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Artigo em Inglês | MEDLINE | ID: mdl-35529769


Feature sizes in integrated circuits have decreased substantially over time, and it has become increasingly difficult to three-dimensionally image these complex circuits after fabrication. This can be important for process development, defect analysis, and detection of unexpected structures in externally sourced chips, among other applications. Here, we report on a non-destructive, tabletop approach that addresses this imaging problem through x-ray tomography, which we uniquely realize with an instrument that combines a scanning electron microscope (SEM) with a transition-edge sensor (TES) x-ray spectrometer. Our approach uses the highly focused SEM electron beam to generate a small x-ray generation region in a carefully designed target layer that is placed over the sample being tested. With the high collection efficiency and resolving power of a TES spectrometer, we can isolate x-rays generated in the target from background and trace their paths through regions of interest in the sample layers, providing information about the various materials along the x-ray paths through their attenuation functions. We have recently demonstrated our approach using a 240 Mo/Cu bilayer TES prototype instrument on a simplified test sample containing features with sizes of ∼ 1 µm. Currently, we are designing and building a 3000 Mo/Au bilayer TES spectrometer upgrade, which is expected to improve the imaging speed by factor of up to 60 through a combination of increased detector number and detector speed.

J Inherit Metab Dis ; 43(2): 297-308, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31339582


Transport And Golgi Organization protein 2 (TANGO2) deficiency has recently been identified as a rare metabolic disorder with a distinct clinical and biochemical phenotype of recurrent metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis, arrhythmias, and encephalopathy with cognitive decline. We report nine subjects from seven independent families, and we studied muscle histology, respiratory chain enzyme activities in skeletal muscle and proteomic signature of fibroblasts. All nine subjects carried autosomal recessive TANGO2 mutations. Two carried the reported deletion of exons 3 to 9, one homozygous, one heterozygous with a 22q11.21 microdeletion inherited in trans. The other subjects carried three novel homozygous (c.262C>T/p.Arg88*; c.220A>C/p.Thr74Pro; c.380+1G>A), and two further novel heterozygous (c.6_9del/p.Phe6del); c.11-13delTCT/p.Phe5del mutations. Immunoblot analysis detected a significant decrease of TANGO2 protein. Muscle histology showed mild variation of fiber diameter, no ragged-red/cytochrome c oxidase-negative fibers and a defect of multiple respiratory chain enzymes and coenzyme Q10 (CoQ10 ) in two cases, suggesting a possible secondary defect of oxidative phosphorylation. Proteomic analysis in fibroblasts revealed significant changes in components of the mitochondrial fatty acid oxidation, plasma membrane, endoplasmic reticulum-Golgi network and secretory pathways. Clinical presentation of TANGO2 mutations is homogeneous and clinically recognizable. The hemizygous mutations in two patients suggest that some mutations leading to allele loss are difficult to detect. A combined defect of the respiratory chain enzymes and CoQ10 with altered levels of several membrane proteins provides molecular insights into the underlying pathophysiology and may guide rational new therapeutic interventions.

Encefalopatias Metabólicas/genética , Doenças Mitocondriais/genética , Debilidade Muscular/genética , Mutação , Proteômica/métodos , Rabdomiólise/genética , Encefalopatias Metabólicas/diagnóstico , Ácidos Graxos/metabolismo , Feminino , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Homozigoto , Humanos , Lactente , Masculino , Doenças Mitocondriais/diagnóstico , Fosforilação Oxidativa , Fenótipo , Rabdomiólise/diagnóstico , Sequenciamento Completo do Genoma
Neuromuscul Disord ; 27(9): 861-872, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28624464


Dominant mutations in STIM1 are a cause of three allelic conditions: tubular aggregate myopathy, Stormorken syndrome (a complex phenotype including myopathy, hyposplenism, hypocalcaemia and bleeding diathesis), and a platelet dysfunction disorder, York platelet syndrome. Previous reports have suggested a genotype-phenotype correlation with mutations in the N-terminal EF-hand domain associated with tubular aggregate myopathy, and a common mutation at p.R304W in a coiled coil domain associated with Stormorken syndrome. In this study individuals with STIM1 variants were identified by exome sequencing or STIM1 direct sequencing, and assessed for neuromuscular, haematological and biochemical evidence of the allelic disorders of STIM1. STIM1 mutations were investigated by fibroblast calcium imaging and 3D modelling. Six individuals with STIM1 mutations, including two novel mutations (c.262A>G (p.S88G) and c.911G>A (p.R304Q)), were identified. Extra-neuromuscular symptoms including thrombocytopenia, platelet dysfunction, hypocalcaemia or hyposplenism were present in 5/6 patients with mutations in both the EF-hand and CC domains. 3/6 patients had psychiatric disorders, not previously reported in STIM1 disease. Review of published STIM1 patients (n = 49) confirmed that neuromuscular symptoms are present in most patients. We conclude that the phenotype associated with activating STIM1 mutations frequently includes extra-neuromuscular features such as hypocalcaemia, hypo-/asplenia and platelet dysfunction regardless of mutation domain.

Transtornos Plaquetários/genética , Dislexia/genética , Estudos de Associação Genética , Ictiose/genética , Transtornos de Enxaqueca/genética , Miose/genética , Mutação/genética , Miopatias Congênitas Estruturais/genética , Proteínas de Neoplasias/genética , Baço/anormalidades , Molécula 1 de Interação Estromal/genética , Adulto , Cálcio/metabolismo , Técnicas de Cultura de Células , Análise Mutacional de DNA , Eritrócitos Anormais , Saúde da Família , Feminino , Fibroblastos/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Modelos Moleculares , Fadiga Muscular/genética , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , NAD/metabolismo