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1.
Proc Natl Acad Sci U S A ; 120(9): e2221109120, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36812203

RESUMO

Certain long non-coding RNAs (lncRNAs) are known to contain small open reading frames that can be translated. Here we describe a much larger 25 kDa human protein, "Ribosomal IGS Encoded Protein" (RIEP), that remarkably is encoded by the well-characterized RNA polymerase (RNAP) II-transcribed nucleolar "promoter and pre-rRNA antisense" lncRNA (PAPAS). Strikingly, RIEP, which is conserved throughout primates but not found in other species, predominantly localizes to the nucleolus as well as mitochondria, but both exogenously expressed and endogenous RIEP increase in the nuclear and perinuclear regions upon heat shock (HS). RIEP associates specifically with the rDNA locus, increases levels of the RNA:DNA helicase Senataxin, and functions to sharply reduce DNA damage induced by heat shock. Proteomics analysis identified two mitochondrial proteins, C1QBP and CHCHD2, both known to have mitochondrial and nuclear functions, that we show interact directly, and relocalize following heat shock, with RIEP. Finally, it is especially notable that the rDNA sequences encoding RIEP are multifunctional, giving rise to an RNA that functions both as RIEP messenger RNA (mRNA) and as PAPAS lncRNA, as well as containing the promoter sequences responsible for rRNA synthesis by RNAP I. Our work has thus not only shown that a nucleolar "non-coding" RNA in fact encodes a protein, but also established a novel link between mitochondria and nucleoli that contributes to the cellular stress response.


Assuntos
RNA Longo não Codificante , Animais , Humanos , RNA Longo não Codificante/metabolismo , Transcrição Gênica , DNA Ribossômico/genética , Nucléolo Celular/metabolismo , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , Proteínas Ribossômicas/metabolismo , RNA não Traduzido/metabolismo , RNA Ribossômico/genética , Proteínas de Transporte/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo
2.
J Med Genet ; 60(12): 1235-1244, 2023 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-37558402

RESUMO

BACKGROUND: Current clinical testing methods used to uncover the genetic basis of rare disease have inherent limitations, which can lead to causative pathogenic variants being missed. Within the rare disease arm of the 100 000 Genomes Project (100kGP), families were recruited under the clinical indication 'single autosomal recessive mutation in rare disease'. These participants presented with strong clinical suspicion for a specific autosomal recessive disorder, but only one suspected pathogenic variant had been identified through standard-of-care testing. Whole genome sequencing (WGS) aimed to identify cryptic 'second-hit' variants. METHODS: To investigate the 31 families with available data that remained unsolved following formal review within the 100kGP, SVRare was used to aggregate structural variants present in <1% of 100kGP participants. Small variants were assessed using population allele frequency data and SpliceAI. Literature searches and publicly available online tools were used for further annotation of pathogenicity. RESULTS: Using these strategies, 8/31 cases were solved, increasing the overall diagnostic yield of this cohort from 10/41 (24.4%) to 18/41 (43.9%). Exemplar cases include a patient with cystic fibrosis harbouring a novel exonic LINE1 insertion in CFTR and a patient with generalised arterial calcification of infancy with complex interlinked duplications involving exons 2-6 of ENPP1. Although ambiguous by short-read WGS, the ENPP1 variant structure was resolved using optical genome mapping and RNA analysis. CONCLUSION: Systematic examination of cryptic variants across a multi-disease cohort successfully identifies additional pathogenic variants. WGS data analysis in autosomal recessive rare disease should consider complex structural and small intronic variants as potentially pathogenic second hits.


Assuntos
Doenças Raras , Humanos , Mutação/genética , Sequência de Bases , Éxons , Mapeamento Cromossômico
3.
Mol Cell Proteomics ; 20: 100016, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33556866

RESUMO

In all cells, proteins are continuously synthesized and degraded to maintain protein homeostasis and modify gene expression levels in response to stimuli. Collectively, the processes of protein synthesis and degradation are referred to as protein turnover. At a steady state, protein turnover is constant to maintain protein homeostasis, but in dynamic responses, proteins change their rates of synthesis and degradation to adjust their proteomes to internal or external stimuli. Thus, probing the kinetics and dynamics of protein turnover lends insight into how cells regulate essential processes such as growth, differentiation, and stress response. Here, we outline historical and current approaches to measuring the kinetics of protein turnover on a proteome-wide scale in both steady-state and dynamic systems, with an emphasis on metabolic tracing using stable isotope-labeled amino acids. We highlight important considerations for designing proteome turnover experiments, key biological findings regarding the conserved principles of proteome turnover regulation, and future perspectives for both technological and biological investigation.


