Telethonin protein expression in neuromuscular disorders

Telethonin is a 19-kDa sarcomeric protein, localized to the Z-disc of skeletal and cardiac muscles. Mutations in the telethonin gene cause limb-girdle muscular dystrophy type 2G (LGMD2G). We investigated the sarcomeric integrity of muscle fibers in LGMD2G patients, through double immunofluorescence analysis for telethonin with three sarcomeric proteins: titin, alpha-actinin-2, and myotilin and observed the typical cross striation pattern, suggesting that the Z-line of the sarcomere is apparently preserved, despite the absence of telethonin. Ultrastructural analysis confirmed the integrity of the sarcomeric architecture. the possible interaction of telethonin with other proteins responsible for several forms of neuromuscular disorders was also analyzed. Telethonin was clearly present in the rods in nemaline myopathy (NM) muscle fibers, confirming its localization to the Z-line of the sarcomere. Muscle from patients with absent telethonin showed normal expression for the proteins dystrophin, sarcoglycans, dysferlin, and calpain-3. Additionally, telethonin showed normal localization in muscle biopsies from patients with LGMD2A, LGMD2B, sarcoglycanopathies, and Duchenne muscular dystrophy (DMD). Therefore, the primary deficiency of calpain-3, dysferlin, sarcoglycans, and dystrophin do not seem to alter telethonin expression. (C) 2002 Elsevier Science B.V. All rights reserved.Univ São Paulo, Ctr Study Human Genome, Dept Biol, IBUSP, BR-05508900 São Paulo, BrazilInt Ctr Genet Engn & Biotechnol, Tieste, ItalyUniv Padua, CRIBI Biotechnol Ctr, I-35121 Padua, ItalyHarvard Univ, Childrens Hosp, Sch Med, Div Genet, Boston, MA 02115 USAUniv Helsinki, Helsinki, FinlandUNIFESP, Dept Neurol, São Paulo, BrazilFMUSP, Dept Neurol, São Paulo, BrazilFMUSP, Dept Pathol, São Paulo, BrazilUNIFESP, Dept Neurol, São Paulo, BrazilWeb of Scienc

Functional study of the type XVIII collagen

A síndrome de Knobloch (SK) é uma doença autossômica recessiva rara, caracterizada por problemas oculares e presença de encefalocele occipital, porém o quadro clínico é variável. Os pacientes apresentam principalmente miopia de grau elevado, degeneração vítreo-retiniana e descolamento de retina; o grau de comprometimento da alteração no occipital também é variável. Nossos estudos mostraram que a SK é causada por mutações no gene COL18A1, que codifica o colágeno tipo XVIII. Esse colágeno, uma proteoglicana da matriz extracelular, tem sido estudado principalmente por liberar a endostatina, um fragmento de 20 kDa clivado proteoliticamente de sua porção C-terminal e que possui atividade inibidora da angiogênese. O colágeno XVIII possui três isoformas conhecidas, as quais diferem entre si apenas na porção N-terminal e apresentam padrões de expressão distintos nos tecidos, mesmo estando ubiquamente presentes nas membranas basais epiteliais e endoteliais. Além da endostatina, o colágeno XVIII apresenta outros motivos com funções ainda desconhecidas: um domínio trombospondina, presente em todas as isoformas e um domínio frizzled, encontrado apenas na forma mais longa da proteína. O espectro de variação clinica na SK ainda é incerto, assim como os mecanismos moleculares que levam ao fenótipo. Nosso trabalho teve como objetivos principais a identificação de mutações no COL18A1 em um número maior de famílias com a SK, estabelecimento de novos protocolos que possam auxiliar no diagnóstico clínico e a avaliação do efeito funcional de variações encontradas na endostatina, domínio de maior conservação do colágeno XVIII. Propusemo-nos ainda a identificar proteínas que interagem com o domínio trombospondina. Apresentamos aqui a caracterização de sete novas mutações no colágeno XVIII em pacientes com SK, permitindo assim uma melhor determinação do espectro de variação fenotípica da SK. Com base na identificação dessas mutações pudemos incluir problemas neurológicos nos possíveis sinais clínicos presentes na SK ao apresentar pela primeira vez alterações de migração neuronal em pacientes com a síndrome. A ausência de mutações detectadas em três famílias sugere ainda a existência de heterogeneidade genética na SK. Também propomos neste trabalho a utilização da imunohistoquímica em biópsias de pele como teste diagnóstico para essa doença. Nossos resultados mostram também que a variação A48T da endostatina leva a alterações em sua interação com proteínas da matriz extracelular, enquanto a variação polimórfica D104N, previamente associada ao desenvolvimento de câncer de próstata, não leva a um efeito sobre a interação com as proteínas testadas. E, por último, o método de duplo híbrido não foi eficaz para a identificação de proteínas que possam interagir com o domínio trombospondina do colágeno XVIII.Knobloch syndrome (KS) is an autosomal recessive disorder characterized by ophthalmological defects and presence of an occipital encephalocele. Clinical variability is present, however, all patients present high grade myopia, vitreoretinal degeneration and in most cases, retinal detachment; the occipital defect is also variable. Studies show that the KS is caused by mutations in COL18A1, the gene that codes for type XVIII collagen. This collagen is an extracellular matrix proteoglycan and has been the focus of a great number of studies due to its C-terminal domain, endostatin. Endostatin is a 20 kDa fragment that is proteolytically cleaved and possesses a high antiangiogenic activity. Type XVIII collagen is known to be expressed in three isoforms, different among themselves in the N-terminal region. These isoforms have distinct expression patterns, but are present in most basement membranes. Besides endostatin, type XVIII collagen also presents other domains with unknown functions: a thrombospondin domain, found in all isoforms; a frizzled domain, present in the longest isoform. The clinical variability spectrum in KS and the molecular mechanisms that lead to the phenotype are still uncertain. The aim of this study was to identify novel mutations in COL18A1 in additional KS families, to develop biochemical diagnostic tests that could allow the screening of a larger number of patients and to evaluate the effect of naturally found variants in the function of endostatin. We also performed a two-hybrid screening in order to identify proteins that can interact with the thrombospondin domain. The characterization of seven novel mutations in KS patients allowed us to better determine the clinical variability of KS. This work shows for the first time the presence of neuronal migration defects in some KS patients. The lack of detected pathogenic mutations in three families led us to propose the genetic heterogeneity of this syndrome. We demonstrate the possibility to use immunohistochemistry in skin biopsies as a diagnosis method. Our results also show the altered properties of T48 endostatin in its interaction with some extracellular matrix proteins. The N104 variant, that has been previously associated with prostate cancer, do not present any change in its interaction to the tested molecules. Finally, the two-hybrid system was not a good method to detect interacting proteins with the thrombospodin domain of collagen XVIII

