The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms ...for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state "catch or present" model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS-Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.
Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne Muscular Dystrophy (DMD) locus. Its regulation is of therapeutic interest as its overexpression can compensate for ...dystrophin's absence in animal models of DMD. The tissue distribution and transcriptional regulation of utrophin have been characterized extensively, and more recently translational control mechanisms that may underlie its complex expression patterns have begun to be identified.
Using a variety of bioinformatic, molecular and cell biology techniques, we show that the muscle isoform utrophin-A is predominantly suppressed at the translational level in C2C12 myoblasts. The extent of translational inhibition is estimated to be ~99% in C2C12 cells and is mediated by both the 5'- and 3'-UTRs of the utrophin-A mRNA. In this study we identify five miRNAs (let-7c, miR-150, miR-196b, miR-296-5p, miR-133b) that mediate the repression, and confirm repression by the previously identified miR-206. We demonstrate that this translational repression can be overcome by blocking the actions of miRNAs, resulting in an increased level of utrophin protein in C2C12 cells.
The present study has identified key inhibitory mechanisms featuring miRNAs that regulate utrophin expression, and demonstrated that these mechanisms can be targeted to increase endogenous utrophin expression in cultured muscle cells. We suggest that miRNA-mediated inhibitory mechanisms could be targeted by methods similar to those described here as a novel strategy to increase utrophin expression as a therapy for DMD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally ...compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use.
We developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells.
We have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A method of choosing swarm optimization algorithms and using swarm intelligence for solving a certain class of optimization tasks in industry-specific geographic information systems was developed ...considering the stationarity characteristic of such systems. The method consists of 8 stages. Classes of swarm algorithms were studied. It is shown which classes of swarm algorithms should be used depending on the stationarity, quasi-stationarity or dynamics of the task solved by an industry geographic information system. An information model of geodata that consists in a formalized combination of their spatial and attributive components, which allows considering the relational, semantic and frame models of knowledge representation of the attributive component, was developed. A method of choosing optimization methods designed to work as part of a decision support system within an industry-specific geographic information system was developed. It includes conceptual information modeling, optimization criteria selection, and objective function analysis and modeling. This method allows choosing the most suitable swarm optimization method (or a set of methods).
The mathematical model of grammatically augmented ontology was intro-duced to address this issue. This model was used for grammatical analysis of Ukrainian sentences. Domain specific language named ...GAODL for description of grammatically augmented ontology was developed. The grammar of the language was defined by means of Xtext extension for Eclipse. The developed language was used as an auxiliary part of the infor-mation technology for bidirectional Ukrainian sign language translation.
Variants in
, which encodes pre-mRNA processing factor 31 homolog, are known to cause autosomal-dominant retinitis pigmentosa (adRP) with incomplete penetrance. However, the majority of mutations ...cause null alleles, with only two proven pathogenic missense mutations. We identified a novel missense mutation in
in a family with adRP.
We performed whole exome sequencing to identify possible pathogenic mutations in the proband of a family with adRP. Available affected family members had a full ophthalmological evaluation including kinetic and two-colour dark adapted static perimetry, electroretinography and multimodal imaging of the retina. Two patients had evaluations covering nearly 20 years. We carried out segregation analysis of the probable mutation,
c.590T>C. We evaluated the cellular localisation of the PRPF31 variant (p.Leu197Pro) compared with the wildtype PRPF31 protein.
c.590T>C segregated with the disease in this four-generation autosomal dominant pedigree. There was intrafamilial variability in disease severity. Nyctalopia and mid-peripheral scotomas presented from the second to the fourth decade of life. There was severe rod >cone dysfunction. Visual acuity (VA) was relatively intact and was maintained until later in life, although with marked interocular asymmetries. Laboratory studies showed that the mutant PRPF31 protein (p.Leu197Pro) does not localise to the nucleus, unlike the wildtype PRPF31 protein. Instead, mutant protein resulted in punctate localisation to the cytoplasm.
c.590T>C is a novel pathogenic variant in
causing adRP with incomplete penetrance. Disease may be due to protein misfolding and associated abnormal protein trafficking to the nucleus.
Accurate clinical diagnosis and prognosis of retinal degeneration can be aided by the identification of the disease-causing genetic variant. It can confirm the clinical diagnosis as well as inform ...the clinician of the risk for potential involvement of other organs such as kidneys. It also aids in genetic counseling for affected individuals who want to have a child. Finally, knowledge of disease-causing variants informs laboratory investigators involved in translational research. With the advent of next-generation sequencing, identifying pathogenic mutations is becoming easier, especially the identification of novel pathogenic variants.
We used whole exome sequencing on a cohort of 69 patients with various forms of retinal degeneration and in whom screens for previously identified disease-causing variants had been inconclusive. All potential pathogenic variants were verified by Sanger sequencing and, when possible, segregation analysis of immediate relatives. Potential variants were identified by using a semi-masked approach in which rare variants in candidate genes were identified without knowledge of the clinical diagnosis (beyond "retinal degeneration") or inheritance pattern. After the initial list of genes was prioritized, genetic diagnosis and inheritance pattern were taken into account.
We identified the likely pathogenic variants in 64% of the subjects. Seven percent had a single heterozygous mutation identified that would cause recessive disease and 13% had no obviously pathogenic variants and no family members available to perform segregation analysis. Eleven subjects are good candidates for novel gene discovery. Two de novo mutations were identified that resulted in dominant retinal degeneration.
Whole exome sequencing allows for thorough genetic analysis of candidate genes as well as novel gene discovery. It allows for an unbiased analysis of genetic variants to reduce the chance that the pathogenic mutation will be missed due to incomplete or inaccurate family history or analysis at the early stage of a syndromic form of retinal degeneration.
This article discusses the application of effective methodologies and strategies for planning, monitoring and optimizing budgets and costs in IT projects. Tasks related to achieving this goal include ...identifying key factors that contribute to successful budget and cost control, studying existing tools and techniques, and proposing innovative solutions that can be implemented by startups in the IT sector. Therefore, effective budgeting and cost control are becoming increasingly important for the successful implementation of IT projects. Such well-known budgeting methods as incremental budgeting, activity-based budgeting, value proposition-based budgeting, and zero budgeting are considered. The use of the analytic hierarchy process for decision-making on effective budget management in IT projects is described.
The information system for translation into sign language on mobile devices is discussed. The system comprises of dictionaries, ontology database, weighted affix context-free grammar (WACFG) parser, ...algorithm for transformation of constituency tree into dependency tree, and an algorithm for synthesis of Ukrainian sign language glosses. Simple video sequencing is utilized for sign language synthesis, however any other sign animation tool can be used. Tasks that require further research are defined.
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