Assuntos
Proteoma , Aminoácidos , Animais , Humanos , Marcação por Isótopo , Luz , Preparações Farmacêuticas , Proteômica , Radioisótopos
4.
BMC Public Health ; 23(1): 2169, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37932707

RESUMO

BACKGROUND: Early reports raised alarms that intimate partner violence (IPV) increased during the COVID-19 pandemic, but initial studies showed that visits to emergency departments (EDs) decreased. This study assessed the impact of the prolonged pandemic and its associated restrictions on both rates of urgent care-seeking and injury severity for IPV. METHODS: Data from the Kingston Health Sciences Centre's (KHSC) ED were utilized to compare IPV presentations during 'Pre-COVID' (December 17, 2018 - March 16, 2020) and 'COVID' (March 17, 2020 - June 16, 2021), as well as three periods of heightened local restrictions: 'Lockdown-1' (March 17 - June 12, 2020), 'Lockdown-2' (December 26, 2020 - February 10, 2021) and 'Lockdown-3' (April 8 - June 2, 2021). The primary outcomes were incidence rate of IPV visits and injury severity, which was assessed using the Clinical Injury Extent Score (CIES) and Injury Severity Score (ISS). RESULTS: A total of 128 individuals were included. This sample had mean age of 34 years, was comprised of mostly women (97%), and represented a variety of intimate relationship types. Some individuals presented multiple times, resulting in a total of 139 acute IPV presentations. The frequency of IPV visits during COVID was similar to the Pre-COVID time period (67 vs. 72; p = 0.67). Incidence rate was 13% higher during COVID, though this difference was non-significant (6.66 vs. 5.90; p = 0.47). IPV visit frequency varied across lockdown periods (11 in Lockdown-1, 12 in Lockdown-2 and 6 in Lockdown-3), with the highest incidence rate during Lockdown-2 (12.71). There were more moderate and severe injuries during COVID compared to Pre-COVID, but mean CIES was not statistically significantly different (1.91 vs. 1.69; p = 0.29), nor was mean ISS (11.88 vs. 12.52; p = 0.73). CONCLUSIONS: During the 15-months following the start of COVID-19, there were small, but non-significant increases in both incidence rate and severity of IPV presentations to the KHSC ED. This may reflect escalation of violence as pandemic restrictions persisted and requires further investigation.


Assuntos
COVID-19 , Violência por Parceiro Íntimo , Humanos , Feminino , Adulto , Masculino , Estudos Retrospectivos , Pandemias , Canadá , Controle de Doenças Transmissíveis , Assistência Ambulatorial
5.
FASEB J ; 34(9): 11546-11561, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32767602

RESUMO

Osteoarthritis (OA) is a degenerative joint disease, and inflammation within an arthritic joint plays a critical role in disease progression. Pro-inflammatory cytokines, specifically IL-1 and TNF-α, induce aberrant expression of catabolic and degradative enzymes and inflammatory cytokines in OA and result in a challenging environment for cartilage repair and regeneration. MicroRNAs (miRNAS) are small noncoding RNAs and are important regulatory molecules that act by binding to target messenger RNAs (mRNAs) to reduce protein synthesis and have been implicated in many diseases, including OA. The goal of this study was to understand the mechanisms of miRNA regulation of the transcriptome of tissue-engineered cartilage in response to IL-1ß and TNF-α using an in vitro murine induced pluripotent stem cell (miPSC) model system. We performed miRNA and mRNA sequencing to determine the temporal and dynamic responses of genes to specific inflammatory cytokines as well as miRNAs that are differentially expressed (DE) in response to both cytokines or exclusively to IL-1ß or TNF-α. Through integration of mRNA and miRNA sequencing data, we created networks of miRNA-mRNA interactions which may be controlling the response to inflammatory cytokines. Within the networks, hub miRNAs, miR-29b-3p, miR-17-5p, and miR-20a-5p, were identified. As validation of these findings, we found that delivery of miR-17-5p and miR-20a-5p mimics significantly decreased degradative enzyme activity levels while also decreasing expression of inflammation-related genes in cytokine-treated cells. This study utilized an integrative approach to determine the miRNA interactome controlling the response to inflammatory cytokines and novel mediators of inflammation-driven degradation in tissue-engineered cartilage.