Identifying genes that mediate anthracyline toxicity in immune cells

The role of the immune system in response to chemotherapeutic agents remains elusive. The interpatient variability observed in immune and chemotherapeutic cytotoxic responses is likely, at least in part, due to complex genetic differences. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at identifying genes underlying these chemotherapeutic cytotoxic effects on immune cells. Using genome-wide association studies (GWAS), we identified four genome-wide significant quantitative trait loci (QTL) that contributed to the sensitivity of doxorubicin and idarubicin in immune cells. Of particular interest, a locus on chromosome 16 was significantly associated with cell viability following idarubicin administration (p = 5.01x10-8). Within this QTL lies App, which encodes amyloid beta precursor protein. Comparison of dose-response curves verified that T-cells in App knockout mice were more sensitive to idarubicin than those of C57BL/6J control mice (p < 0.05).In conclusion, the cellular screening approach coupled with GWAS led to the identification and subsequent validation of a gene involved in T-cell viability after idarubicin treatment. Previous studies have suggested a role for App in in vitro and in vivo cytotoxicity to anticancer agents; the overexpression of App enhances resistance, while the knockdown of this gene is deleterious to cell viability. Thus, further investigations should include performing mechanistic studies, validating additional genes from the GWAS, including Ppfia1 and Ppfibp1, and ultimately translating the findings to in vivo and human studies

Data quality monitors of vertex detectors at the start of the Belle II experiment

The Belle II experiment features a substantial upgrade of the Belle detector and will operate at the SuperKEKB energy-asymmetric e+e− collider at KEK in Tsukuba, Japan. The accelerator completed its first phase of commissioning in 2016, and the Belle II detector saw its first electron-positron collisions in April 2018. Belle II features a newly designed silicon vertex detector based on double-sided strip layers and DEPFET pixel layers. A subset of the vertex detector was operated in 2018 to determine background conditions (Phase 2 operation). The collaboration completed full detector installation in January 2019, and the experiment started full data taking. This paper will report on the final arrangement of the silicon vertex detector part of Belle II with a focus on online monitoring of detector conditions and data quality, on the design and use of diagnostic and reference plots, and on integration with the software framework of Belle II. Data quality monitoring plots will be discussed with a focus on simulation and acquired cosmic and collision data

Alignment for the first precision measurements at Belle II

International audienceOn March 25th 2019, the Belle II detector recorded the first collisions delivered by the SuperKEKB accelerator. This marked the beginning of the physics run with vertex detector.The vertex detector was aligned initially with cosmic ray tracks without magnetic field simultaneously with the drift chamber. The alignment method is based on Millepede II and the General Broken Lines track model and includes also the muon system or primary vertex position alignment. To control weak modes, we employ sensitive validation tools and various track samples can be used as alignment input, from straight cosmic tracks to mass-constrained decays.With increasing luminosity and experience, the alignment is approaching the target performance, crucial for the first physics analyses in the era of Super-BFactories. We will present the software framework for the detector calibration and alignment, the results from the first physics run and the prospects in view of the experience with the first data

Alignment for the first precision measurements at Belle II

On March 25th 2019, the Belle II detector recorded the first collisions delivered by the SuperKEKB accelerator. This marked the beginning of the physics run with vertex detector. The vertex detector was aligned initially with cosmic ray tracks without magnetic field simultaneously with the drift chamber. The alignment method is based on Millepede II and the General Broken Lines track model and includes also the muon system or primary vertex position alignment. To control weak modes, we employ sensitive validation tools and various track samples can be used as alignment input, from straight cosmic tracks to mass-constrained decays. With increasing luminosity and experience, the alignment is approaching the target performance, crucial for the first physics analyses in the era of Super-BFactories. We will present the software framework for the detector calibration and alignment, the results from the first physics run and the prospects in view of the experience with the first data

Data quality monitors of vertex detectors at the start of the Belle II experiment

The Belle II experiment features a substantial upgrade of the Belle detector and will operate at the SuperKEKB energy-asymmetric e+e− collider at KEK in Tsukuba, Japan. The accelerator completed its first phase of commissioning in 2016, and the Belle II detector saw its first electron-positron collisions in April 2018. Belle II features a newly designed silicon vertex detector based on double-sided strip layers and DEPFET pixel layers. A subset of the vertex detector was operated in 2018 to determine background conditions (Phase 2 operation). The collaboration completed full detector installation in January 2019, and the experiment started full data taking. This paper will report on the final arrangement of the silicon vertex detector part of Belle II with a focus on online monitoring of detector conditions and data quality, on the design and use of diagnostic and reference plots, and on integration with the software framework of Belle II. Data quality monitoring plots will be discussed with a focus on simulation and acquired cosmic and collision data