RNA-binding-incompetent FUS mutants with and without ALS-causing mutations (R518K or R521C). We found that mutating the aforementioned four phenylalanine (F) amino acids to leucines (L) (4F-L) eliminates FUS RNA binding. We observed that these RNA-binding mutations block neurodegenerative phenotypes seen in the fly brains, eyes and motor neurons compared with the expression of RNA-binding-competent FUS carrying ALS-causing mutations. Interestingly, RNA-binding-deficient FUS strongly localized to the nucleus of Drosophila motor neurons and mammalian neuronal cells, whereas FUS carrying ALS-linked mutations was distributed to the nucleus and cytoplasm. Importantly, we determined that incorporation of mutant FUS into the SG compartment is dependent on the RNA-binding ability of FUS. In summary, we demonstrate that the RNA-binding ability of FUS is essential for the neurodegenerative phenotype in vivo of mutant FUS (either through direct contact with RNA or through interactions with other RBPs). Mutations in fused in sarcoma (FUS), a DNA/RNA binding protein, have been associated with familial amyotrophic lateral sclerosis (fALS), which is a fatal neurodegenerative disease that causes progressive muscular weakness and has overlapping clinical and pathologic characteristics with frontotemporal lobar degeneration. However, the role of autophagy in regulation of FUS-positive stress granules (SGs) and aggregates remains unclear. We found that the ALS-linked FUS(R521C) mutation causes accumulation of FUS-positive SGs under oxidative stress, leading to a disruption in the release of FUS from SGs in cultured neurons. Autophagy controls the quality of proteins or organelles; therefore, we checked whether autophagy regulates FUS(R521C)-positive SGs. Interestingly, FUS(R521C)-positive SGs were colocalized to RFP-LC3-positive autophagosomes. Furthermore, FUS-positive SGs accumulated in atg5(-/-) mouse embryonic fibroblasts (MEFs) and in autophagy-deficient neurons. However, FUS(R521C) expression did not significantly impair autophagic degradation. Moreover, autophagy activation with rapamycin reduced the accumulation of FUS-positive SGs in an autophagy-dependent manner. Rapamycin further reduced neurite fragmentation and cell death in neurons expressing mutant FUS under oxidative stress. Overall, we provide a novel pathogenic mechanism of ALS associated with a FUS mutation under oxidative stress, as well as therapeutic insight regarding FUS pathology associated with excessive SGs. In amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, TAR DNA binding protein 43 (TDP-43) accumulates in the cytoplasm of affected neurons and glia, where it associates with stress granules (SGs) and forms large inclusions. SGs form in response to cellular stress, including endoplasmic reticulum (ER) stress, which is induced in both familial and sporadic forms of ALS. Here we demonstrate that pharmacological induction of ER stress causes TDP-43 to accumulate in the cytoplasm, where TDP-43 also associates with SGs. Furthermore, treatment with salubrinal, an inhibitor of dephosphorylation of eukaryotic initiation factor 2-α, a key modulator of ER stress, potentiates ER stress-mediated SG formation. Inclusions of C-terminal fragment TDP-43, reminiscent of disease-pathology, form in close association with ER and Golgi compartments, further indicating the involvement of ER dysfunction in TDP-43-associated disease. Consistent with this notion, over-expression of ALS-linked mutant TDP-43, and to a lesser extent wildtype TDP-43, triggers several ER stress pathways in neuroblastoma cells. Similarly, we found an interaction between the ER chaperone protein disulphide isomerase and TDP-43 in transfected cell lysates and in the spinal cords of mutant A315T TDP-43 transgenic mice. This study provides evidence for ER stress as a pathogenic pathway in TDP-43-mediated disease. Fused in sarcoma (FUS) belongs to the group of RNA-binding proteins implicated as underlying factors in amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. Multiple FUS gene mutations have been linked to hereditary forms, and aggregation of FUS protein is believed to play an important role in pathogenesis of these diseases. In cultured cells, FUS variants with disease-associated amino acid substitutions or short deletions affecting nuclear localization signal (NLS) and causing cytoplasmic mislocalization can be sequestered into stress granules (SGs). We demonstrated that disruption of motifs responsible for RNA recognition and binding not only prevents SG recruitment, but also dramatically increases the protein propensity to aggregate in the cell cytoplasm with formation of juxtanuclear structures displaying typical features of aggresomes. Functional RNA-binding domains from TAR DNA-binding protein of 43 kDa (TDP-43) fused to highly aggregation-prone C-terminally truncated FUS protein restored the ability to enter SGs and prevented aggregation of the chimeric protein. Truncated FUS was also able to trap endogenous FUS molecules in the cytoplasmic aggregates. Our data indicate that RNA binding and recruitment to SGs protect cytoplasmic FUS from aggregation, and loss of this protection may trigger its pathological aggregation in vivo. Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). In order to identify binding partners for FUS/TLS, we performed a yeast two-hybrid screening and found that protein arginine methyltransferase 1 (PRMT1) is one of binding partners primarily in the nucleus. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. Although co-localized primarily in the nucleus in normal condition, FUS/TLS and PRMT1 were partially recruited to the cytoplasmic granules under oxidative stress, which were merged with stress granules (SGs) markers in SH-SY5Y cell. C-terminal truncated form of FUS/TLS (FUS-dC), which lacks C-terminal nuclear localization signal (NLS), formed cytoplasmic inclusions like ALS-linked FUS mutants and was partially co-localized with PRMT1. Furthermore, conditional over-expression of PRMT1 reduced the FUS-dC-mediated SGs formation and the detergent-insoluble aggregates in HEK293 cells. These findings indicate that PRMT1-mediated arginine methylation could be implicated in the nucleus-cytoplasmic shuttling of FUS/TLS and in the SGs formation and the detergent-insoluble inclusions of ALS-linked FUS/TLS mutants.

Does TGF-beta play a role in cardiac regeneration after myocardial infarction?

TGFβ signaling orchestrates the beneficial interplay between scar-based repair and cardiomyocyte-based regeneration to achieve complete heart regeneration.

[18985280, 17322368, 19966054, 15883211, 23293297, 10198196, 16842199, 12374778, 22513374]

Macrophages have been suggested to be beneficial for myocardial wound healing. We investigated the role of macrophages in myocardial wound healing by inhibition of macrophage infiltration after myocardial injury. We used a murine cryoinjury model to induce left ventricular damage. Infiltrating macrophages were depleted during the 1st week after cryoinjury by serial intravenous injections of clodronate-containing liposomes. After injury, the presence of macrophages, which secreted high levels of transforming growth factor-beta and vascular endothelial growth factor-A, led to rapid removal of cell debris and replacement by granulation tissue containing inflammatory cells and blood vessels, followed by myofibroblast infiltration and collagen deposition. In macrophage-depleted hearts, nonresorbed cell debris was still observed 4 weeks after injury. Secretion of transforming growth factor-beta and vascular endothelial growth factor-A as well as neovascularization, myofibroblast infiltration, and collagen deposition decreased. Moreover, macrophage depletion resulted in a high mortality rate accompanied by increased left ventricular dilatation and wall thinning. In conclusion, infiltrating macrophage depletion markedly impairs wound healing and increases remodeling and mortality after myocardial injury, identifying the macrophage as a key player in myocardial wound healing. Based on these findings, we propose that increasing macrophage numbers early after myocardial infarction could be a clinically relevant option to promote myocardial wound healing and subsequently to reduce remodeling and heart failure. In addition to mediating cell-to-cell electrical coupling, gap junctions are important in tissue repair, wound healing, and scar formation. The expression and distribution of connexin43 (Cx43), the major gap junction protein expressed in the heart, are altered substantially after myocardial infarction (MI); however, the effects of Cx43 remodeling on wound healing and the attendant ventricular dysfunction are incompletely understood. Cx43-deficient and wild-type mice were subjected to proximal ligation of the anterior descending coronary artery and followed for 6 days or 4 wk to test the hypothesis that reduced expression of Cx43 influences wound healing, fibrosis, and ventricular remodeling after MI. We quantified the progression of infarct healing by measuring neutrophil expression, collagen content, and myofibroblast expression. We found significantly reduced transformation of fibroblasts to myofibroblasts at 6 days and significantly reduced collagen deposition both in the infarct at 6 days and at 4 wk in the noninfarcted region of Cx43-deficient mice. As expected, transforming growth factor (TGF)-beta, a profibrotic cytokine, was dramatically upregulated in MI hearts, but its phosphorylated comediator (pSmad) was significantly downregulated in the nuclei of Cx43-deficient hearts post-MI, suggesting that downstream signaling of TGF-beta is diminished substantially in Cx43-deficient hearts. This diminution in profibrotic TGF-beta signaling resulted in the attenuation of adverse structural remodeling as assessed by echocardiography. These findings suggest that efforts to enhance the expression of Cx43 to maintain intercellular coupling or reduce susceptibility to arrhythmias should be met with caution until the role of Cx43 in infarct healing is fully understood. BACKGROUND: Recent studies have shown that bone marrow-derived stem cells differentiate into the phenotype of cardiomyocytes in vivo and in vitro. We tried to regenerate infarcted myocardium by implanting ex vivo transforming growth factor (TGF)-beta-preprogrammed CD117 (c-kit)-positive (CD117+) stem cells intramyocardially. METHODS AND RESULTS: CD117+ cells were isolated from the bone marrow mononuclear cells of GFP-transgenic or normal C57/BL6 mice. The myogenic differentiation of CD117+ cells was achieved by cultivation with TGF-beta. Using an acute myocardial infarction model, we also tried to regenerate infarcted myocardium by implanting untreated (newly isolated) or preprogrammed (24 hours of cultivation with 5 ng/mL TGF-beta1) CD117+ cells intramyocardially. TGF-beta increased the cellular expression of myosin, troponins, connexin-43, GATA-4, and NKx-2.5, which suggested that it induced the myogenic differentiation of CD117+ cells. Compared with the effects of PBS injection only, the microvessel density in the infarcted myocardium was increased significantly 3 months after the implantation of either TGF-beta-preprogrammed or untreated CD117+ cells. Moreover, many of the TGF-beta-preprogrammed CD117+ cells were stained positively for myosin, whereas few of the untreated CD117+ cells were. Histological analysis revealed newly regenerated myocardium in the left ventricular anterior wall after the implantation of TGF-beta-preprogrammed cells but not untreated cells. Furthermore, the left ventricular percent fraction shortening was significantly higher after the implantation of TGF-beta-preprogrammed cells than after the implantation of untreated CD117+ cells. CONCLUSIONS: TGF-beta conducted the myogenic differentiation of CD117+ stem cells by upregulating GATA-4 and NKx-2.5 expression. Therefore, the intramyocardial implantation of TGF-beta-preprogrammed CD117+ cells effectively assisted the myocardial regeneration and induced therapeutic angiogenesis, contributing to functional cardiac regeneration. Healing of myocardial infarcts depends on an inflammatory cascade that ultimately results in clearance of dead cells and matrix debris and formation of a scar. Myocardial necrosis activates complement, Nuclear Factor (NF)-kappaB and Toll-like Receptor (TLR)-dependent pathways, and generates free radicals, triggering an inflammatory response. Chemokines and cytokines are markedly induced in the infarct and mediate recruitment and activation of neutrophils and mononuclear cells. Extravasation of platelets and plasma proteins, such as fibrinogen and fibronectin, results in formation of a clot, consisting of platelets embedded in a mesh of crosslinked fibrin. This provisional matrix provides a scaffold for migration of cells into the infarct. Monocytes differentiate into macrophages and secrete fibrogenic and angiogenic growth factors inducing formation of granulation tissue, containing myofibroblasts and neovessels. Repression of proinflammatory cytokine and chemokine synthesis, mediated in part through Transforming Growth Factor (TGF)-beta and Interleukin (IL)-10, is critical for resolution of the inflammatory infiltrate and transition to fibrous tissue deposition. Infarct myofibroblasts deposit extracellular matrix proteins and a collagen-based scar is formed. As the wound matures, fibroblasts undergo apoptosis and neovessels regress, resulting in formation of a scar with a low cellular content containing dense, cross-linked collagen. The pathologic and structural changes associated with infarct healing directly influence ventricular remodeling and affect prognosis in patients with myocardial infarction. Understanding the mechanisms involved in the regulation of the post-infarction inflammatory response, and the spatial and temporal parameters of wound healing is necessary in order to identify specific molecular targets for therapeutic intervention. Members of the transforming growth factor beta1 (TGF-beta) superfamily--namely, TGF-beta and BMP2--applied to undifferentiated murine embryonic stem cells up-regulated mRNA of mesodermal (Brachyury) and cardiac specific transcription factors (Nkx2.5, MEF2C). Embryoid bodies generated from stem cells primed with these growth factors demonstrated an increased potential for cardiac differentiation with a significant increase in beating areas and enhanced myofibrillogenesis. In an environment of postmitotic cardiomyocytes, stem cells engineered to express a fluorescent protein under the control of a cardiac promoter differentiated into fluorescent ventricular myocytes beating in synchrony with host cells, a process significantly enhanced by TGF-beta or BMP2. In vitro, disruption of the TGF-beta/BMP signaling pathways by latency-associated peptide and/or noggin prevented differentiation of stem cells. In fact, only host cells that secrete a TGF-beta family member induced a cardiac phenotype in stem cells. In vivo, transplantation of stem cells into heart also resulted in cardiac differentiation provided that TGF-beta/BMP2 signaling was intact. In infarcted myocardium, grafted stem cells differentiated into functional cardiomyocytes integrated with surrounding tissue, improving contractile performance. Thus, embryonic stem cells are directed to differentiate into cardiomyocytes by signaling mediated through TGF-beta/BMP2, a cardiac paracrine pathway required for therapeutic benefit of stem cell transplantation in diseased heart.