Assuntos
Condrócitos/efeitos dos fármacos , Citocinas/farmacologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mediadores da Inflamação/farmacologia , MicroRNAs/genética , RNA Mensageiro/genética , Animais , Cartilagem Articular/citologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Interleucina-1beta/farmacologia , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Engenharia Tecidual/métodos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Fator de Necrose Tumoral alfa/farmacologia
6.
Qual Health Res ; 30(3): 448-457, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31451052

RESUMO

To address the risks associated with polypharmacy, health care providers are investigating the feasibility of deprescribing programs as part of routine medical care to reduce medication burden to older adults. As older adults are enrolled in these programs, they are confronted with two dominant and legitimate accounts of medications, labeled the medication paradox: medications keep you healthy but they might be making you sick. We investigated how the medication paradox operates in the lives of older adults. In-depth qualitative interviews were conducted and analyzed with older adults aged 70+ to identify the various paradoxes that seniors live through regarding their medications and the narratives that they engage to negotiate these contradictions. Older adults were found to have established interpretative repertoires to make sense of the incongruent narratives of the medication paradox. In this article, we demonstrate older adults' efforts to carve out their unique place in the dichotomized institution of medicine.


Assuntos
Envelhecimento/psicologia , Desprescrições , Polimedicação , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Entrevistas como Assunto , Masculino , Negociação , Preferência do Paciente , Pesquisa Qualitativa , Fatores Socioeconômicos
7.
Biochemistry ; 58(30): 3225-3231, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31298844

RESUMO

A library of natural products and their derivatives was screened for inhibition of protein tyrosine phosphatase (PTP) 1B, which is a validated drug target for the treatment of obesity and type II diabetes. Of those active in the preliminary assay, the most promising was compound 2 containing a novel pyrrolopyrazoloisoquinolone scaffold derived by treating radicicol (1) with hydrazine. This nitrogen-atom augmented radicicol derivative was found to be PTP1B selective relative to other highly homologous nonreceptor PTPs. Biochemical evaluation, molecular docking, and mutagenesis revealed 2 to be an allosteric inhibitor of PTP1B with a submicromolar Ki. Cellular analyses using C2C12 myoblasts indicated that 2 restored insulin signaling and increased glucose uptake.


Assuntos
Inibidores Enzimáticos/química , Macrolídeos/química , Nitrogênio/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Animais , Inibidores Enzimáticos/metabolismo , Macrolídeos/metabolismo , Camundongos , Nitrogênio/metabolismo , Ligação Proteica/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo
8.
Bioorg Med Chem Lett ; 29(14): 1689-1693, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31129054

RESUMO

Glucose-regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.


Assuntos
Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Hexaclorofeno/uso terapêutico , Ensaios de Triagem em Larga Escala/métodos , Chaperona BiP do Retículo Endoplasmático , Hexaclorofeno/farmacologia , Humanos
9.
Proc Natl Acad Sci U S A ; 113(31): E4513-22, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27432980

RESUMO

Biological resurfacing of entire articular surfaces represents an important but challenging strategy for treatment of cartilage degeneration that occurs in osteoarthritis. Not only does this approach require anatomically sized and functional engineered cartilage, but the inflammatory environment within an arthritic joint may also inhibit chondrogenesis and induce degradation of native and engineered cartilage. The goal of this study was to use adult stem cells to engineer anatomically shaped, functional cartilage constructs capable of tunable and inducible expression of antiinflammatory molecules, specifically IL-1 receptor antagonist (IL-1Ra). Large (22-mm-diameter) hemispherical scaffolds were fabricated from 3D woven poly(ε-caprolactone) (PCL) fibers into two different configurations and seeded with human adipose-derived stem cells (ASCs). Doxycycline (dox)-inducible lentiviral vectors containing eGFP or IL-1Ra transgenes were immobilized to the PCL to transduce ASCs upon seeding, and constructs were cultured in chondrogenic conditions for 28 d. Constructs showed biomimetic cartilage properties and uniform tissue growth while maintaining their anatomic shape throughout culture. IL-1Ra-expressing constructs produced nearly 1 µg/mL of IL-1Ra upon controlled induction with dox. Treatment with IL-1 significantly increased matrix metalloprotease activity in the conditioned media of eGFP-expressing constructs but not in IL-1Ra-expressing constructs. Our findings show that advanced textile manufacturing combined with scaffold-mediated gene delivery can be used to tissue engineer large anatomically shaped cartilage constructs that possess controlled delivery of anticytokine therapy. Importantly, these cartilage constructs have the potential to provide mechanical functionality immediately upon implantation, as they will need to replace a majority, if not the entire joint surface to restore function.


