Repeat Contraction Research Articles

Localization to the nuclear pore complex is required for stabilizing CAG repeats

CAG repeats are structure-forming repetitive DNA sequences which can interfere with DNA replication and DNA repair. CAG repeats are prone to breakage, and repair of the breaks can cause repeat expansions or contractions. Expansion beyond a threshold of ~35 CAG repeats is the cause of more than 14 human diseases including Huntington's disease and myotonic dystrophy. Studies in yeast have shown that irreparable DNA double strand breaks or collapsed replication forks localize to the nuclear periphery to promote proper repair. We tagged an expanded CAG repeat locus with GFP (green fluorescent protein) on a yeast chromosome, and visualized the localization of the repeats in the nucleus by using fluorescent microscopy. Our data showed that expanded CAG-70 and -130 repeats are more likely than a control to localize to the nuclear periphery during S-phase, in a tract length dependent manner. Through a mobility analysis, we also showed that the mobility of expanded CAG repeats decreases due to this localization. By chromatin immunoprecipitation (ChIP), we found that expanded CAG repeats interact with the Nup84 component of the nuclear pore complex. Moreover, Nup84 and its associated SUMO targeting ubiquitin ligase (STUbL), Slx5/Slx8, are important for preventing CAG repeat instability and fragility. Our results indicate that lesions associated with structure-forming repeats trigger a repair or fork restart event that occurs at the nuclear periphery, and that this event is crucial for the maintenance of genome stability. This study was funded by Tufts University.

Incentives for Repeated Contracts in Public Sector: Empirical Study of Gasoline Procurement in Russia

This paper analyzes the phenomenon of repeated procurements made by public sector customers from the same supplier. The previous surveys of “relational contracts” gave different explanations for the possible implications of such repeated procurements, but those surveys dealt mostly with goods and services, with quality difficult to verify at the point of delivery. This work studies the impact of repeated procurements on the price of a simple homogeneous product. We presume that the downward price shift of such a product during repeated procurements can be the consequence of transaction costs reduction in the framework of the bona fide behavior of a customer and supplier. An upward shift in the prices as compared to the market average can, on the contrary, be interpreted as an indirect indication of corrupt collusion between them. Using a huge dataset on procurements of AI-92 gasoline in Russia in 2011, we show that the price difference between repeated and one-time contracts can be explained by the type of procurement procedures providing different opportunities for corrupt behavior. Less transparent procedures (single-sourcing and requests for quotations) are more suitable for corrupt collusion. This might explain why the prices of repeat contracts in this case were higher. On the contrary, the prices of repeat contracts were lower compared to one-time procurement in the case of more transparent e-auctions.

Effect of plyometric training on vertical jump height in high school basketball players: A randomised control trial

Background: Plyometric involve high intensity eccentric contraction immediately after a powerful concentric contraction. A vertical leap in basketball also involves rapid & repeated muscle contraction & stretching. Various methods have been used to improve the vertical leap in players, but only few studies mention about plyometrics. Aim: To determine the effect of Plyometric training on vertical jump height in high school basketball players & compare them with their untrained counterparts. Methods and Materials: 144 students were randomly selected & distributed in Group I (Pre-pubertal) & Group II (Pubertal) which was further divided into Group A (trained players) & Group B (untrained students). A gender wise distribution followed this. Plyometric training of 6 weeks was conducted & the vertical jump height pre & post training were recorded & compared. Results: Vertical jump height improved significantly post Plyometric in Group Bcompared to Group A. Boys showed improvement in Group B, however girls were better in Group A. Correlation of BMI with vertical jump height was negative & significant in Group B. Conclusion: Plyometric training brought significant change in untrained students. Boys gained more jump height while girls showed significant increase in jump height during pubertal growth spurt. Also, increased BMI reduced jump height