Is there a genetic component for happiness?

Results of studies on genetic factors indicated an average effectiveness of genetic about 35 -50 percent on happiness. The MAOA gene predicts happiness in women. The heritability of happiness was estimated at 22% for males and 41% in females.

[20440640, 19728071, 26060713, 20981772, 20397744, 23769682, 24690898, 22885141, 19569406]

Subjective Wellbeing (SWB) can be assessed with distinct measures that have been hypothesized to represent different domains of SWB. The current study assessed SWB with four different measures in a genetically informative sample of adolescent twins and their siblings aged 13-28 years (N = 5,024 subjects from 2,157 families). Multivariate genetic modeling was applied to the data to explore the etiology of individual differences in SWB measures and the association among them. Developmental trends and sex differences were examined for mean levels and the variance-covariance structure. Mean SWB levels were equal in men and women. A small negative effect of age on mean levels of SWB was found. Individual differences in SWB were accounted for by additive and non-additive genetic influences, and non-shared environment. The broad-sense heritabilities were estimated between 40 and 50%. The clustering of the four different measures (quality of life in general, satisfaction with life, quality of life at present, and subjective happiness) was explained by an underlying additive genetic factor and an underlying non-additive genetic factor. The effect of these latent genetic factors on the phenotypes was not moderated by either age or sex. The Angelman syndrome is clinically delineated by the combination of seizures, absent speech, hypermotoric and ataxic movements and certain remarkable behaviors. Those with the syndrome have a predisposition toward apparent happiness and paroxysms of laughter, and this finding helps distinguish Angelman syndrome from other ones involving severe developmental handicap. In this review the core neurological features of the syndrome are discussed with a focus on those behaviors that make Angelman syndrome a prototypical genetic disorder expressing a behavioral phenotype. Causes of individual differences in happiness, as assessed with the Subjective Happiness Scale, are investigated in a large of sample twins and siblings from the Netherlands Twin Register. Over 12,000 twins and siblings, average age 24.7 years (range 12 to 88), took part in the study. A genetic model with an age by sex design was fitted to the data with structural equation modeling in Mx. The heritability of happiness was estimated at 22% for males and 41% in females. No effect of age was observed. To identify the genomic regions contributing to this heritability, a genome-wide linkage study for happiness was conducted in sibling pairs. A subsample of 1157 offspring from 441 families was genotyped with an average of 371 micro-satellite markers per individual. Phenotype and genotype data were analyzed in MERLIN with multipoint variance component linkage analysis and age and sex as covariates. A linkage signal (logarithm of odds score 2.73, empirical p value 0.095) was obtained at the end of the long arm of chromosome 19 for marker D19S254 at 110 cM. A second suggestive linkage peak was found at the short arm of chromosome 1 (LOD of 2.37) at 153 cM, marker D1S534 (empirical p value of .209). These two regions of interest are not overlapping with the regions found for contrasting phenotypes (such as depression, which is negatively associated with happiness). Further linkage and future association studies are warranted. BACKGROUND: Investigating genetic modulation of emotion processing may contribute to the understanding of heritable mechanisms of emotional disorders. The aim of the present study was to test the effects of catechol-O-methyltransferase (COMT) val158met and serotonin-transporter-linked promoter region (5-HTTLPR) polymorphisms on facial emotion processing in healthy individuals. METHODS: Two hundred and seventy five (167 female) participants were asked to complete a computerized facial affect recognition task, which involved four experimental conditions, each containing one type of emotional face (fearful, angry, sad or happy) intermixed with neutral faces. Participants were asked to indicate whether the face displayed an emotion or was neutral. The COMT-val158met and 5-HTTLPR polymorphisms were genotyped. RESULTS: Met homozygotes (COMT) showed a stronger bias to perceive neutral faces as expressions of anger, compared with val homozygotes. However, the S-homozygotes (5-HTTLPR) showed a reduced bias to perceive neutral faces as expressions of happiness, compared to L-homozygotes. No interaction between 5-HTTLPR and COMT was found. CONCLUSIONS: These results add to the knowledge of individual differences in social cognition that are modulated via serotonergic and dopaminergic systems. This potentially could contribute to the understanding of the mechanisms of susceptibility to emotional disorders. Happiness has been viewed as a temporary emotional state (e.g., pleasure) and a relatively stable state of being happy (subjective happiness level). As previous studies demonstrated that individuals with high subjective happiness level rated their current affective states more positively when they experience positive events, these two aspects of happiness are interrelated. According to a recent neuroimaging study, the cytosine to thymine single-nucleotide polymorphism of the human cannabinoid receptor 1 gene is associated with sensitivity to positive emotional stimuli. Thus, we hypothesized that our genetic traits, such as the human cannabinoid receptor 1 genotypes, are closely related to the two aspects of happiness. In Experiment 1, 198 healthy volunteers were used to compare the subjective happiness level between cytosine allele carriers and thymine-thymine carriers of the human cannabinoid receptor 1 gene. In Experiment 2, we used positron emission tomography with 20 healthy participants to compare the brain responses to positive emotional stimuli of cytosine allele carriers to that of thymine-thymine carriers. Compared to thymine-thymine carriers, cytosine allele carriers have a higher subjective happiness level. Regression analysis indicated that the cytosine allele is significantly associated with subjective happiness level. The positive mood after watching a positive film was significantly higher for the cytosine allele carriers compared to the thymine-thymine carriers. Positive emotion-related brain region such as the medial prefrontal cortex was significantly activated when the cytosine allele carriers watched the positive film compared to the thymine-thymine carriers. Thus, the human cannabinoid receptor 1 genotypes are closely related to two aspects of happiness. Compared to thymine-thymine carriers, the cytosine allele carriers of the human cannabinoid receptor 1 gene, who are sensitive to positive emotional stimuli, exhibited greater magnitude positive emotions when they experienced positive events and had a higher subjective happiness level. Psychologists, quality of life and well-being researchers have grown increasingly interested in understanding the factors that are associated with human happiness. Although twin studies estimate that genetic factors account for 35-50% of the variance in human happiness, knowledge of specific genes is limited. However, recent advances in molecular genetics can now provide a window into neurobiological markers of human happiness. This investigation examines association between happiness and monoamine oxidase A (MAOA) genotype. Data were drawn from a longitudinal study of a population-based cohort, followed for three decades. In women, low expression of MAOA (MAOA-L) was related significantly to greater happiness (0.261 SD increase with one L-allele, 0.522 SD with two L-alleles, P=0.002) after adjusting for the potential effects of age, education, household income, marital status, employment status, mental disorder, physical health, relationship quality, religiosity, abuse history, recent negative life events and self-esteem use in linear regression models. In contrast, no such association was found in men. This new finding may help explain the gender difference on happiness and provide a link between MAOA and human happiness. Although there is considerable evidence linking success -- including wealth, marriage, and friendships -- to happiness, this relationship might not reflect, as is often assumed, the effects of the proximate environment on well-being. Such an interpretation is contravened by evidence that both happiness and the environment are influenced by genetic factors and family upbringing. Using the National Survey of Midlife Development in the United States, which includes a subsample of twins, this study evaluates the relationship between happiness and various features of success before and after eliminating the influence of endowments. The results suggest that many putative indicators of the environment are highly heritable and, indeed, that the same genes that affect the environment may affect happiness as well. Yet the results also suggest that the role of genetic endowments varies considerably across different features of success, suggesting complex patterns of selection, reinforcement, and causation among genes and the environment.

What enzyme is inhibied by Opicapone?

