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1.
Clin Chem Lab Med ; 62(2): 322-331, 2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-37702323

RESUMO

OBJECTIVES: Neurofilament light chain (NfL) has emerged as a promising biomarker for detecting and monitoring axonal injury. Until recently, NfL could only be reliably measured in cerebrospinal fluid, but digital single molecule array (Simoa) technology has enabled its precise measurement in blood samples where it is typically 50-100 times less abundant. We report development and multi-center validation of a novel fully automated digital immunoassay for NfL in serum for informing axonal injury status. METHODS: A 45-min immunoassay for serum NfL was developed for use on an automated digital analyzer based on Simoa technology. The analytical performance (sensitivity, precision, reproducibility, linearity, sample type) was characterized and then cross validated across 17 laboratories in 10 countries. Analytical performance for clinical NfL measurement was examined in individual patients with relapsing remitting multiple sclerosis (RRMS) after 3 months of disease modifying treatment (DMT) with fingolimod. RESULTS: The assay exhibited a lower limit of detection (LLoD) of 0.05 ng/L, a lower limit of quantification (LLoQ) of 0.8 ng/L, and between-laboratory imprecision <10 % across 17 validation sites. All tested samples had measurable NfL concentrations well above the LLoQ. In matched pre-post treatment samples, decreases in NfL were observed in 26/29 RRMS patients three months after DMT start, with significant decreases detected in a majority of patients. CONCLUSIONS: The sensitivity characteristics and reproducible performance across laboratories combined with full automation make this assay suitable for clinical use for NfL assessment, monitoring in individual patients, and cross-comparisons of results across multiple sites.


Assuntos
Filamentos Intermediários , Neurônios , Humanos , Reprodutibilidade dos Testes , Imunoensaio , Proteínas de Neurofilamentos , Biomarcadores , Testes Hematológicos
2.
PLoS Comput Biol ; 11(4): e1004161, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25894653

RESUMO

The search for genes that regulate stem cell self-renewal and differentiation has been hindered by a paucity of markers that uniquely label stem cells and early progenitors. To circumvent this difficulty we have developed a method that identifies cell-state regulators without requiring any markers of differentiation, termed Perturbation-Expression Analysis of Cell States (PEACS). We have applied this marker-free approach to screen for transcription factors that regulate mammary stem cell differentiation in a 3D model of tissue morphogenesis and identified RUNX1 as a stem cell regulator. Inhibition of RUNX1 expanded bipotent stem cells and blocked their differentiation into ductal and lobular tissue rudiments. Reactivation of RUNX1 allowed exit from the bipotent state and subsequent differentiation and mammary morphogenesis. Collectively, our findings show that RUNX1 is required for mammary stem cells to exit a bipotent state, and provide a new method for discovering cell-state regulators when markers are not available.


Assuntos
Diferenciação Celular/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Glândulas Mamárias Humanas/citologia , Células-Tronco/citologia , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Perfilação da Expressão Gênica , Humanos , Organoides/citologia , Organoides/metabolismo , Biologia de Sistemas
3.
ACS Chem Biol ; 2024 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-39418577

RESUMO

Mutations in creatine transporter SLC6A8 cause creatine transporter deficiency (CTD), which is responsible for 2% of all cases of X-linked intellectual disability. CTD has no current treatments and has a high unmet medical need. Inspired by the transformational therapeutic impact of small molecule "correctors" for the treatment of cystic fibrosis, which bind to mutated versions of the CFTR ion channel to promote its trafficking to the cell surface, we sought to identify small molecules that could stabilize SLC6A8 as a potential treatment for CTD. We leveraged a novel chemoproteomic technology for ligand discovery, reactive affinity probe interaction discovery, to identify small-molecule fragments with photoaffinity handles that bind to SLC6A8 in a cellular environment. We synthesized a library of irreversible covalent analogs of these molecules to characterize in functional assays, which revealed molecules that could promote the trafficking of mutant SLC6A8 variants to the cell surface. Further medicinal chemistry was able to identify reversible drug-like small molecules that both promoted trafficking of the transporter and also rescued creatine uptake. When profiled across the 27 most prevalent SLC6A8 missense variants, we found that 10-20% of patient mutations were amenable to correction by our molecules. These results were verified in an endogenous setting using the CRISPR knock-in of selected missense alleles. We established in vivo proof-of-mechanism for correctors in a novel CTD mouse model with the P544L patient-defined variant knocked in to the SLC6A8 locus, where treatment with our orally bioavailable and brain penetrant tool corrector increased brain creatine levels in heterozygous female mice, validating correctors as a potential therapeutic approach for CTD.