Diagnostic approach for FSHD revisited: SMCHD1 mutations cause FSHD2 and act as modifiers of disease severity in FSHD1.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder with a wide clinical variability. Contractions of the D4Z4 macrosatellite repeat on chromosome 4q35 are the molecular basis of the pathophysiology. Recently, in a subset of patients without D4Z4 repeat contractions, variants in the SMCHD1 gene have been identified that lead to hypomethylation of D4Z4 and thus DUX4 transcription, which causes FSHD type 2. In this study, we have screened 55 FSHD1-negative and 40 FSHD1-positive patients from unrelated families for potentially pathogenic variants in SMCHD1 by next-generation sequencing (NGS). We identified variants in SMCHD1 in 11 index patients, including missense, splice site and non-sense mutations. We developed a pyrosequencing assay to determine the methylation status of the D4Z4 repeat array and found significantly lower methylation levels for FSHD2 patients than for healthy controls and FSHD1 patients. Two out of eleven SMCHD1 mutation carriers had moderately contracted D4Z4 alleles thus these patients are suffering from FSHD1 and 2. Comparing the phenotype of patients, all FSHD2 patients were relatively mildly affected while patients with FSHD1+2 were much more severely affected than expected from their D4Z4 copy number. Our findings confirm the role of SMCHD1 mutations in FSHD2 and as a modifier of disease severity. With SMCHD1 variants found in 16.4% of phenotypic FSHD patients without D4Z4 repeat contractions, the incidence of FSHD2 is rather high and hence we suggest including sequencing of SMCHD1, haplotyping and methylation analysis in the workflow of molecular FSHD diagnostics.

G.P.11: SMCHD1 mutations cause FSHD type 2 and act as modifiers of disease severity

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder of wide clinical variability. Contractions of the D4Z4 macro-satellite repeat on the permissive chromosome 4qA haplotype are the molecular basis of the disease. D4Z4 repeats with less than 11 copies are associated with chromatin decondensation and hypomethylation allowing for expression of the flanking DUX4 gene which is highly cytotoxic and causes skeletal muscle cell death. Recently, mutations in the SMCHD1 gene have been identified in a subset of patients without D4Z4 repeat contractions (FSHD2). Haploinsufficiency of the SMCHD1 protein results in hypomethylation of D4Z4 and a more open chromatin structure permitting activation of DUX4. We have screened phenotypic FSHD patients from 95 unrelated families for mutations in SMCHD1 by next generation sequencing (NGS). We identified mutations (deletions, missense, splice site and nonsense) in nine index patients without D4Z4 contractions. We developed a pyrosequencing assay to determine the methylation status of the D4Z4 repeat array and found significantly lower methylation levels for FSHD2 patients than for healthy controls and FSHD1 patients. SMCHD1 mutations were also found in two patients with moderately contracted D4Z4 alleles thus suffering from FSHD1 and 2. Comparing the phenotype of patients, all FSHD2 patients were relatively mildly affected while patients with FSHD1+2 were much more severely affected than expected from their D4Z4 copy number. Our findings confirm the digenic inheritance of FSHD2 with mutations in SMCHD1 and hypomethylation of D4Z4. In cases of “double trouble” with FSHD1 and 2, SMCHD1 mutations seem to act as a modifier of disease severity. With SMCHD1 mutations found in 16.4% of phenotypic FSHD patients, FSHD2 cannot be considered to be excessively rare. Hence, we suggest including sequencing of SMCHD1, haplotyping and methylation analysis in the workflow of molecular FSHD diagnostics.

Genetic and Epigenetic Characteristics of FSHD-Associated 4q and 10q D4Z4 that are Distinct from Non-4q/10q D4Z4 Homologs

Facioscapulohumeral dystrophy (FSHD) is one of the most prevalent muscular dystrophies. The majority of FSHD cases are linked to a decreased copy number of D4Z4 macrosatellite repeats on chromosome 4q (FSHD1). Less than 5% of FSHD cases have no repeat contraction (FSHD2), most of which are associated with mutations of SMCHD1. FSHD is associated with the transcriptional derepression of DUX4 encoded within the D4Z4 repeat, and SMCHD1 contributes to its regulation. We previously found that the loss of heterochromatin mark (i.e., histone H3 lysine 9 tri-methylation (H3K9me3)) at D4Z4 is a hallmark of both FSHD1 and FSHD2. However, whether this loss contributes to DUX4 expression was unknown. Furthermore, additional D4Z4 homologs exist on multiple chromosomes, but they are largely uncharacterized and their relationship to 4q/10q D4Z4 was undetermined. We found that the suppression of H3K9me3 results in displacement of SMCHD1 at D4Z4 and increases DUX4 expression in myoblasts. The DUX4 open reading frame (ORF) is disrupted in D4Z4 homologs and their heterochromatin is unchanged in FSHD. The results indicate the significance of D4Z4 heterochromatin in DUX4 gene regulation and reveal the genetic and epigenetic distinction between 4q/10q D4Z4 and the non-4q/10q homologs, highlighting the special role of the 4q/10q D4Z4 chromatin and the DUX4 ORF in FSHD.