Opicapone is a novel catechol-O-methyltransferase (COMT) inhibitor to be used as adjunctive therapy in levodopa-treated patients with Parkinson's disease

[24925090, 23248072, 24148813, 23336248, 24271646]

BACKGROUND AND OBJECTIVES: Opicapone is a novel third generation catechol-O-methyltransferase (COMT) inhibitor. The purpose of this study was to compare the levodopa pharmacokinetic profile throughout a day driven by the COMT inhibition either following repeated doses of opicapone or concomitant administration with entacapone. METHODS: A randomized, double-blind, gender-balanced, parallel-group study was performed in 4 groups of 20 healthy subjects each. Four subjects in each group received placebo during the entire study. Sixteen subjects in one group received placebo once daily for 11 days and on day 12, 200 mg entacapone concomitantly with each levodopa/carbidopa dose (three times separated by a 5-h interval). Sixteen subjects in each of the remaining three groups received respectively 25, 50, and 75 mg opicapone once daily for 11 days and on day 12, placebo concomitantly with each levodopa/carbidopa dose. RESULTS: Levodopa minimum plasma concentration (Cmin) for each levodopa/carbidopa dose and for the mean of all levodopa/carbidopa doses increased substantially with all active treatments (entacapone and opicapone) when compared to the control group (placebo), with values ranging from 1.7-fold (200 mg entacapone) to 3.3-fold (75 mg opicapone). No statistical difference was found for levodopa peak of systemic exposure (as assessed by maximum observed plasma concentration (Cmax)) between all active treatments and placebo. A significant increase in the levodopa extent of systemic exposure (as assessed by concentration-time curve (AUC)) occurred with all opicapone treatments in relation to placebo. No statistical difference was found for levodopa AUC when entacapone was compared to placebo. When compared to entacapone, both 50 and 75 mg opicapone presented a significant increase for the levodopa AUC. All active treatments significantly inhibited both peak (as assessed by Emax) and extent (as assessed by effect-time curve (AUEC)) of the COMT activity in relation to placebo. When compared to entacapone, all opicapone treatments significantly decreased the extent (AUEC) of the COMT activity due to a long-lasting and sustained effect. The tolerability profile was favorable for all active treatments. CONCLUSION: Opicapone, a novel third generation COMT inhibitor, when compared to entacapone, provides a superior response upon the bioavailability of levodopa associated to more pronounced, long-lasting, and sustained COMT inhibition. The tolerability profile was favorable. On the basis of the results presented in this study and along with the earlier pharmacology studies, it is anticipated that opicapone adjunct therapy at the dosages of 25 and 50 mg will provide an enhancement in levodopa availability that will translate into clinical benefit for Parkinson's disease patients. BACKGROUND AND OBJECTIVES: Opicapone is a novel catechol-O-methyltransferase (COMT) inhibitor. The purpose of this study was to evaluate the tolerability, pharmacokinetics (including the effect of food) and pharmacodynamics (effect on COMT activity) following single oral doses of opicapone in young healthy male volunteers. METHODS: Single rising oral doses of opicapone (10, 25, 50, 100, 200, 400, 800 and 1,200 mg) were administered to eight groups of eight subjects per group (two subjects randomized to placebo and six subjects to opicapone), under a double-blind, randomized, placebo-controlled design. In an additional group of 12 subjects, a 50 mg single dose of opicapone was administered on two occasions, once having fasted overnight and once with a high-fat high-calorie meal. RESULTS: Opicapone was well tolerated at all doses tested. The extent of systemic exposure (area under the plasma concentration-time curve and maximum plasma concentration) to opicapone and metabolites increased in an approximately dose-proportional manner and showed a decrease following concomitant ingestion of a high-fat high-calorie meal. The apparent terminal elimination half-life of opicapone was 0.8-3.2 h. Sulphation appeared to be the main metabolic pathway for opicapone, and both opicapone and the main sulphated metabolite are likely excreted by the biliary route. Maximum COMT inhibition by opicapone was dose dependent, ranged from 36.1% (10 mg) to 100% (200 mg and above), and reached statistical significance at all doses tested. The long duration of COMT inhibition by opicapone, however, tended to be independent from the dose taken. The observed half-life of opicapone-induced COMT inhibition in human erythrocytes was 61.6 h (standard deviation [SD] = 37.6 h), which reflects an underlying dissociative process with a kinetic rate constant of 3.1 × 10(-6) s(-1) (SD = 1.9 × 10(-6) s(-1)). Such a process compares well to the estimated dissociation rate constant (k(off)) of the COMT-opicapone molecular complex (k(off) = 1.9 × 10(-6) s(-1)). CONCLUSIONS: Opicapone was well-tolerated and presented dose-proportional kinetics. Opicapone demonstrated marked and sustained inhibition of erythrocyte soluble COMT activity. Based on the observation that the half-life of COMT inhibition is independent of the dose and that it reflects an underlying kinetic process that is consistent with the k(off) value of the COMT-opicapone complex, we propose that the sustained COMT inhibition, far beyond the observable point of clearance of circulating drug, is due to the long residence time of the reversible complex formed between COMT and opicapone. Globally, these promising results provide a basis for further clinical development of opicapone. OBJECTIVE: The present study aimed at evaluating the effect of opicapone, a third generation nitrocatechol catechol-O-methyltransferase (COMT) inhibitor, on the systemic and central bioavailability of 3,4-dihydroxy-l-phenylalanine (levodopa) and related metabolites in the cynomolgus monkey. METHODS: Four monkeys, implanted with guiding cannulas for microdialysis probes, in the substantia nigra, dorsal striatum and prefrontal cortex, were randomized in two groups that received, in a crossover design, vehicle or 100 mg/kg opicapone for 14 days. Twenty-three hours after last administration of vehicle or opicapone, animals were challenged with levodopa/benserazide (12/3 mg/kg). Extracellular dialysate and blood samples were collected over 360 min (at 30 min intervals) for the assays of catecholamine and COMT activity. RESULTS: Opicapone increased levodopa systemic exposure by 2-fold not changing Cmax values and reduced both 3-O-methyldopa (3-OMD) exposure and Cmax values by 5-fold. These changes were accompanied by ∼76-84% reduction in erythrocyte COMT activity. In dorsal striatum and substantia nigra, opicapone increased levodopa exposure by 1.7- and 1.4-fold, respectively, reducing 3-OMD exposure by 5- and 7-fold respectively. DOPAC exposure was increased by 4-fold in the substantia nigra. In the prefrontal cortex, opicapone increased levodopa exposure and reduced 3-OMD levels by 2.3- and 2.4-fold, respectively. CONCLUSIONS: Opicapone behaved as long-acting COMT inhibitor that markedly increased systemic and central levodopa bioavailability. Opicapone is a strong candidate to fill the unmet need for COMT inhibitors that lead to more sustained levodopa levels in Parkinson's disease patients. AIMS: The aim of this study was to assess the tolerability, pharmacokinetics and inhibitory effect on erythrocyte soluble catechol-O-methyltransferase (S-COMT) activity following repeated doses of opicapone. METHODS: This randomized, placebo-controlled, double-blind study enrolled healthy male subjects who received either once daily placebo or opicapone 5, 10, 20 or 30 mg for 8 days. RESULTS: Opicapone was well tolerated. Its systemic exposure increased in an approximately dose-proportional manner with an apparent terminal half-life of 1.0 to 1.4 h. Sulphation was the main metabolic pathway. Opicapone metabolites recovered in urine accounted for less than 3% of the amount of opicapone administered suggesting that bile is likely the main route of excretion. Maximum S-COMT inhibition (Emax ) ranged from 69.9% to 98.0% following the last dose of opicapone. The opicapone-induced S-COMT inhibition showed a half-life in excess of 100 h, which was dose-independent and much longer than plasma drug exposure. Such a half-life translates into a putative underlying rate constant that is comparable with the estimated dissociation rate constant of the COMT-opicapone complex. CONCLUSION: Despite its short elimination half-life, opicapone markedly and sustainably inhibited erythrocyte S-COMT activity making it suitable for a once daily regimen. PURPOSE: Opicapone (OPC) is a novel catechol-O-methyltransferase (COMT) inhibitor to be used as adjunctive therapy in levodopa-treated patients with Parkinson's disease. The purpose of this study was to evaluate the effect of moderate liver impairment on the pharmacokinetics (PK) and pharmacodynamics (PD; effect on COMT activity) of OPC. METHODS: An open-label, parallel-group study in patients (n = 8) with moderate liver impairment (Child-Pugh category B, score of 7 to 9) and matched healthy subjects (n = 8, control) with normal liver function. All subjects received a single 50-mg oral dose of OPC, with plasma and urine concentrations of opicapone and its metabolites measured up to 72 h post-dose, including soluble COMT (S-COMT) activity. A one-way analysis of variance (ANOVA) was used to compare the main PK and PD parameters between groups. Point estimates (PE) of geometric mean ratios (GMR) and corresponding 90 % confidence intervals (90%CI) for the ratio hepatic/control subjects of each parameter were calculated and compared with the reference interval (80-125 %). RESULTS: Exposure to opicapone (AUC and Cmax) increased significantly in patients with moderate hepatic impairment (PE [90%CI]: AUC0-∞, 184 % [135-250 %]; Cmax, 189 % [144-249 %]). Although apparent total clearance (CL/F) of opicapone was decreased by ∼35 %, similar elimination half-life and unbound/bound fractions of opicapone were observed between the two groups. Both rate and extent of exposure to BIA 9-1103 were higher in the hepatically impaired group, but not statistically significant compared with the control group. Similar to the parent (opicapone), the observed increase in exposure to BIA 9-1106 was statistically significant in the hepatically impaired group over the control group. BIA 9-1106 was the only metabolite detected in urine and its urine PK parameters were in accordance with plasma data. Maximum S-COMT inhibition (Emax) occurred earlier for the hepatically impaired group with values of 100 % and 91.2 % for the hepatically impaired and control groups respectively. Both Emax and AUEC for the hepatically impaired group reached statistical significance over the control group. OPC was well tolerated in both hepatically impaired and control groups. CONCLUSION: The bioavailability of an orally administered single dose of 50 mg OPC was significantly higher in patients with moderate chronic hepatic impairment, perhaps by a reduced first-pass effect. As the tolerability profile of OPC was favourable under the conditions of this study and its exposure is completely purged from systemic circulation before the subsequent dose administration, no OPC dose adjustment is needed in patients with mild to moderate chronic hepatic impairment. However, as OPC is under clinical development for use as adjunctive therapy in levodopa-treated patients with Parkinson's disease, an adjustment of levodopa and/or OPC regimens in patients should be carefully considered based on a potentially enhanced levodopa dopaminergic response and the associated tolerability.

What kind of affinity purification would you use in order to isolate soluble lysosomal proteins?

The rationale for purification of the soluble lysosomal proteins resides in their characteristic sugar, the mannose-6-phosphate (M6P), which allows an easy purification by affinity chromatography on immobilized M6P receptors.