4.
Nat Commun ; 12(1): 7116, 2021 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-34893587

RESUMO

Mammary morphogenesis is an orchestrated process involving differentiation, proliferation and organization of cells to form a bi-layered epithelial network of ducts and lobules embedded in stromal tissue. We have engineered a 3D biomimetic human breast that makes it possible to study how stem cell fate decisions translate to tissue-level structure and function. Using this advancement, we describe the mechanism by which breast epithelial cells build a complex three-dimensional, multi-lineage tissue by signaling through a collagen receptor. Discoidin domain receptor tyrosine kinase 1 induces stem cells to differentiate into basal cells, which in turn stimulate luminal progenitor cells via Notch signaling to differentiate and form lobules. These findings demonstrate how human breast tissue regeneration is triggered by transmission of signals from the extracellular matrix through an epithelial bilayer to coordinate structural changes that lead to formation of a complex ductal-lobular network.


Assuntos
Mama/citologia , Mama/fisiologia , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Receptor com Domínio Discoidina 1/metabolismo , Materiais Biocompatíveis , Engenharia Biomédica , Linhagem Celular , Receptor com Domínio Discoidina 1/genética , Células Epiteliais/citologia , Matriz Extracelular , Humanos , Regeneração , Transdução de Sinais , Células-Tronco/citologia
5.
Open Biol ; 7(2)2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28202626

RESUMO

Phenotypic heterogeneity in cancers is associated with invasive progression and drug resistance. This heterogeneity arises in part from the ability of cancer cells to switch between phenotypic states, but the dynamics of this cellular plasticity remain poorly understood. Here we apply DNA barcodes to quantify and track phenotypic plasticity across hundreds of clones in a population of cancer cells exhibiting epithelial or mesenchymal differentiation phenotypes. We find that the epithelial-to-mesenchymal cell ratio is highly variable across the different clones in cancer cell populations, but remains stable for many generations within the progeny of any single clone-with a heritability of 0.89. To estimate the effects of combination therapies on phenotypically heterogeneous tumours, we generated quantitative simulations incorporating empirical data from our barcoding experiments. These analyses indicated that combination therapies which alternate between epithelial- and mesenchymal-specific treatments eventually select for clones with increased phenotypic plasticity. However, this selection could be minimized by increasing the frequency of alternation between treatments, identifying designs that may minimize selection for increased phenotypic plasticity. These findings establish new insights into phenotypic plasticity in cancer, and suggest design principles for optimizing the effectiveness of combination therapies for phenotypically heterogeneous tumours.


Assuntos
Antineoplásicos/farmacologia , DNA de Neoplasias/genética , Transição Epitelial-Mesenquimal , Neoplasias/genética , Linhagem Celular Tumoral , Plasticidade Celular , Código de Barras de DNA Taxonômico , Farmacorresistência Bacteriana , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células HEK293 , Humanos , Invasividade Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fenótipo
6.
South Med J ; 98(12): 1169-72, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16440916

RESUMO

OBJECTIVES: Previous studies evaluating the use of CT in the diagnosis of appendicitis have taken place at university-based institutions where surgical bedside consultation seems prudent before radiological study. In the private hospital setting, the emergency department (ED) physician is responsible for diagnosis. We attempt to assess if this process is detrimental to patient care. METHODS: Retrospective review of 150 patient's records admitted through the ED was performed with the discharge diagnosis of appendicitis between March 1998 and May 2000. Data was stratified for analysis based on age (< 15, 15-50, > 50) and gender. Using Graph Pad Prism software the groups were compared for complications based on whether or not CT was obtained. Chi-square, number needed to treat (NNT), absolute risk reduction (ARR), relative risk reduction (RRR) and respective confidence intervals were calculated for each group. RESULTS: No significant differences overall were obtained between CT and no CT groups at P < 0.05. A significant benefit is demonstrated at P = 0.017 in females of childbearing age while a detrimental trend is found for those over the age of 50 years. CONCLUSIONS: Contrary to our initial hypothesis, no increased incidence of appendiceal perforation or abscess was demonstrated based on the ED physician's decision to perform CT without surgical consultation.


Assuntos
Apendicite/diagnóstico por imagem , Serviço Hospitalar de Emergência , Hospitais Comunitários , Tomografia Computadorizada por Raios X , Adolescente , Adulto , Idoso , Antibacterianos/uso terapêutico , Apendicectomia , Apendicite/terapia , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Processos e Resultados em Cuidados de Saúde , Estudos Retrospectivos , Fatores de Tempo
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