Nanoscale Operando and 3D In Situ Imaging of Micron-Sized Ge Anode Particles in Li Batteries

Ge anodes have very high theoretical lithium capacity (1600 mAhg-1) and are promising anodes for Li batteries. Ge is more electronically conductive than Si and has a higher room temperature Li-ion diffusivity. However, because of their high capacity, Ge anodes suffer from large volume changes (up to 400%; similar to Si, at 4200 mAhg-1) during electrochemical cycling. Repeated expansion and contraction of these materials lead to fracturing and eventual pulverization resulting in the loss of electrical contact with the current collector and ultimately battery failure.In order to better understand these failures we have conducted the first 3D nanoscale in situ study of a Li battery system. We imaged Ge anodes with hard X-rays (11.2 keV, above the Ge K-edge) with transmission X-ray microscopy at 30 nm resolution. 2D images in operando were collected at several regions within the particles, and 3D tomography data was also collected at different time points within the fully operating battery (in situ) to further examine the crack formation and density changes that occur with cycling. We found that volume expansion during lithiation is particle size dependent, but the subsequent volume contraction is not. Moreover, we found that only the largest particles actively participate in the second (de)lithiation cycle. From the in situ tomographic information we quantified the volume change in particles and measured the changes in porosity or density as particles (de)lithiate. Our results demonstrate the significant value of high resolution 3D imaging of batteries under operating conditions.

Making the invisible public service visible? Exploring data on the supply of policy and management consultancies inCanada

AbstractThe use of external policy and management consultants in government has been receiving increasing attention in many countries, includingCanada. We explore new data on management consulting compiled from information released since the creation of theFederalAccountabilityAct to address the supply side of contracting. We find several large multi‐year contracts have taken up a larger percentage of contracting activity while the number of smaller contracts has declined. The data suggest a pattern of oligopsonic demand concentrated mostly in a handful of very heavy users and an increasingly oligopolistic supply pattern where less than 5% of companies accounted for 80% of total contract values and where repeat contracts are the norm. Measures of accountability and transparency need to extend to the “invisible” public service of contract consultants.

Highly specific contractions of a single CAG/CTG trinucleotide repeat by TALEN in yeast.

Trinucleotide repeat expansions are responsible for more than two dozens severe neurological disorders in humans. A double-strand break between two short CAG/CTG trinucleotide repeats was formerly shown to induce a high frequency of repeat contractions in yeast. Here, using a dedicated TALEN, we show that induction of a double-strand break into a CAG/CTG trinucleotide repeat in heterozygous yeast diploid cells results in gene conversion of the repeat tract with near 100% efficacy, deleting the repeat tract. Induction of the same TALEN in homozygous yeast diploids leads to contractions of both repeats to a final length of 3–13 triplets, with 100% efficacy in cells that survived the double-strand breaks. Whole-genome sequencing of surviving yeast cells shows that the TALEN does not increase mutation rate. No other CAG/CTG repeat of the yeast genome showed any length alteration or mutation. No large genomic rearrangement such as aneuploidy, segmental duplication or translocation was detected. It is the first demonstration that induction of a TALEN in an eukaryotic cell leads to shortening of trinucleotide repeat tracts to lengths below pathological thresholds in humans, with 100% efficacy and very high specificity.

Base Excision Repair of Chemotherapeutically-Induced Alkylated DNA Damage Predominantly Causes Contractions of Expanded GAA Repeats Associated with Friedreich's Ataxia

Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER). We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA)20 repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5'-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5'-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.