[15789345, 17258946, 18370023, 18507433, 11079561, 16145712, 16399764, 22158965, 18977398, 19383612, 16709564]

The lysosome is a membrane delimited cytoplasmic organelle that contains at least 50 hydrolytic enzymes and associated cofactors. The biomedical importance of these enzymes is highlighted by the many lysosomal storage disorders that are associated with mutations in genes encoding lysosomal proteins, and there is also evidence that lysosomal activities may be involved in more widespread human diseases. The aim of this study was to characterize the human brain lysosomal proteome with the goal of establishing a reference map to investigate human diseases of unknown etiology and to gain insights into the cellular function of the lysosome. Proteins containing mannose 6-phosphate (Man6-P), a carbohydrate modification used for targeting resident soluble lysosomal proteins to the lysosome, were affinity-purified using immobilized Man6-P receptor. Fractionation by two-dimensional electrophoresis resolved a complex mixture comprising approximately 800 spots. Constituent proteins in each spot were identified using a combination of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (both peptide mass fingerprinting and tandem mass spectrometry) [corrected] on in-gel tryptic digests and N-terminal sequencing. In a complementary analysis, we also analyzed a tryptic digest of the unfractionated mixture by liquid chromatography MS/MS. In total, 61 different proteins were identified. Seven were likely contaminants associated with true Man6-P glycoproteins. Forty-one were known lysosomal proteins of which 11 have not previously been reported to contain Man6-P. An additional nine proteins were either uncharacterized or proteins not previously reported to have lysosomal function. We found that the human brain Man6-P-containing lysosomal proteome is highly complex and contains more proteins with a much greater number of individual isoforms than found in previous studies of Man6-P glycoproteomes. Glycoproteins containing the mannose 6-phosphate (Man-6-P) modification represent a class of proteins of considerable biomedical importance. They include over sixty different soluble lysosomal hydrolases and accessory proteins, deficiencies of which result in over forty different known human genetic diseases. In addition, there are patients with lysosomal storage diseases of unknown etiology and lysosomal proteins have been implicated in pathophysiological processes associated with Alzheimer disease, arthritis, and cancer. The aim of this study was to explore urine as a source for the proteomic investigation of lysosomal storage disorders as well as for biomarker studies on the role of Man-6-P containing proteins in other human diseases. To this end, urinary proteins were affinity purified on immobilized Man-6-P receptors, digested with trypsin, and analyzed using nanospray LC/MS/MS. This resulted in identification of 67 proteins, including 48 known lysosomal proteins and 9 proteins that may be lysosomal. The identification of a large proportion of the known set of soluble lysosomal proteins with relatively few contaminants suggests that urine represents a promising substrate for the development of comparative proteomic methods for the investigation of lysosomal disorders and other diseases involving Man-6-P glycoproteins. This chapter describes the process of production, purification, separation, and mass spectrometry identification of soluble lysosomal proteins. The rationale for purification of these proteins resides in their characteristic sugar, the mannose-6-phosphate (M6P), which allows an easy purification by affinity chromatography on immobilized M6P receptor (MPR). The secretion of M6P proteins (essentially soluble lysosomal proteins) from cells in culture is induced by adding a weak base in the culture medium. Secreted proteins are ammonium sulfate precipitated, dialyzed, and loaded onto the immobilized MPR column. After specific elution and collection of the M6P proteins, these are resolved by either bidimensional or monodimensional gel electrophoresis (designated as 2-DE or 1-DE, respectively). Mass spectrometry analysis is performed on spots excised from the 2-DE gel, or on discrete bands covering altogether the whole length of the 1-DE gel lane: these spots or bands are in-gel digested with trypsin and protein identification is obtained, thanks to peptide mass fingerprints [provided by analysis of the digests by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS)] or peptide amino acid sequences (provided by analysis of the digests by the coupling between liquid chromatography and tandem mass spectrometry, LC-MS/MS). Most luminal lysosomal proteins are synthesized as precursors containing mannose 6-phosphate (Man6-P) and a number of recent studies have conducted affinity purification of Man6-P containing proteins as a step toward defining the composition of the lysosome. Approximately 60 known lysosomal proteins have been found in such studies as well as many other Man-6-P glycoproteins, some of which represent new lysosomal proteins. The latter are of considerable interest from cell-biological and biomedical perspectives, but differentiating between them and other proteins remains a significant challenge. The aim of this study was to conduct a global analysis of the mammalian Man6-P glycoproteome, implementing technical and biostatistical methods to aid in the discovery and validation of lysosomal candidates. We purified Man6-P glycoproteins from 17 individual rat tissues. To distinguish nonspecific contaminants (i.e., abundant or "sticky" proteins that are not fully removed during purification) from specifically purified proteins, we conducted a semiquantitative mass spectrometric comparison of protein levels in nonspecific mock eluates versus specific affinity chromatography eluates to identify those proteins that are specifically purified. We identified 60 known lysosomal proteins, representing nearly all that are currently known to contain Man-6-P. We also find 136 other proteins that are specifically purified but which are not known to have lysosomal function. This approach provides a list of candidate lysosomal proteins and also provides insights into the relative distribution of Man6-P glycoproteins. The lysosomal compartment of human monocytic cells has never been investigated by a proteomic approach. By a combination of one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis, protein identification by N-terminal sequencing, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting and tandem mass spectrometry (MS/MS) peptide sequence analysis, we initiated an exhaustive study of the human lyososomal proteome, which aims at establishing a 2-D reference map of human soluble lyososomal proteins. Human monocytic U937 cells were induced to secrete lysosomal soluble hydrolases by addition of NH4Cl in the culture medium. Since lysosomal soluble proteins are characterized by the presence of mannose-6-phosphate, they were purified on an affinity support bearing mannose-6-phosphate receptor. Analysis of the purified fraction led to the preliminary identification of fifteen proteins, among which twelve are well-known lysosomal hydrolases, one is assumed to be lysosomal on the basis of sequence homology to cysteine proteinases of the papain family, and two (leukocystatin and the human cellular repressor of E1A-stimulated genes) are described here for the first time as mannose-6-phosphate-containing proteins. The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins. Most newly synthesized soluble lysosomal proteins contain mannose 6-phosphate (Man-6-P), a specific carbohydrate modification that is recognized by Man-6-P receptors (MPRs) that direct targeting to the lysosome. A number of proteomic studies have focused on lysosomal proteins, exploiting the fact that Man-6-P-containing forms can be purified by affinity chromatography on immobilized MPRs. These studies have identified many known lysosomal proteins as well as many proteins not previously classified as lysosomal. The latter are of considerable biological interest with potential implications for lysosomal function and as candidates for lysosomal storage diseases of unknown etiology. However, a significant problem in interpreting the biological relevance of such proteins has been in distinguishing true Man-6-P glycoproteins from simple contaminants and from proteins associated with true Man-6-P glycoproteins (e.g. protease inhibitors and lectins). In this report, we describe a mass spectrometric approach to the verification of Man-6-phosphorylation based upon LC-MS of MPR-purified proteolytic glycopeptides. This provided a useful tool in validating novel MPR-purified proteins as true Man-6-P glycoproteins and also allowed identification of low abundance components not observed in the analysis of the total Man-6-P glycoprotein mixture. In addition, this approach allowed the global mapping of 99 Man-6-phosphorylation sites from 44 known lysosomal proteins purified from mouse and human brain. This information is likely to provide useful insights into protein determinants for this modification and may be of significant value in protein engineering approaches designed to optimize protein delivery to the lysosome in therapeutic applications such as gene and enzyme replacement therapies. Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient receptor-dependent transport of soluble lysosomal proteins to lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both phosphatases (Acp2/Acp5(-/-)) to examine their role in dephosphorylation of Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P affinity chromatography and mass spectrometry. Depending on the presence of Acp2 or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively charged lysosomal membranes at acidic pH. Correspondingly, unesterified cholesterol was found to accumulate in lysosomes of cultured hepatocytes of Acp2/Acp5(-/-) mice. The data demonstrate that dephosphorylation of Man6P-containing lysosomal proteins requires the concerted action of Acp2 and Acp5 and is needed for hydrolysis and removal of degradation products. Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.

Which are the genes thought to be regulated by EWS/FLI?

The EWS/FLI translocation product is the causative oncogene in Ewing sarcoma and acts as an aberrant transcription factor. EWS/FLI dysregulates gene expression during tumorigenesis by abnormally activating or repressing genes. The expression levels of a significant number of genes are affected in Ewing sarcoma, some of which are known to be directly or indirectly regulated by EWS/FLI. Such genes are BCL11B, NRoB1, GSTM4, NKX2.2 and p53.

[23527175, 16697960, 17114343, 18927503, 15492248, 19920188, 19718047]