Assuntos
Cartilagem Articular/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Osteoartrite/metabolismo , Engenharia Tecidual/métodos , Tecido Adiposo/citologia , Adulto , Células-Tronco Adultas/metabolismo , Cartilagem Articular/citologia , Células Cultivadas , Condrócitos/metabolismo , Condrogênese , Feminino , Humanos , Proteína Antagonista do Receptor de Interleucina 1/genética , Pessoa de Meia-Idade , Osteoartrite/genética , Osteoartrite/terapia , Reprodutibilidade dos Testes , Alicerces Teciduais
10.
Am J Hum Genet ; 97(2): 343-52, 2015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26235985

RESUMO

Intellectual disability (ID) affects approximately 1%-3% of humans with a gender bias toward males. Previous studies have identified mutations in more than 100 genes on the X chromosome in males with ID, but there is less evidence for de novo mutations on the X chromosome causing ID in females. In this study we present 35 unique deleterious de novo mutations in DDX3X identified by whole exome sequencing in 38 females with ID and various other features including hypotonia, movement disorders, behavior problems, corpus callosum hypoplasia, and epilepsy. Based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%-3% of unexplained ID in females. Although no de novo DDX3X mutations were identified in males, we present three families with segregating missense mutations in DDX3X, suggestive of an X-linked recessive inheritance pattern. In these families, all males with the DDX3X variant had ID, whereas carrier females were unaffected. To explore the pathogenic mechanisms accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants, we used canonical Wnt defects in zebrafish as a surrogate measure of DDX3X function in vivo. We demonstrate a consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway, and we further show a differential effect by gender. The differential activity possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDX3X mutations.


Assuntos
RNA Helicases DEAD-box/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto/genética , Fenótipo , Caracteres Sexuais , Via de Sinalização Wnt/genética , Substituição de Aminoácidos/genética , Animais , Sequência de Bases , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Exoma/genética , Feminino , Dosagem de Genes/genética , Humanos , Deficiência Intelectual/patologia , Masculino , Dados de Sequência Molecular , Análise de Sequência de DNA , Peixe-Zebra
11.
Bioorg Med Chem Lett ; 27(17): 4082-4085, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28757063

RESUMO

Activation of translation initiation is a common trait of cancer cells. Formation of the heterotrimeric eukaryotic initiation factor F (eIF4F) complex is the rate-limiting step in 5' m7GpppN cap-dependent translation. This trimeric complex includes the eIF4E cap binding protein, the eIF4G scaffolding protein, and the DEAD box RNA helicase eIF4A. eIF4A is an ATP-dependent helicase and because it is the only enzyme in the eIF4F complex, it has been shown to be a potential therapeutic target for a variety of malignancies. To this end, we have used a simple ATPase biochemical screen to survey several hundred marine and terrestrial derived natural products. Herein, we report the discovery of two natural products from marine sources, elisabatin A (1) and allolaurinterol (2), which show low µM inhibition of eIF4A ATPase activity. Enzymological analyses revealed 1 and 2 to be ATP-competitive, and cellular evaluations showed reasonable cytotoxicity against A549 (lung cancer) and MDA-MA-468 (breast cancer) cell lines. However, only compound 2 showed potent inhibition of helicase activity congruent with its ATPase inhibitory activity.


Assuntos
Trifosfato de Adenosina/metabolismo , Produtos Biológicos/farmacologia , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Fator de Iniciação 4A em Eucariotos/metabolismo , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
12.
J Med Genet ; 51(10): 659-68, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25125236

RESUMO

BACKGROUND: Cornelia de Lange syndrome (CdLS) is a multisystem disorder with distinctive facial appearance, intellectual disability and growth failure as prominent features. Most individuals with typical CdLS have de novo heterozygous loss-of-function mutations in NIPBL with mosaic individuals representing a significant proportion. Mutations in other cohesin components, SMC1A, SMC3, HDAC8 and RAD21 cause less typical CdLS. METHODS: We screened 163 affected individuals for coding region mutations in the known genes, 90 for genomic rearrangements, 19 for deep intronic variants in NIPBL and 5 had whole-exome sequencing. RESULTS: Pathogenic mutations [including mosaic changes] were identified in: NIPBL 46 [3] (28.2%); SMC1A 5 [1] (3.1%); SMC3 5 [1] (3.1%); HDAC8 6 [0] (3.6%) and RAD21 1 [0] (0.6%). One individual had a de novo 1.3 Mb deletion of 1p36.3. Another had a 520 kb duplication of 12q13.13 encompassing ESPL1, encoding separase, an enzyme that cleaves the cohesin ring. Three de novo mutations were identified in ANKRD11 demonstrating a phenotypic overlap with KBG syndrome. To estimate the number of undetected mosaic cases we used recursive partitioning to identify discriminating features in the NIPBL-positive subgroup. Filtering of the mutation-negative group on these features classified at least 18% as 'NIPBL-like'. A computer composition of the average face of this NIPBL-like subgroup was also more typical in appearance than that of all others in the mutation-negative group supporting the existence of undetected mosaic cases. CONCLUSIONS: Future diagnostic testing in 'mutation-negative' CdLS thus merits deeper sequencing of multiple DNA samples derived from different tissues.