A single intake of capsiate improves mechanical performance and bioenergetics efficiency in contracting mouse skeletal muscle

Capsiate is known to increase whole body oxygen consumption possibly via the activation of uncoupling processes, but its effect at the skeletal muscle level remains poorly documented and conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated in mice 2 h after a single intake of either vehicle (control) or purified capsiate (at 10 or 100 mg/kg body wt) through a multidisciplinary approach combining in vivo and in vitro measurements. Mechanical performance and energy pathway fluxes were assessed strictly noninvasively during a standardized electrostimulation-induced exercise, using an original device implementing 31-phosphorus magnetic resonance spectroscopy, and mitochondrial respiration was evaluated in isolated saponin-permeabilized fibers. Compared with control, both capsiate doses produced quantitatively similar effects at the energy metabolism level, including an about twofold decrease of the mitochondrial respiration sensitivity for ADP. Interestingly, they did not alter either oxidative phosphorylation or uncoupling protein 3 gene expression at rest. During 6 min of maximal repeated isometric contractions, both doses reduced the amount of ATP produced from glycolysis and oxidative phosphorylation but increased the relative contribution of oxidative phosphorylation to total energy turnover (+28 and +21% in the 10- and 100-mg groups, respectively). ATP cost of twitch force generation was further reduced in the 10- (-35%) and 100-mg (-45%) groups. Besides, the highest capsiate dose also increased the twitch force-generating capacity. These data present capsiate as a helpful candidate to enhance both muscle performance and oxidative phosphorylation during exercise, which could constitute a nutritional approach for improving health and preventing obesity and associated metabolic disorders.

Effects of neuromuscular electrical stimulation on arterial hemodynamic properties and body composition in paretic upper extremities of patients with subacute stroke.

Neuromuscular electric stimulation (NMES) induces repeated muscular contraction, possibly promoting the perfusion/oxygenation of the regional tissues. It remains unclear how NMES influences vascular hemodynamic property and segmental fluid distribution/composition in paretic extremities of hemiplegic patients. Eleven hemiplegic patients aged 62.6 ± 12.5 years in the subacute stage of stroke received NMES for paretic wrist extensor and flexor muscles 30 min daily, 5 days per week for 4 weeks. The non-paretic upper extremities (NPUE) that did not receive NMES served as control. Distribution of fluid to intra/extracellular milieu and arterial hemodynamic properties were determined by using the multi-frequency bioelectrical impedance and pulse wave analysis, respectively. Compared with NPUE without NMES, paretic upper extremity (PUE) with NMES revealed a significantly less decrease in arterial blood flow, impedance quotient, slope quotient, and less increase in crest width and crest time of arterial pulse wave. NMES for 4 weeks increased body cell mass in PUE. Furthermore, NPUE without NMES reduced intracellular water, whereas PUE with NMES retarded loss of intracellular water after stroke. NMES therapy increases body cell mass, attenuates reduction of intracellular water, and alleviates arterial hemodynamic disturbance in PUE in subacute stroke. However, stroke-related physical deconditioning may negatively regulate body composition and impair hemodynamic function in NPUE.

The FSHD2 Gene SMCHD1 Is a Modifier of Disease Severity in Families Affected by FSHD1

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array on chromosome 4 to a size of 1-10 units. The residual number of D4Z4 units inversely correlates with clinical severity, but significant clinical variability exists. Each unit contains a copy of the DUX4 retrogene. Repeat contractions are associated with changes in D4Z4 chromatin structure that increase the likelihood of DUX4 expression in skeletal muscle, but only when the repeat resides in a genetic background that contains a DUX4 polyadenylation signal. Mutations in the structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) gene, encoding a chromatin modifier of D4Z4, also result in the increased likelihood of DUX4 expression in individuals with a rare form of FSHD (FSHD2). Because SMCHD1 directly binds to D4Z4 and suppresses somatic expression of DUX4, we hypothesized that SMCHD1 may act as a genetic modifier in FSHD1. We describe three unrelated individuals with FSHD1 presenting an unusual high clinical severity based on their upper-sized FSHD1 repeat array of nine units. Each of these individuals also carries a mutation in the SMCHD1 gene. Familial carriers of the FSHD1 allele without the SMCHD1 mutation were only mildly affected, suggesting a modifier effect of the SMCHD1 mutation. Knocking down SMCHD1 in FSHD1 myotubes increased DUX4 expression, lending molecular support to a modifier role for SMCHD1 in FSHD1. We conclude that FSHD1 and FSHD2 share a common pathophysiological pathway in which the FSHD2 gene can act as modifier for disease severity in families affected by FSHD1.