The EWS/FLI translocation product is the causative oncogene in Ewing sarcoma and acts as an aberrant transcription factor. EWS/FLI dysregulates gene expression during tumorigenesis by abnormally activating or repressing genes. The expression levels of thousands of genes are affected in Ewing sarcoma, however, it is unknown which of these genes contribute to the transformed phenotype. Here we characterize BCL11B as an up-regulated EWS/FLI target that is necessary for the maintenance of transformation in patient derived Ewing sarcoma cells lines. BCL11B, a zinc finger transcription factor, acts as a transcriptional repressor in Ewing's sarcoma and contributes to the EWS/FLI repressed gene signature. BCL11B repressive activity is mediated by the NuRD co-repressor complex. We further demonstrate that re-expression of SPRY1, a repressed target of BCL11B, limits the transformation capacity of Ewing sarcoma cells. These data define a new pathway downstream of EWS/FLI required for oncogenic maintenance in Ewing sarcoma. Our understanding of Ewing's sarcoma development mediated by the EWS/FLI fusion protein has been limited by a lack of knowledge regarding the tumor cell of origin. To circumvent this, we analyzed the function of EWS/FLI in Ewing's sarcoma itself. By combining retroviral-mediated RNA interference with reexpression studies, we show that ongoing EWS/FLI expression is required for the tumorigenic phenotype of Ewing's sarcoma. We used this system to define the full complement of EWS/FLI-regulated genes in Ewing's sarcoma. Functional analysis revealed that NKX2.2 is an EWS/FLI-regulated gene that is necessary for oncogenic transformation in this tumor. Thus, we developed a highly validated transcriptional profile for the EWS/FLI fusion protein and identified a critical target gene in Ewing's sarcoma development. A number of solid tumors, such as alveolar rhabdomyosarcoma, synovial sarcoma, and myxoid liposarcoma, are associated with recurrent translocation events that encode fusion proteins. Ewing's sarcoma is a pediatric tumor that serves as a prototype for this tumor class. Ewing's sarcomas usually harbor the (11;22)(q24;q12) translocation. The t(11;22) encodes the EWS/FLI fusion oncoprotein. EWS/FLI functions as an aberrant transcription factor, but the key target genes that are involved in oncogenesis are largely unknown. Although some target genes have been defined, many of these have been identified in heterologous model systems with uncertain relevance to the human disease. To understand the function of EWS/FLI and its targets in a more clinically relevant system, we used retroviral-mediated RNAi to "knock-down" the fusion protein in patient-derived Ewing's sarcoma cell lines. By combining transcriptional profiling data from three of these lines, we identified a conserved transcriptional response to EWS/FLI. The gene that was most reproducibly up-regulated by EWS/FLI was NR0B1. NR0B1 is a developmentally important orphan nuclear receptor with no previously defined role in oncogenesis. We validated NR0B1 as an EWS/FLI-dysregulated gene and confirmed its expression in primary human tumor samples. Functional studies revealed that ongoing NR0B1 expression is required for the transformed phenotype of Ewing's sarcoma. These studies define a new role for NR0B1 in oncogenic transformation and emphasize the utility of analyzing the function of EWS/FLI in Ewing's sarcoma cells. Ewing's sarcoma is a solid tumor of the bone that primarily occurs in children and young adults. Most cases harbor the (11;22) (q24;q12) chromosomal translocation that encodes the EWS/FLI oncoprotein. EWS/FLI is an aberrant ETS-type transcription factor that dysregulates a number of genes important in the development of Ewing's sarcoma. Because EWS/FLI is the key oncoprotein in this tumor and ETS proteins are often dysregulated in various human cancers, Ewing's sarcoma serves as a useful paradigm for ETS-mediated oncogenesis. We recently showed that EWS/FLI interacts with GGAA-microsatellites to regulate some of its target genes, including NR0B1, an EWS/FLI-regulated gene that is required for the oncogenic phenotype of Ewing's sarcoma. While microsatellites typically have no ascribed function, and are sometimes considered "junk" DNA, our findings provide a unique role for microsatellites in cancer development. Furthermore, these findings may indicate a novel mechanism for normal ETS protein function as well. Finally, it is tempting to speculate that microsatellite polymorphisms may confer differences in susceptibility to Ewing's sarcoma, both between individuals and between populations, and other diseases mediated by ETS transcription factors. The observation of microsatellites as transcriptional response elements for EWS/FLI suggest that these elements may not be "junk" after all. One hallmark of Ewing's sarcoma/peripheral neuroectodermal tumors is the presence of the Ews/Fli-1 chimeric oncogene. Interestingly, infection of neuroblastoma tumor cell lines with Ews/Fli-1 switches the differentiation program of neuroblastomas to Ewing's sarcoma/peripheral neuroectodermal tumors. Here we examined the status of cytoplasmically sequestered wt-p53 in neuroblastomas after stable expression of Ews/Fli-1. Immunofluorescence revealed that in the neuroblastoma-Ews/Fli-1 infectant cell lines, p53 went from a punctate-pattern of cytoplasmic sequestration to increased nuclear localization. Western blot analysis revealed that PARC was down-regulated in one neuroblastoma cell line but not expressed in the second. Therefore, decreased PARC expression could not fully account for relieving p53 sequestration in the neuroblastoma tumor cells. Neuroblastoma-Ews/Fli-1 infectant cell lines showed marked increases in p53 protein expression without transcriptional up-regulation. Interestingly, p53 was primarily phosphorylated, without activation of its downstream target p21(WAF1). Western blot analysis revealed that whereas MDM2 gene expression does not change, p14(ARF), a negative protein regulator of MDM2, increases. These observations suggest that the downstream p53 pathway may be inactivated as a result of abnormal p53. We also found that p53 has an extended half-life in the neuroblastoma-Ews/Fli-1 infectants despite the retention of a wild-type sequence in neuroblastoma-Ews/Fli-1 infectant cell lines. We then tested the p53 response pathway and observed that the neuroblastoma parent cells responded to genotoxic stress, whereas the neuroblastoma-Ews/Fli-1 infectants did not. These results suggest that Ews/Fli-1 can directly abrogate the p53 pathway to promote tumorigenesis. These studies also provide additional insight into the relationship among the p53 pathway proteins. Ewing's sarcoma is a malignant bone-associated tumor of children and young adults. Most cases of Ewing's sarcoma express the EWS/FLI fusion protein. EWS/FLI functions as an aberrant ETS-type transcription factor and serves as the master regulator of Ewing's sarcoma-transformed phenotype. We recently showed that EWS/FLI regulates one of its key targets, NR0B1, through a GGAA-microsatellite in its promoter. Whether other critical EWS/FLI targets are also regulated by GGAA-microsatellites was unknown. In this study, we combined transcriptional analysis, whole genome localization data, and RNA interference knockdown to identify glutathione S-transferase M4 (GSTM4) as a critical EWS/FLI target gene in Ewing's sarcoma. We found that EWS/FLI directly binds the GSTM4 promoter, and regulates GSTM4 expression through a GGAA-microsatellite in its promoter. Reduction of GSTM4 levels caused a loss of oncogenic transformation. Furthermore, reduction of GSTM4 resulted in an increased sensitivity of Ewing's sarcoma cells to chemotherapeutic agents, suggesting a role for this protein in drug resistance. Consistent with this hypothesis, patients with Ewing's sarcoma whose tumors had higher levels of GSTM4 expression had worse outcomes than those with lower expression levels. These data show that GSTM4 contributes to the cancerous behavior of Ewing's sarcoma and define a wider role for GGAA-microsatellites in EWS/FLI function than previously appreciated. These data also suggest a novel therapeutic resistance mechanism, in which the central oncogenic abnormality directly regulates a resistance gene.

Do archaeal genomes contain one or multiple origins of replication?

Some archaea replicate from single origins but most archaea and all eukaryotes replicate using multiple origins.

