RESUMEN

Widespread indications for use of molecular diagnostics in various aspects of clinical medicine have driven proliferation of testing. The rapid adoption and continuous technological evolution of molecular diagnostics have often strained the development and maintenance of a functional underlying framework of coding, coverage, and reimbursement policies, thereby presenting challenges to various stakeholders, including molecular professionals, payers, and patients. A multidisciplinary working group convened by the Association for Molecular Pathology Economic Affairs Committee was tasked to describe the complex landscape of molecular pathology economics and highlight opportunities for member engagement. In this article, on the basis of review and synthesis of government regulations and procedures, published payer policy documents, peer-reviewed literature, and expert consensus, the Working Group navigates the ecosystem of molecular pathology economics in terms of stakeholders, coding systems and processes, coverage policy determination, and pricing mechanisms. The composition and interrelatedness of various working groups and committees are emphasized to highlight the functional underpinnings of the system. Molecular professionals must be conversant in the language and complex inner workings of molecular pathology economics to lead successful, viable laboratories and advocate effectively for policy development on their behalf. This overview is provided to be a resource to molecular professionals as they navigate the reimbursement landscape.

Asunto(s)

Cobertura del Seguro/economía , Reembolso de Seguro de Salud/economía , Técnicas de Diagnóstico Molecular/economía , Costos y Análisis de Costo/métodos , Regulación Gubernamental , Humanos , Laboratorios de Hospital/economía , Medicare/economía , Participación de los Interesados , Estados Unidos

RESUMEN

The increasing use of advanced nucleic acid sequencing technologies for clinical diagnostics and therapeutics has made vital understanding the costs of performing these procedures and their value to patients, providers, and payers. The Association for Molecular Pathology invested in a cost and value analysis of specific genomic sequencing procedures (GSPs) newly coded by the American Medical Association Current Procedural Terminology Editorial Panel. Cost data and work effort, including the development and use of data analysis pipelines, were gathered from representative laboratories currently performing these GSPs. Results were aggregated to generate representative cost ranges given the complexity and variability of performing the tests. Cost-impact models for three clinical scenarios were generated with assistance from key opinion leaders: impact of using a targeted gene panel in optimizing care for patients with advanced non-small-cell lung cancer, use of a targeted gene panel in the diagnosis and management of patients with sensorineural hearing loss, and exome sequencing in the diagnosis and management of children with neurodevelopmental disorders of unknown genetic etiology. Each model demonstrated value by either reducing health care costs or identifying appropriate care pathways. The templates generated will aid laboratories in assessing their individual costs, considering the value structure in their own patient populations, and contributing their data to the ongoing dialogue regarding the impact of GSPs on improving patient care.

Asunto(s)

Genómica/economía , Genómica/métodos , Análisis de Secuencia de ADN/economía , Análisis de Secuencia de ADN/métodos , Biomarcadores , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Costos y Análisis de Costo , Exoma , Costos de la Atención en Salud , Pérdida Auditiva Sensorineural/diagnóstico , Pérdida Auditiva Sensorineural/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Modelos Económicos , Trastornos del Neurodesarrollo/diagnóstico , Trastornos del Neurodesarrollo/genética

RESUMEN

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these 'proto-chromatosomes' are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.

Asunto(s)

ADN/metabolismo , Histonas/metabolismo , Nucleosomas/química , Animales , ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Femenino , Expresión Génica , Histonas/deficiencia , Histonas/genética , Hígado/metabolismo , Ratones , Nucleasa Microcócica/química , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo

RESUMEN

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion within the N-terminal region of the ataxin-7 protein, a known subunit of the SAGA complex. Although the mechanisms of SCA7 pathogenesis remain poorly understood, previous studies have shown perturbations in SAGA histone acetyltransferase function and transcriptional alterations. We sought to determine whether and how polyQ-expanded ataxin-7 affects SAGA catalytic activity. Here, we determined that polyQ-expanded ataxin-7 directly bound the Gcn5 catalytic core of SAGA while in association with its regulatory proteins, Ada2 and Ada3. This caused a significant decrease in Gcn5 histone acetyltransferase activity in vitro and in vivo at two SAGA-regulated galactose genes, GAL1 and GAL7. However, Gcn5 occupancy at the GAL1 and GAL7 promoters was increased in these cells, revealing a dominant-negative phenotype of the polyQ-expanded ataxin-7-incorporated, catalytically inactive SAGA. These findings suggest a dominant mechanism of polyQ-mediated SAGA inhibition that potentially contributes to SCA7 disease pathogenesis.