Lack of myostatin impairs mechanical performance and ATP cost of contraction in exercising mouse gastrocnemius muscle in vivo

Although it is well established that the lack of myostatin (Mstn) promotes skeletal muscle hypertrophy, the corresponding changes regarding force generation have been studied mainly in vitro and remain conflicting. Furthermore, the metabolic underpinnings of these changes are very poorly documented. To clarify this issue, we have investigated strictly noninvasively in vivo the impact of the lack of Mstn on gastrocnemius muscle function and energetics in Mstn-targeted knockout (Mstn-/-) mice using ¹H-magnetic resonance (MR) imaging and ³¹P-MR spectroscopy during maximal repeated isometric contractions induced by transcutaneous electrostimulation. In Mstn-/- animals, although body weight, gastrocnemius muscle volume, and absolute force were larger (+38, +118, and +34%, respectively) compared with wild-type (Mstn+/+) mice, specific force (calculated from MR imaging measurements) was significantly lower (-36%), and resistance to fatigue was decreased. Besides, Mstn deficiency did not affect phosphorylated compound concentrations and intracellular pH at rest but caused a large increase in ATP cost of contraction (up to +206% compared with Mstn+/+) throughout the stimulation period. Further, Mstn deficiency limits the shift toward oxidative metabolism during muscle activity despite the fact that oxidative ATP synthesis capacity was not altered. Our data demonstrate in vivo that the absence of Mstn impairs both mechanical performance and energy cost of contraction in hypertrophic muscle. These findings must be kept in mind when considering Mstn as a potential therapeutic target for increasing muscle mass in patients suffering from muscle-wasting disorders.

Hypothesis: Local dNTP depletion as the cause of microsatellite repeat instability during replication (Comment on DOI 10.1002/bies.201200128)

Nucleotide depletion in vivo is correlated with global genomic instability and tumorigenesis 1-3. In contrast, localized instability of low complexity microsatellite sequences causes multiple neurodegenerative diseases (e.g. Huntington's disease, myotonic dystrophy, spinocerebellar ataxias), depending on the host gene. CNG microsatellites are particularly prone to the formation of imperfectly base paired hairpin structures in vitro 4 and in vivo 5, and are sites of genome instability exacerbated by replication fork slowing. Current models of microsatellite instability invoke the formation of hairpin structures during replication, repair or transcription. In replication or repair models, the microsatellite sequence or structure directly inhibits DNA polymerase movement. Contractions arise via two mechanisms: (i) slowing of the polymerase and consequent increase in hairpin formation in extended leading or lagging strand template ssDNA at the replication fork; (ii) threading of the leading strand template through the MCM helicase without concurrent polymerization. Expansions are provoked by nascent strand hairpins, because the repeat sequence directly causes polymerase ‘stuttering’ and primer slippage. In contrast, the new model presented in the current issue of BioEssays proposes that polymerase slowing and nascent strand hairpins are not direct effects of the repeat on the polymerase; rather they are indirect consequences of local nucleotide depletion, which results from a local stoichiometric imbalance in nucleotide incorporation into the nascent DNA (repeat sequences manifest such imbalanced stoichiometries) 6. The hypothesis predicts that nucleotide diffusion through the cell should be slower than the rate of precursor consumption at an individual replication fork. Although nucleotide diffusion rates have not been determined experimentally, based on the turnover of nucleotide pools in vivo, it is argued that the rate of nucleotide incorporation may be equal to or greater than the rate of diffusion. Additionally, replication is thought to occur in congregated replication forks, or factories: this raises the concern that if several forks simultaneously draw nucleotides from a common pool, imbalanced incorporation by a single fork would not have significant impact. However, the author presents evidence from ultra-high resolution microscopy that individual replication factories may contain only two forks, hence isolating the effects of nucleotide depletion. Trinucleotide repeat expansion or contraction in bacteria or yeast have shown significantly greater instability for CNG repeats than GNC repeats, possibly due to differences in the structure, stability or downstream metabolism of the respective hairpins. Dr. Kuzminov suggests in vitro tests of the nucleotide depletion model by analysis of replication in the response to differing nucleotide doses. Since nucleotide depletion by replication of CTG or GTC repeats should be similar, an alternative in vivo test of whether nucleotide depletion is the immediate cause of fork slowing would be to compare the distribution of replication intermediates by 2D gel electrophoresis during replication of plasmids containing CTG and GTC isomeric repeat sequences. Verification of the hypothesis that local nucleotide depletion potentiates microsatellite instability could lead to novel approaches to prognosis, prevention or therapy of neurodegenerative disorders.