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Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC) that recruits the replicative helicase MCM(2-7) via Cdc6 and Cdt1. We find that the three origins in the single chromosome of the archaeon Sulfolobus islandicus are specified by distinct initiation factors. While two origins are dependent on archaeal homologs of eukaryal Orc1 and Cdc6, the third origin is instead reliant on an archaeal Cdt1 homolog. We exploit the nonessential nature of the orc1-1 gene to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the protein's structure rather than that of the DNA template. Hyperthermus butylicus, a hyperthermophilic neutrophile and anaerobe, is a member of the archaeal kingdom Crenarchaeota. Its genome consists of a single circular chromosome of 1,667,163 bp with a 53.7% G+C content. A total of 1672 genes were annotated, of which 1602 are protein-coding, and up to a third are specific to H. butylicus. In contrast to some other crenarchaeal genomes, a high level of GUG and UUG start codons are predicted. Two cdc6 genes are present, but neither could be linked unambiguously to an origin of replication. Many of the predicted metabolic gene products are associated with the fermentation of peptide mixtures including several peptidases with diverse specificities, and there are many encoded transporters. Most of the sulfur-reducing enzymes, hydrogenases and electron-transfer proteins were identified which are associated with energy production by reducing sulfur to H(2)S. Two large clusters of regularly interspaced repeats (CRISPRs) are present, one of which is associated with a crenarchaeal-type cas gene superoperon; none of the spacer sequences yielded good sequence matches with known archaeal chromosomal elements. The genome carries no detectable transposable or integrated elements, no inteins, and introns are exclusive to tRNA genes. This suggests that the genome structure is quite stable, possibly reflecting a constant, and relatively uncompetitive, natural environment. The "baby machine" provides a means of generating synchronized cultures of minimally perturbed cells. We describe the use of this technique to establish the key cell-cycle parameters of hyperthermophilic archaea of the genus Sulfolobus. The 3 DNA replication origins of Sulfolobus acidocaldarius were mapped by 2D gel analysis to near 0 (oriC2), 579 (oriC1), and 1,197 kb (oriC3) on the 2,226-kb circular genome, and we present a direct demonstration of their activity within the first few minutes of a synchronous cell cycle. We also detected X-shaped DNA molecules at the origins in log-phase cells, but these were not directly associated with replication initiation or ongoing chromosome replication in synchronized cells. Whole-genome marker frequency analyses of both synchronous and log-phase cultures showed that origin utilization was close to 100% for all 3 origins per round of replication. However, oriC2 was activated slightly later on average compared with oriC1 and oriC3. The DNA replication forks moved bidirectionally away from each origin at approximately 88 bp per second in synchronous culture. Analysis of the 3 Orc1/Cdc6 initiator proteins showed a uniformity of cellular abundance and origin binding throughout the cell cycle. In contrast, although levels of the MCM helicase were constant across the cell cycle, its origin localization was regulated, because it was strongly enriched at all 3 origins in early S phase. BACKGROUND: While multiple replication origins have been observed in archaea, considerably less is known about their evolutionary processes. Here, we performed a comparative analysis of the predicted (proved in part) orc/cdc6-associated replication origins in 15 completely sequenced haloarchaeal genomes to investigate the diversity and evolution of replication origins in halophilic Archaea. RESULTS: Multiple orc/cdc6-associated replication origins were predicted in all of the analyzed haloarchaeal genomes following the identification of putative ORBs (origin recognition boxes) that are associated with orc/cdc6 genes. Five of these predicted replication origins in Haloarcula hispanica were experimentally confirmed via autonomous replication activities. Strikingly, several predicted replication origins in H. hispanica and Haloarcula marismortui are located in the distinct regions of their highly homologous chromosomes, suggesting that these replication origins might have been introduced as parts of new genomic content. A comparison of the origin-associated Orc/Cdc6 homologs and the corresponding predicted ORB elements revealed that the replication origins in a given haloarchaeon are quite diverse, while different haloarchaea can share a few conserved origins. Phylogenetic and genomic context analyses suggested that there is an original replication origin (oriC1) that was inherited from the ancestor of archaea, and several other origins were likely evolved and/or translocated within the haloarchaeal species. CONCLUSION: This study provides detailed information about the diversity of multiple orc/cdc6-associated replication origins in haloarchaeal genomes, and provides novel insight into the evolution of multiple replication origins in Archaea. Deviations from Chargaff's 2nd parity rule, according to which A approximately T and G approximately C in single stranded DNA, have been associated with replication as well as with transcription in prokaryotes. Based on observations regarding mainly the transcription-replication co-linearity in a large number of prokaryotic species, we formulate the hypothesis that the replication procedure may follow different modes between genomes throughout which the skews clearly follow different patterns. We draw the conclusion that multiple functional sites of origin of replication may exist in the genomes of most archaea and in some exceptional cases of eubacteria, while in the majority of eubacteria, replication occurs through a single fixed origin. In all three domains of life, DNA replication begins at specialized loci termed replication origins. In bacteria, replication initiates from a single, clearly defined site. In contrast, eukaryotic organisms exploit a multitude of replication origins, dividing their genomes into an array of short contiguous units. Recently, the multiple replication origin paradigm has also been demonstrated within the archaeal domain of life, with the discovery that the hyperthermophilic archaeon Sulfolobus has three replication origins. However, the evolutionary mechanism driving the progression from single to multiple origin usage remains unclear. Here, we demonstrate that Aeropyrum pernix, a distant relative of Sulfolobus, has two origins. Comparison with the Sulfolobus origins provides evidence for evolution of replicon complexity by capture of extrachromosomal genetic elements. We additionally identify a previously unrecognized candidate archaeal initiator protein that is distantly related to eukaryotic Cdt1. Our data thus provide evidence that horizontal gene transfer, in addition to its well-established role in contributing to the information content of chromosomes, may fundamentally alter the manner in which the host chromosome is replicated. DNA replication initiates at defined sites called origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number and structure across the three domains of life and their properties determine the dynamics of chromosome replication. Bacteria and some archaea replicate from single origins, whereas most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in some viruses. Here we show that such mechanisms also operate in archaea. We use deep sequencing to study replication in Haloferax volcanii and identify four chromosomal origins of differing activity. Deletion of individual origins results in perturbed replication dynamics and reduced growth. However, a strain lacking all origins has no apparent defects and grows significantly faster than wild type. Origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA, unlike strains lacking individual origins. Our results demonstrate that homologous recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose replication origins serve and why they have evolved. In bacterial chromosomes, the position of a gene relative to the single origin of replication generally reflects its replication timing, how often it is expressed, and consequently, its rate of evolution. However, because some archaeal genomes contain multiple origins of replication, bias in gene dosage caused by delayed replication should be minimized and hence the substitution rate of genes should associate less with chromosome position. To test this hypothesis, six archaeal genomes from the genus Sulfolobus containing three origins of replication were selected, conserved orthologs were identified, and the evolutionary rates (dN and dS) of these orthologs were quantified. Ortholog families were grouped by their consensus position and designated by their proximity to one of the three origins (O1, O2, O3). Conserved orthologs were concentrated near the origins and most variation in genome content occurred distant from the origins. Linear regressions of both synonymous and nonsynonymous substitution rates on distance from replication origins were significantly positive, the rates being greatest in the region furthest from any of the origins and slowest among genes near the origins. Genes near O1 also evolved faster than those near O2 and O3, which suggest that this origin may fire later in the cell cycle. Increased evolutionary rates and gene dispensability are strongly associated with reduced gene expression caused in part by reduced gene dosage during the cell cycle. Therefore, in this genus of Archaea as well as in many Bacteria, evolutionary rates and variation in genome content associate with replication timing. Halobacterium halobium contains two gas vacuole protein genes that are located in plasmid pHH1 (p-vac) and in the chromosomal DNA (c-vac). The mutation frequency for these genes is different: the constitutively expressed p-vac gene is mutated with a frequency of 10(-2), while the chromosomal gene expressed in the stationary phase of growth is mutated with a frequency of 10(-5). The difference in the mutation susceptibility is due to the dynamics of plasmid pHH1. p-vac gene mutations are caused (i) by the integration of an insertion element or (ii) by a deletion event encompassing the p-vac gene region. In contrast, c-vac mutants analyzed to date incurred neither insertion elements nor deletions. Deletion events within pHH1 occur at high frequencies during the development of a H. halobium culture. The investigation of the fusion regions resulting from deletion events indicates that insertion elements are involved. The analysis of pHH1 deletion variants led to a 4 kilobase pair DNA region containing the origin of replication of the pHH1 plasmid. Genome replication is a crucial and essential process for the continuity of life.In all organisms it starts at a specific region of the genome known as origin of replication (Ori) site. The number of Ori sites varies in prokaryotes and eukaryotes. Replication starts at a single Ori site in bacteria, but in eukaryotes multiple Ori sites are used for fast copying across all chromosomes. The situation becomes complex in archaea, where some groups have single and others have multiple origins of replication. Themococcales, are a hyperthermophilic order of archaea. They are anaerobes and heterotrophs-peptide fermenters, sulphate reducers, methanogens being some of the examples of metabolic types. In this paper we have applied a combination of multiple in silico approaches - Z curve, the cell division cycle (cdc6) gene location and location of consensus origin recognition box (ORB) sequences for location of origin of replication in Thermococcus onnurineus, Thermococcus gammatolerans and other Themococcales and compared the results to that of the well-documented case of Pyrococcus abyssi. The motivation behind this study is to find the number of Ori sites based on the data available for members of this order. Results from this in silico analysis show that the Themococcales have a single origin of replication. This report shows that isochore-like structures can be found not only in warm-blooded animals, some reptiles, fishes and yeast, but also in certain archaeal species. In perfectly shaped isochore-like structures (in "protoisochores") from Sulfolobus acidocaldarius and Thermofilum pendens genomes the difference in 3GC levels between genes from different "protoisochores" is about 30%. In these archaeal species GC-poor "protoisochores" are situated near the origin of replication, while GC-rich "protoisochores" are situated near the terminus of replication. There is a strong linear dependence between position of a gene and its 3GC level in S. acidocaldarius (an