Asunto(s)

Proteínas del Tejido Nervioso/química , Péptidos/química , Factores de Transcripción p300-CBP/química , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ataxina-7 , Proteínas de Unión al ADN , Galectinas/química , Galectinas/genética , Galectinas/metabolismo , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Péptidos/genética , Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismo

RESUMEN

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

Asunto(s)

Acetiltransferasas/química , Acetiltransferasas/metabolismo , Regulación de la Expresión Génica , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Acetilación , Acetiltransferasas/genética , Secuencia de Aminoácidos , Western Blotting , Inmunoprecipitación de Cromatina , Histona Acetiltransferasas/genética , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Transactivadores/genética

RESUMEN

Using robotic automation, a microarray printing protocol for whole bacterial cells was developed for subsequent label-free and nondestructive infrared microspectroscopic detection. Using this contact microspotting system, 24 microorganisms were printed on zinc selenide slides; these were 6 species of Listeria, 10 species of Vibrio, 2 strains of Photobacterium damselae, Yersinia enterocolitica 289, Bacillus cereus ATCC 14529, Staphylococcus aureus, ATCC 19075 (serotype 104 B), Shigella sonnei 20143, Klebsiella pneumoniae KP73, Enterobacter cloacae, Citrobacter freundii 200, and Escherichia coli. Microarrays consisting of separate spots of bacterial deposits gave consistent and reproducible infrared spectra, which were differentiated by unsupervised pattern recognition algorithms. Two multivariate analysis algorithms, principal component analysis and hierarchical cluster analysis, successfully separated most, but not all, the bacteria investigated down to the species level.

Asunto(s)

Bacterias/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Contaminación de Alimentos/análisis , Análisis por Micromatrices/métodos , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Bacterias/clasificación , Bacterias/patogenicidad , Análisis por Conglomerados , Microbiología de Alimentos , Enfermedades Transmitidas por los Alimentos/diagnóstico , Enfermedades Transmitidas por los Alimentos/prevención & control , Microquímica , Análisis Multivariante , Análisis de Componente Principal , Reproducibilidad de los Resultados , Compuestos de Selenio/química , Especificidad de la Especie , Compuestos de Zinc/química

RESUMEN

Activation of the p75 neurotrophin receptor leads to a variety of effects within the nervous system, including neuronal apoptosis. Both c-Jun N-terminal kinase (JNK) and the tumor suppressor p53 have been reported to be critical for this receptor to induce cell death; however, the mechanisms by which p75 activates these pathways is undetermined. Here we report that the neurotrophin receptor interacting factor (NRIF) is necessary for p75-dependent JNK activation and apoptosis. Upon nerve growth factor withdrawal, nrif-/- sympathetic neurons underwent apoptosis, whereas p75-mediated death was completely abrogated. The lack of cell death correlated with a lack of JNK activation in the nrif-/- neurons, suggesting that NRIF is a selective mediator for p75-dependent JNK activation and apoptosis. Moreover, we document that NRIF expression is sufficient to induce cell death through a mechanism that requires p53. Taken together, these results establish NRIF as an essential component of the p75 apoptotic pathway.

Asunto(s)

Apoptosis/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal/fisiología , Fibras Adrenérgicas/metabolismo , Animales , Caspasas/metabolismo , Células Cultivadas , Proteínas de Unión al ADN , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Noqueados , Factor de Crecimiento Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Ratas , Receptor de Factor de Crecimiento Nervioso , Receptores de Factor de Crecimiento Nervioso/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

Neurotrophin signaling through the p75 receptor regulates apoptosis within the nervous system both during development and in response to injury. Whereas a number of p75 interacting factors have been identified, how these upstream factors function in a coordinated manner to mediate receptor signaling is still unclear. Here, we report a functional interaction between TRAF6 and the neurotrophin receptor interacting factor (NRIF), two proteins known to associate with the intracellular domain of the p75 neurotrophin receptor. The association between NRIF and TRAF6 was direct and occurred with both endogenous and ectopically expressed proteins. A KRAB repressor domain of NRIF and the carboxyl-terminal, receptor-binding region of TRAF6 were required for the interaction. Co-expression of TRAF6 increased the levels of NRIF protein and induced its nuclear translocation. Reciprocally, NRIF enhanced TRAF6-mediated activation of the c-Jun NH2-terminal kinase (JNK) by 3-fold, while only modestly increasing the stimulation of NF-kappaB. The expression of both NRIF and TRAF6 was required for reconstituting p75 activation of JNK in HEK293 cells, whereas NRIF mutants lacking the TRAF6 interaction domain were unable to substitute for the full-length protein in facilitating activation of the kinase. These results suggest that NRIF and TRAF6 functionally interact to facilitate neurotrophin signaling through the p75 receptor.

Asunto(s)

Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal , Animales , Línea Celular , Proteínas de Unión al ADN , Humanos , Unión Proteica , Transporte de Proteínas , Receptor de Factor de Crecimiento Nervioso , Factor 6 Asociado a Receptor de TNF