Trinucleotide repeat expansions catalyzed by human cell-free extracts

Trinucleotide repeat expansions cause 17 heritable human neurological disorders. In some diseases, somatic expansions occur in non-proliferating tissues such as brain where DNA replication is limited. This finding stimulated significant interest in replication-independent expansion mechanisms. Aberrant DNA repair is a likely source, based in part on mouse studies showing that somatic expansions are provoked by the DNA repair protein MutSβ (Msh2-Msh3 complex). Biochemical studies to date used cell-free extracts or purified DNA repair proteins to yield partial reactions at triplet repeats. The findings included expansions on one strand but not the other, or processing of DNA hairpin structures thought to be important intermediates in the expansion process. However, it has been difficult to recapitulate complete expansions in vitro, and the biochemical role of MutSβ remains controversial. Here, we use a novel in vitro assay to show that human cell-free extracts catalyze expansions and contractions of trinucleotide repeats without the requirement for DNA replication. The extract promotes a size range of expansions that is similar to certain diseases, and triplet repeat length and sequence govern expansions in vitro as in vivo. MutSβ stimulates expansions in the extract, consistent with aberrant repair of endogenous DNA damage as a source of expansions. Overall, this biochemical system retains the key characteristics of somatic expansions in humans and mice, suggesting that this important mutagenic process can be restored in the test tube.

Generation of Isogenic D4Z4 Contracted and Noncontracted Immortal Muscle Cell Clones from a Mosaic Patient: A Cellular Model for FSHD

In most cases facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of the D4Z4 repeat in the 4q subtelomere. This contraction is associated with local chromatin decondensation and derepression of the DUX4 retrogene. Its complex genetic and epigenetic cause and high clinical variability in disease severity complicate investigations on the pathogenic mechanism underlying FSHD. A validated cellular model bypassing the considerable heterogeneity would facilitate mechanistic and therapeutic studies of FSHD. Taking advantage of the high incidence of somatic mosaicism for D4Z4 repeat contraction in de novo FSHD, we have established a clonal myogenic cell model from a mosaic patient. Individual clones are genetically identical except for the size of the D4Z4 repeat array, being either normal or FSHD sized. These clones retain their myogenic characteristics, and D4Z4 contracted clones differ from the noncontracted clones by the bursts of expression of DUX4 in sporadic nuclei, showing that this burst-like phenomenon is a locus-intrinsic feature. Consequently, downstream effects of DUX4 expression can be observed in D4Z4 contracted clones, like differential expression of DUX4 target genes. We also show their participation to in vivo regeneration with immunodeficient mice, further expanding the potential of these clones for mechanistic and therapeutic studies. These cell lines will facilitate pairwise comparisons to identify FSHD-specific differences and are expected to create new opportunities for high-throughput drug screens.