average difference in 3GC per 100,000 base pairs is equal to 3.6%). Detailed analyses of nucleotide usage biases in genes from leading and lagging strands led us to the suggestion that 3GC in genes situated near terminus of replication grows due to higher rates of thymine oxidation producing T to C transitions in lagging strands. Until recently, the only archaeon for which a bona fide origin of replication was reported was Pyrococcus abyssi, where a single origin was identified. Although several in silico analyses have suggested that some archaeal species might contain more than one origin, this has only been demonstrated recently. Two studies have shown that multiple origins of replication function in two archaeal species. One study identified two origins of replication in the archaeon Sulfolobus solfataricus, whereas a second study used a different technique to show that both S. solfataricus and Sulfolobus acidocaldarius have three functional origins. These are the first reports of archaea having multiple origins. This finding has implications for research on the mechanisms of DNA replication and evolution. We report the construction of a series of replicating shuttle vectors that consist of a low-copy-number cloning vector for Escherichia coli and functional components of the origin of replication (oriC) of the chromosome of the hyperthermophilic archaeon Pyrococcus furiosus. In the process of identifying the minimum replication origin sequence required for autonomous plasmid replication in P. furiosus, we discovered that several features of the origin predicted by bioinformatic analysis and in vitro binding studies were not essential for stable autonomous plasmid replication. A minimum region required to promote plasmid DNA replication was identified, and plasmids based on this sequence readily transformed P. furiosus. The plasmids replicated autonomously and existed in a single copy. In contrast to shuttle vectors based on a plasmid from the closely related hyperthermophile Pyrococcus abyssi for use in P. furiosus, plasmids based on the P. furiosus chromosomal origin were structurally unchanged after transformation and were stable without selection for more than 100 generations. The genomic sequence of the archaeon Methanosarcina mazei has been analyzed by the Z curve method. The Z curve is a three-dimensional curve that uniquely represents the given DNA sequence. The three-dimensional Z curve and its x and y components for the genome of M. mazei show a sharp peak and relatively broad peak, respectively. The cdc6 gene is located exactly at the position of the sharp peak. Based on the known behavior of the Z curves for the archaea whose replication origins have been identified, we hypothesize that the replication origin and termination sites correspond to the positions of the sharp peak and broad peak, respectively. We have located an intergenic region that is between the cdc6 gene (MM1314) and the gene for an adjacent protein (MM1315), which shows strong characteristics of the known replication origins. This region is highly rich in AT and contains multiple copies of consecutive repeats. Our results strongly suggest that the single replication origin of M. mazei is situated at the intergenic region between the cdc6 gene and the gene for the adjacent protein, from 1,564,657 to 1,566,241 bp of the genome. Different patterns of strand asymmetry have been documented in a variety of prokaryotic genomes as well as mitochondrial genomes. Because different replication mechanisms often lead to different patterns of strand asymmetry, much can be learned of replication mechanisms by examining strand asymmetry. Here I summarize the diverse patterns of strand asymmetry among different taxonomic groups to suggest that (1) the single-origin replication may not be universal among bacterial species as the endosymbionts Wigglesworthia glossinidia, Wolbachia species, cyanobacterium Synechocystis 6803 and Mycoplasma pulmonis genomes all exhibit strand asymmetry patterns consistent with the multiple origins of replication, (2) different replication origins in some archaeal genomes leave quite different patterns of strand asymmetry, suggesting that different replication origins in the same genome may be differentially used, (3) mitochondrial genomes from representative vertebrate species share one strand asymmetry pattern consistent with the strand-displacement replication documented in mammalian mtDNA, suggesting that the mtDNA replication mechanism in mammals may be shared among all vertebrate species, and (4) mitochondrial genomes from primitive forms of metazoans such as the sponge and hydra (representing Porifera and Cnidaria, respectively), as well as those from plants, have strand asymmetry patterns similar to single-origin or multi-origin replications observed in prokaryotes and are drastically different from mitochondrial genomes from other metazoans. This may explain why sponge and hydra mitochondrial genomes, as well as plant mitochondrial genomes, evolves much slower than those from other metazoans. The genomic sequence of the halophilic archaeon Halobacterium NRC-1 has been analyzed by the Z curve method. The Z curve is a three-dimensional curve that uniquely represents a given DNA sequence. Based on the known behaviors of the Z curves for the archaea whose replication origins have been identified, the analysis of the Z curve for the genome of Halobacterium NRC-1 strongly suggests that the large genome has two replication origins, oriC1 (921,863-922,014) and oriC2 (1,806,444-1,807,229), which are located at two sharp peaks of the Z curve. These two regions are next to the cdc6 genes and contain multiple copies of stretches of G and C, i.e., ggggtgggg and ccccacccc, which may also be regarded as direct and inverted repeats. Based on the above analysis, a model of replication of Halobacterium NRC-1 with two replication origins and two termini has been proposed. The experimental confirmation of this model would constitute the first example of multiple replication origins of archaea, which will finally provide much insight into the understanding of replication mechanisms of eukaryotic organisms, including human. In addition, the potential multiple replication origins of the archaeon Sulfolobus solfataricus are suggested by the analysis based on the Z curve method. The Z-curve is a three-dimensional curve that constitutes a unique representation of a DNA sequence, i.e., both the Z-curve and the given DNA sequence can be uniquely reconstructed from the other. We employed Z-curve analysis to identify one replication origin in the Methanocaldococcus jannaschii genome, two replication origins in the Halobacterium species NRC-1 genome and one replication origin in the Methanosarcina mazei genome. One of the predicted replication origins of Halobacterium species NRC-1 is the same as a replication origin later identified by in vivo experiments. The Z-curve analysis of the Sulfolobus solfataricus P2 genome suggested the existence of three replication origins, which is also consistent with later experimental results. This review aims to summarize applications of the Z-curve in identifying replication origins of archaeal genomes, and to provide clues about the locations of as yet unidentified replication origins of the Aeropyrum pernix K1, Methanococcus maripaludis S2, Picrophilus torridus DSM 9790 and Pyrobaculum aerophilum str. IM2 genomes. BACKGROUND: Species of the crenarchaeon Sulfolobus harbour three replication origins in their single circular chromosome that are synchronously initiated during replication. RESULTS: We demonstrate that global gene expression in two Sulfolobus species is highly biased, such that early replicating genome regions are more highly expressed at all three origins. The bias by far exceeds what would be anticipated by gene dosage effects alone. In addition, early replicating regions are denser in archaeal core genes (enriched in essential functions), display lower intergenic distances, and are devoid of mobile genetic elements. CONCLUSION: The strong replication-biased structuring of the Sulfolobus chromosome implies that the multiple replication origins serve purposes other than simply shortening the time required for replication. The higher-level chromosomal organisation could be of importance for minimizing the impact of DNA damage, and may also be linked to transcriptional regulation. The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle. HF2 is a haloarchaeal virus infecting two Halorubrum species (Family Halobacteriaceae). It is lytic, has a head-and-tail morphology and belongs to the Myoviridae (contractile tails). The linear double-stranded DNA genome was sequenced and found to be 77 670 bp in length, with a mol% G+C of 55.8. A total of 121 likely open reading frames (ORFs) were identified, of which 37 overlapped at start and stop codons. The predicted proteins were usually acidic (average pI of 4.8), and less than about 12% of them had homologues in the sequence databases. Four complete tRNA-like sequences (tRNA-Arg, -Asx, -Pro and -Tyr) and an incomplete tRNA-Thr were detected. A transcription map showed that most of the genome was transcribed and that the synthesis of transcripts occurred in a highly organized and reproducible pattern over a 5 h infection cycle. Transcripts often spanned multiple ORFs, suggesting that viral genes were organized into operons. The predicted ORF and observed transcript directions matched well and showed that transcription is mainly directed inwards from the genome termini, meeting at about 45-48 kb, and this was also a turning point in a cumulative GC-skew plot. The low point in cumulative GC-skew, near the left end, was a region rich in short repeats and lacking ORFs, which is likely to be an origin of replication. The HF2 genome is a mosaic of components from widely different sources, demonstrating clearly that viruses of haloarchaea, like their bacteriophage counterparts, are vectors for the exchange and transmission of genetic material between wide taxonomic distances, even across domains. We report here a comparative analysis of the genome sequence of Methanosarcina barkeri with those of Methanosarcina acetivorans and Methanosarcina mazei. The genome of M. barkeri is distinguished by having an organization that is well conserved with respect to the other Methanosarcina spp. in the region proximal to the origin of replication, with interspecies gene similarities as high as 95%. However, it is disordered and marked by increased transposase frequency and decreased gene synteny and gene density in the distal semigenome. Of the 3,680 open reading frames (ORFs) in M. barkeri, 746 had homologs with better than 80% identity to both M. acetivorans and M. mazei, while 128 nonhypothetical ORFs were unique (nonorthologous) among these species, including a complete formate dehydrogenase operon, genes required for N-acetylmuramic acid synthesis, a 14-gene gas vesicle cluster, and a bacterial-like P450-specific ferredoxin reductase cluster not previously observed or characterized for this genus. A cryptic 36-kbp plasmid sequence that contains an orc1 gene flanked by a presumptive origin of replication consisting of 38 tandem repeats of a 143-nucleotide motif was detected in M. barkeri. Three-way comparison of these genomes reveals differing mechanisms for the accrual of changes. Elongation of the relatively large M. acetivorans genome is the result of uniformly distributed multiple gene scale insertions and duplications, while the M. barkeri genome is characterized by localized inversions associated with the loss of gene content. In contrast, the short M. mazei genome most closely approximates the putative ancestral organizational state of these species. Replication origins were mapped in hyperthermophilic crenarchaea, using high-throughput sequencing-based marker frequency analysis. We confirm previous origin mapping in Sulfolobus acidocaldarius, and demonstrate that the single chromosome of Pyrobaculum calidifontis contains four replication origins, the highest number detected in a prokaryotic organism. The relative positions of the origins in both organisms coincided with regions enriched in highly conserved (core) archaeal genes. We show that core gene distribution provides a useful tool for origin identification in archaea, and predict multiple replication origins in a range of species. One of the P. calidifontis origins was mapped in detail, and electrophoretic mobility shift assays demonstrated binding of the Cdc6/Orc1 replication initiator protein to a repeated sequence element, denoted Orb-1, within the origin. The high-throughput sequencing approach also allowed for an annotation update of both genomes, resulting in the restoration of open reading frames encoding proteins involved in, e.g., sugar, nitrate and energy metabolism, as well as in glycosylation and DNA repair.

Which pathological conditions are caused by mutations in the CYLD gene?

Since loss of CYLD expression can be observed in different types of human cancer, it is now well established that CYLD acts as a tumor suppressor gene. Pathogenic mutations in CYLD can be identified in patients affected with Brooke-Spiegler syndrome, (Familial) Cylindromatosis or multiple familial trichoepithelioma. CYLD expression has also been reported to be dramatically downregulated in basal cell carcinoma (BCC), the most common cancer in humans.

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Multiple familial trichoepitheliomas (MFT) constitute an autosomally inherited syndrome possibly related to Brooke-Spiegler syndrome (BSS). Although some early studies suggested a role for the PTCH gene on chromosome 9q22.3 in the etiopathogenesis of MFT, recent studies of occasional patients with the MFT clinical phenotype identified mutations in the CYLD gene on chromosome 16q12-q13, a gene responsible for BSS. A systematic investigation of PTCH and CYLD mutations in patients with MFT has never been performed. Our main objective was to collect a reasonably large series of patients with MFT to (1) study the clinicopathological spectrum of the disease, (2) determine whether the PTCH gene is implicated in the pathogenesis of MFT, and if so (3) determine the relative frequency of CYLD and PTCH mutations, (4) establish if there may be any possible genotype-phenotype correlations, and (5) study the spectrum of somatic mutations. Clinical analysis including family histories, histopathological investigations, and molecular genetic studies were performed. There were 9 female and 7 male patients ranging in age from 11 to 63 years. They presented with multiple, small, discrete and sometimes confluent, skin-colored to pink, asymptomatic nodules preferentially located on the face, being especially prominent and confluent in the nasolabial folds and inner aspects of the eyebrows. A total of 66 conventional trichoepitheliomas (TEs) were studied microscopically. Aside from typical features of TE, some also exhibited variant morphological patterns including areas reminiscent of other benign adnexal neoplasms and melanocytic hyperplasia. In none of the 9 patients tested was a germline mutation of the PTCH gene identified. Germline CYLD mutations were detected in 6 of 13 patients tested (identical in 2 unrelated patients) including 2 novel mutations, whereas the remaining 7 individuals showed wild-type alleles. Two patients with germline wild-type CYLD showed, however, a somatic mutation in the gene (1 duplication, 1 substitution mutation). Neither CYLD nor PTCH germline mutations were found in the 5 patients in whom both genes were analyzed. MFT seems to be a phenotypic variant of BSS. The PTCH gene is rarely, if ever, involved in the pathogenesis of MFT. Absence of a germline mutation of the CYLD gene in cases harboring a somatic mutation may be explained by large deletions in the gene or by mutation in intronic sequences or in the promoter region. Considering our 5 patients with no mutation in either gene, the final possibility is that another, as yet undescribed gene (neither CYLD nor PTCH) is implicated in the pathogenesis of some patients with MFT. Familial cylindromatosis (turban tumor syndrome; Brooke-Spiegler syndrome) (OMIM numbers 123850, 132700, 313100, and 605041) is a rare autosomal dominantly inherited tumor syndrome. The disorder can present with cutaneous adnexal tumors such as cylindromas, trichoepitheliomas, and spiradenomas, and tumors preferably develop in hairy areas of the body such as head and neck. In affected families, mutations have been demonstrated in the CYLD gene located on chromosome 16q12-13 and reveal the characteristic attributes of a tumor suppressor. Here, we studied familial cylindromatosis in a multigeneration family of German origin. Clinically, some individuals only revealed discrete small skin-colored tumors localized in the nasolabial region whereas one family member showed expansion of multiple big tumors on the trunk and in a turban-like fashion on the scalp. Histologically, cylindromas as well as epithelioma adenoides cysticum were found. We detected a frameshift mutation in the CYLD gene, designated 2253delG, underlying the disorder and were able to show that a single mutation can result in distinct clinical and histologic expression in familial cylindromatosis. The reasons for different expression patterns of the same genetic defect in this disease remain elusive, however. Identification of mutations in the CYLD gene enable us to rapidly confirm putative diagnoses on the genetic level and to provide affected families with genetic counseling. Individuals with germline mutations in the tumour-suppressor gene CYLD are at high risk of developing disfiguring cutaneous appendageal tumours, the defining tumour being the highly organised cylindroma. Here, we analysed CYLD mutant tumour genomes by array comparative genomic hybridisation and gene expression microarray analysis. CYLD mutant tumours were characterised by an absence of copy-number aberrations apart from LOH chromosome 16q, the genomic location of the CYLD gene. Gene expression profiling of CYLD mutant tumours showed dysregulated tropomyosin kinase (TRK) signalling, with overexpression of TRKB and TRKC in tumours when compared with perilesional skin. Immunohistochemical analysis of a tumour microarray showed strong membranous TRKB and TRKC staining in cylindromas, as well as elevated levels of ERK phosphorylation and BCL2 expression. Membranous TRKC overexpression was also observed in 70% of sporadic BCCs. RNA interference-mediated silencing of TRKB and TRKC, as well as treatment with the small-molecule TRK inhibitor lestaurtinib, reduced colony formation and proliferation in 3D primary cell cultures established from CYLD mutant tumours. These results suggest that TRK inhibition could be used as a strategy to treat tumours with loss of functional CYLD. The pleiomorphic adenoma gene 1 (PLAG1) gene is activated in a subset of pleomorphic adenomas of the salivary gland by gene fusion. Germ‑line mutation in cylindromatosis (CYLD), a tumor suppressor gene, causes familial cylindromatosis and Brook‑Spiegler syndrome. In the present study, aberrations in PLAG1 and CYLD were investigated in adenoid cystic carcinoma (ACC) of the salivary gland. Reverse‑transcription PCR and PCR direct sequencing were performed to detect gene fusion of PLAG1 and mutation of CYLD in 34 ACC tissues. No PLAG1 fusion was detected in ACC. However, silent mutation of CYLD was detected in 2 cases of ACC, but no missense mutation was detected in ACC. These results suggest that PLAG1 and CYLD do not play a role in ACC tumorigenesis. We report a patient with multiple trichoepitheliomas whose biopsy material also demonstrated a range of other neoplasms with follicular differentiation, including small nodular trichoblastoma, small nodular basal cell carcinoma (BCC), and areas resembling infundibulocystic BCC/basaloid follicular hamartoma. These were all intimately associated with otherwise typical trichoepitheliomas that dominated the microscopic appearances. Peripheral blood and tumor tissues of the patient and his 2 daughters, who apparently had a milder phenotype, were studied for alterations in the CYLD and PTCH genes, but mutations or loss of heterozygosity was not found in either gene. The occurrence of multiple follicular neoplasms within a single lesion adds evidence that, although in most cases BCC and trichoblastoma are distinct lesions, the 2 neoplasms do encompass a morphological spectrum of follicular differentiation, which is probably more overtly expressed in syndromic patients. Multiple familial trichoepithelioma (MFT) is an autosomal dominant disease characterized by numerous skin-coloured papules on the central face. Mutations in the CYLD gene, which is also the gene responsible for familial cylindromatosis, have been reported recently. Recent studies indicate that CYLD is a tumour-suppressor gene. The CYLD protein is a negative regulator of the activation of transcription factor nuclear factor-kappaB, and loss of CYLD contributes to oncogenesis. We report a novel splicing mutation (IVS12 + 1 G-->A) in the CYLD gene in a Taiwanese pedigree with MFT, and discuss new developments in treatment options. Brooke-Spiegler syndrome, familial cylindromatosis, and familial trichoepithelioma are autosomal-dominant genetic predispositions for benign tumors of skin appendages caused by mutations in the CYLD gene localized on chromosome 16q12-q13. The encoded protein functions as ubiquitin-specific protease (UBP), which negatively regulates NF-kappaB and c-Jun N-terminal kinase (JNK) signaling. We investigated five families affected with these skin neoplasms and identified four premature stop codons and the novel missense mutation D681G in a family in which 11 of 12 investigated tumors were trichoepitheliomas. CYLD protein harboring this missense mutation had a significant reduced ability to inhibit TNF receptor-associated factor (TRAF)2- and TRAF6-mediated NF-kappaB activation, tumor necrosis factor-alpha (TNFalpha)-induced JNK signaling, and to deubiquitinate TRAF2. CYLD-D681G was coimmunoprecipitated by TRAF2, but was unable to cleave K63-linked polyubiquitin chains. Aspartic acid 681 is highly conserved in CYLD homologues and other members of the UBP family, but does not belong to the Cys and His boxes providing the CYLD catalytic triad (Cys601, His871, and Asp889). As reported previously, the homologous residue D295 of HAUSP/USP-7 forms a hydrogen bond with the C-terminal end of ubiquitin and is important for the enzymatic activity. These results underline that D681 in CYLD is required for cleavage of K63-linked polyubiquitin chains. The authors report a case of Brooke-Spiegler syndrome (BSS) with a novel germline CYLD mutation and various somatic mutations identified in the lesional tissues. The patient was a 46-year-old man with multiple lesions on the face. The available histopathological material included 24 trichoepitheliomas, 2 large nodular basal cell carcinomas (BCCs), 2 spiradenomas, 1 spiradenocylindroma and 1 trichoblastoma composed of large and small nodules with prominent clear cell differentiation. Whereas one of the two BCCs manifested a conventional morphology, the second neoplasm additionally showed foci with high grade cytological features characterized by marked pleomorphism and numerous mitotic figures. There were also numerous signet ring cells and cells containing intracytoplasmic eosinophilic inclusions. The germline mutation was a substitution mutation c.1684 + 1G> A. Somatic mutations were investigated in eight tissue blocks from which high quality genomic DNA had been successfully extracted. Somatic mutations included loss of heterozygosity (LOH) in four lesions and a single sequence mutation, namely a single base deletion c. 2322delA causing a frameshift mutation E774DfsX2. LOH occurred in both BCCs, one trichoepithelioma and one spiradenoma. In the remaining three lesions, the somatic event remained undetected. Brooke-Spiegler syndrome represents an autosomal dominant disease characterized by the occurrence of multiple cylindromas, trichoepitheliomas and (sporadically) spiroadenomas. Patients with Brooke-Spiegler syndrome are also at risk of developing tumors of the major and minor salivary glands. Patients with Brooke-Spiegler syndrome have various mutations in the CYLD gene, a tumor-suppressor gene located on chromosome 16q. To date, 68 unique CYLD mutations have been identified. We describe two families with Brooke-Spiegler syndrome, one with familial cylindromatosis and one with multiple familial trichoepithelioma, which showed wide inter-family phenotypic variability. Analysis of germline mutations of the CYLD and PTCH genes was performed using peripheral blood. In addition, formalin-fixed paraffin-embedded tumor samples were analyzed for PTCH somatic mutations and cylindroma cell cultures were obtained directly from patients for further growth and analysis. Clinically, the major features of Brooke-Spiegler syndrome include the presence of heterogeneous skin tumors and wide inter- and intra-familial phenotypic variability. Histopathologically, both cylindromas and trichoepitheliomas were found in affected individuals. Mutations or loss of heterozygosity was not found in CYLD and PTCH genes. In CYLD and PTCH mutation-negative patients, other genes may be affected and further studies are needed to clarify whether these patients may be affected by de novo germline mutations. Pathogenic mutations in CYLD can be identified in patients affected with Brooke-Spiegler syndrome, (Familial) Cylindromatosis or multiple familial trichoepithelioma. To date, only technologies which are able to identify small point mutations in CYLD, such as sequence and WAVE analysis, were used. Here we describe the identification of a larger rearrangement identified by Quantitative PCR analysis of CYLD, indicating that a combination of these technologies is necessary when searching for pathogenic mutations in CYLD. The authors report a case of basaloid carcinoma involving the anus and rectum of a 57-year-old woman. Microscopically, the tumor showed unusual morphologic features strongly resembling a spiradenocylindroma because it consisted, in most parts, of basaloid cell nodules arranged in a jigsaw-puzzle fashion containing or surrounded by eosinophilic basal membrane material; in addition, there were intratumoral lymphocytes. The overlying squamous epithelium manifested dysplastic changes compatible with in situ squamous carcinoma that gradually became invasive and blended with basaloid cell islands; additionally, there were koilocytes in the squamous epithelium. A molecular biology study identified HPV-16 in the lesional tissue. Analysis of the CYLD gene did not prove any mutation. Familial cylindromatosis is a rare dominantly inherited disease characterized by the development of multiple benign tumours of the skin appendages, including cylindromas, trichoepitheliomas and spiradenomas. The gene responsible was positionally cloned recently, and was designated CYLD. We describe a family with cylindromatosis, in which affected individuals have an inherited R758X nonsense mutation of CYLD. Affected members of this family manifest a relatively mild tumour phenotype; the largest tumour was only 30 mm in diameter. Thus far, there is no evident genotype-phenotype relationship in cylindromatosis, although the number of families reported with both phenotypic and genotypic data remains small. Brooke-Spiegler syndrome (BSS) is an autosomal dominant disease characterized by cylindromas, trichoepitheliomas and occasionally spiradenomas. The disease gene was mapped to 16q12-13, and mutations in the CYLD gene were identified in families with BSS. In the present report, we describe a large consanguineous Chinese family with BSS showing an intra-family phenotypic variability. Clinically, some affected individuals only revealed discrete small skin-coloured tumors whereas the proband showed an expansion of multiple large tumors on the back of nose and numerous dome-shaped papules on her scalp. Histologically, both trichoepitheliomas and cylindromas were found in the affected individuals. By sequence analysis, we identified a recurrent mutation 2272C>T (R758X) of the CYLD gene in the affected individuals of this family, which was previously identified in other ethnic families with familial cylindromatosis. Our result provided additional information for phenotype-genotype correlation in BSS. Brooke-Spiegler syndrome (BSS), familial cylindromatosis (FC), and multiple familial trichoepithelioma (MFT), originally described as distinct inherited disorders, are characterized by a variety of skin appendage neoplasms. Mutations in the CYLD gene are found in individuals with these syndromes. We describe a single family with affected members exhibiting either the FC or the MFT phenotypes associated with a mutation in the CYLD gene. These findings support the notion that BSS, FC, and MFT represent phenotypic variation of a single defect. Of interest, one of the affected individuals described in this report exhibits a severe phenotype illustrating the morbidity of the disorder. Multiple familial trichoepithelioma (MFT) and familial cylindromatosis are two clinically distinct cancer syndromes. MFT patients developed mostly trichoepithelioma in the face while cylindromatosis patients developed cylindromas predominantly (approximately 90%) on the head and neck. However, multiple familial trichoepithelioma is occasionally associated with familial cylindromatosis while cylindromatosis patients can also develop trichoepithelioma. This has led to the speculation that the 2 types of dermatoses may be caused by dysfunction of a common pathway. Previously, a candidate MTF locus has been mapped to 9p21 while disease gene for familial cylindromatosis, the CYLD gene located on 16q21-13 has been identified. Here, we show that mutations in the CYLD gene are also the genetic basis for three different Chinese families with MFT. Sequence analysis reveal a single nucleotide deletion, c.1462delA (P.Ile488fsX9) in exon 9, a nonsense mutation, c.2128C>T (p. Gln710X) in exon 17, and a missense mutation, c.2822A>T (p. Asp941Val) in exon 21 in each of the three families respectively. This provides direct evidence that the mutations in CYLD can cause two clinically distinct cancer syndromes. Survival of the malignant Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) is dependent on constitutive activation of the nuclear factor kappaB (NF-kappaB) transcription factor. The deubiquitinating enzyme CYLD is a negative regulator of NF-kappaB and known to function as a tumor suppressor. To determine whether CYLD mutations play a role in cHL pathogenesis, we sequenced the gene in cHL cell lines and microdissected HRS cells obtained from lymph-node biopsies. A biallelic inactivation by mutations was found in the cHL cell-line KM-H2. However, the other seven cHL cell lines analyzed and HRS cells of 10 primary cHL cases did not show any mutations. By interphase cytogenetics, a (sub)clonal biallelic CYLD deletion was observed by interphase cytogenetics in 1 of 29 primary cHL, whereas signal patterns indicating decreased CYLD copy numbers were observed in a total of 10 of 29 primary cases. Our results suggest that biallelic CYLD mutations are rarely involved in cHL pathogenesis. Nevertheless, it is remarkable that KM-H2 cells, besides the CYLD mutations, also carry inactivating mutations in the genes of two other NF-kappaB inhibitors, that is, NFKBIA and TNFAIP3, exemplifying that multiple lesions in regulators of this signaling pathway can likely cooperatively contribute to the strong NF-kappaB activity of these cells. We present a case of Brooke-Spiegler syndrome with a germline deep intronic mutation in the CYLD gene leading to intronic exonization. Additionally, diverse somatic mutations were identified, namely loss of heterozygosity, a recurrent nonsense mutation, and a sequence mutation causing exon skipping. These somatic aberrations were identified in 4 different cylindromas that had been removed from the patient. Additionally, we microscopically studied a spiradenocylindroma that showed unusual histology, including foci of follicular differentiation. A deep intronic mutation resulting in exonization and a somatic sequence mutations causing exon skipping are hitherto unreported genetic mechanisms involving the CYLD gene in patients with Brooke-Spiegler syndrome. CYLD is a deubiquitination enzyme that regulates different cellular processes, such as cell proliferation and cell survival. Mutation and loss of heterozygosity of the CYLD gene causes development of cylindromatosis, a benign tumour originating from the skin. Our study shows that CYLD expression is dramatically downregulated in basal cell carcinoma (BCC), the most common cancer in humans. Reduced CYLD expression in basal cell carcinoma was mediated by GLI1-dependent activation of the transcriptional repressor Snail. Inhibition of GLI1 restored the CYLD expression-mediated Snail signaling pathway, and caused a significant delay in the G1 to S phase transition, as well as proliferation. Our data suggest that GLI1-mediated suppression of CYLD has a significant role in basal cell carcinoma progression. The authors report a 64-year-old female with Brooke-Spiegler syndrome who presented with multiple cutaneous nodules and tumors mostly involving the scalp. Histopathological examination of one of the lesions located in a periauricular area revealed a typical cylindroma. In some neoplastic nodules ductal differentiation and occasional bilayered glands composed of the dark abluminal basal/myoepithelial cells and luminal mucinous cells might be recognized. Apocrine secretion was focally noted. Molecular biologic study of the CYLD gene performed from the peripheral blood identified a novel splice site c.2041+1 G>T mutation. This new germline mutation in the CYLD gene of a Slovak patient with Brooke-Spiegler syndrome extends the catalogue of known CYLD germline mutations in this condition.