EFFICIENT DATA STRUCTURE FOR REPRESENTING AND SIMPLIFYING SIMPLICIAL COMPLEXES IN HIGH DIMENSIONS

We study the simplification of simplicial complexes by repeated edge contractions. First, we extend to arbitrary simplicial complexes the statement that edges satisfying the link condition can be contracted while preserving the homotopy type. Our primary interest is to simplify flag complexes such as Rips complexes for which it was proved recently that they can provide topologically correct reconstructions of shapes. Flag complexes (sometimes called clique complexes) enjoy the nice property of being completely determined by the graph of their edges. But, as we simplify a flag complex by repeated edge contractions, the property that it is a flag complex is likely to be lost. Our second contribution is to propose a new representation for simplicial complexes particularly well adapted for complexes close to flag complexes. The idea is to encode a simplicial complex K by the graph G of its edges together with the inclusion-minimal simplices in the set difference Flag (G)\ K. We call these minimal simplices blockers. We prove that the link condition translates nicely in terms of blockers and give formulae for updating our data structure after an edge contraction. Finally, we observe in some simple cases that few blockers appear during the simplification of Rips complexes, demonstrating the efficiency of our representation in this context.

Different Molecular Signatures in Magnetic Resonance Imaging-Staged Facioscapulohumeral Muscular Dystrophy Muscles

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies and is characterized by a non-conventional genetic mechanism activated by pathogenic D4Z4 repeat contractions. By muscle Magnetic Resonance Imaging (MRI) we observed that T2-short tau inversion recovery (T2-STIR) sequences identify two different conditions in which each muscle can be found before the irreversible dystrophic alteration, marked as T1-weighted sequence hyperintensity, takes place. We studied these conditions in order to obtain further information on the molecular mechanisms involved in the selective wasting of single muscles or muscle groups in this disease.MethodsHistopathology, gene expression profiling and real time PCR were performed on biopsies from FSHD muscles with different MRI pattern (T1-weighted normal/T2-STIR normal and T1-weighted normal/T2-STIR hyperintense). Data were compared with those from inflammatory myopathies, dysferlinopathies and normal controls. In order to validate obtained results, two additional FSHD samples with different MRI pattern were analyzed.ResultsMyopathic and inflammatory changes characterized T2-STIR hyperintense FSHD muscles, at variance with T2-STIR normal muscles. These two states could be easily distinguished from each other by their transcriptional profile. The comparison between T2-STIR hyperintense FSHD muscles and inflammatory myopathy muscles showed peculiar changes, although many alterations were shared among these conditions.ConclusionsAt the single muscle level, different stages of the disease correspond to the two MRI patterns. T2-STIR hyperintense FSHD muscles are more similar to inflammatory myopathies than to T2-STIR normal FSHD muscles or other muscular dystrophies, and share with them upregulation of genes involved in innate and adaptive immunity. Our data suggest that selective inflammation, together with perturbation in biological processes such as neoangiogenesis, lipid metabolism and adipokine production, may contribute to the sequential bursts of muscle degeneration that involve individual muscles in an asynchronous manner in this disease.

Complete Chloroplast Genome Sequence of a Major Invasive Species, Crofton Weed (Ageratina adenophora)

BackgroundCrofton weed (Ageratina adenophora) is one of the most hazardous invasive plant species, which causes serious economic losses and environmental damages worldwide. However, the sequence resource and genome information of A. adenophora are rather limited, making phylogenetic identification and evolutionary studies very difficult. Here, we report the complete sequence of the A. adenophora chloroplast (cp) genome based on Illumina sequencing.Methodology/Principal FindingsThe A. adenophora cp genome is 150, 689 bp in length including a small single-copy (SSC) region of 18, 358 bp and a large single-copy (LSC) region of 84, 815 bp separated by a pair of inverted repeats (IRs) of 23, 755 bp. The genome contains 130 unique genes and 18 duplicated in the IR regions, with the gene content and organization similar to other Asteraceae cp genomes. Comparative analysis identified five DNA regions (ndhD-ccsA, psbI-trnS, ndhF-ycf1, ndhI-ndhG and atpA-trnR) containing parsimony-informative characters higher than 2%, which may be potential informative markers for barcoding and phylogenetic analysis. Repeat structure, codon usage and contraction of the IR were also investigated to reveal the pattern of evolution. Phylogenetic analysis demonstrated a sister relationship between A. adenophora and Guizotia abyssinica and supported a monophyly of the Asterales.ConclusionWe have assembled and analyzed the chloroplast genome of A. adenophora in this study, which was the first sequenced plastome in the Eupatorieae tribe. The complete chloroplast genome information is useful for plant phylogenetic and evolutionary studies within this invasive species and also within the Asteraceae family.