Assuntos
Síndrome de Cornélia de Lange/genética , Heterogeneidade Genética , Mosaicismo , Face/patologia , Estudos de Associação Genética , Humanos , Mutação , Fenótipo
13.
Nat Genet ; 37(10): 1135-40, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16170314

RESUMO

The evolutionarily conserved planar cell polarity (PCP) pathway (or noncanonical Wnt pathway) drives several important cellular processes, including epithelial cell polarization, cell migration and mitotic spindle orientation. In vertebrates, PCP genes have a vital role in polarized convergent extension movements during gastrulation and neurulation. Here we show that mice with mutations in genes involved in Bardet-Biedl syndrome (BBS), a disorder associated with ciliary dysfunction, share phenotypes with PCP mutants including open eyelids, neural tube defects and disrupted cochlear stereociliary bundles. Furthermore, we identify genetic interactions between BBS genes and a PCP gene in both mouse (Ltap, also called Vangl2) and zebrafish (vangl2). In zebrafish, the augmented phenotype results from enhanced defective convergent extension movements. We also show that Vangl2 localizes to the basal body and axoneme of ciliated cells, a pattern reminiscent of that of the BBS proteins. These data suggest that cilia are intrinsically involved in PCP processes.


Assuntos
Síndrome de Bardet-Biedl/patologia , Proteínas Associadas aos Microtúbulos/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/metabolismo , Animais , Síndrome de Bardet-Biedl/genética , Polaridade Celular/genética , Cílios/química , Cóclea/patologia , Células Epiteliais/química , Pálpebras/fisiopatologia , Chaperoninas do Grupo II , Camundongos , Camundongos Mutantes , Mutação , Proteínas do Tecido Nervoso/análise , Defeitos do Tubo Neural/patologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
14.
Nutrients ; 16(10)2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38794715

RESUMO

Obesity in the United States and Western countries represents a major health challenge associated with an increased risk of metabolic diseases, including cardiovascular disease, hypertension, diabetes, and certain cancers. Our past work revealed a more pronounced obesity-cancer link in certain ethnic groups, motivating us to develop a tailored dietary intervention called the Healthy Diet and Lifestyle 2 (HDLS2). The study protocol is described herein for this randomized six-month trial examining the effects of intermittent energy restriction (5:2 Diet) plus the Mediterranean dietary pattern (IER + MED) on visceral adipose tissue (VAT), liver fat, and metabolic biomarkers, compared to a standard MED with daily energy restriction (DER + MED), in a diverse participant group. Using MRI and DXA scans for body composition analysis, as well as metabolic profiling, this research aims to contribute to nutritional guidelines and strategies for visceral obesity reduction. The potential benefits of IER + MED, particularly regarding VAT reduction and metabolic health improvement, could be pivotal in mitigating the obesity epidemic and its metabolic sequelae. The ongoing study will provide essential insights into the efficacy of these energy restriction approaches across varied racial/ethnic backgrounds, addressing an urgent need in nutrition and metabolic health research. Registered Trial, National Institutes of Health, ClinicalTrials.gov (NCT05132686).


Assuntos
Restrição Calórica , Dieta Mediterrânea , Gordura Intra-Abdominal , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Biomarcadores/sangue , Composição Corporal , Restrição Calórica/métodos , Dieta Saudável/métodos , Gordura Intra-Abdominal/metabolismo , Estilo de Vida , Obesidade Abdominal/dietoterapia , Ensaios Clínicos Controlados Aleatórios como Assunto
15.
Cell Rep Methods ; 4(5): 100760, 2024 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-38677284

RESUMO

The role of protein turnover in pancreatic ductal adenocarcinoma (PDA) metastasis has not been previously investigated. We introduce dynamic stable-isotope labeling of organoids (dSILO): a dynamic SILAC derivative that combines a pulse of isotopically labeled amino acids with isobaric tandem mass-tag (TMT) labeling to measure proteome-wide protein turnover rates in organoids. We applied it to a PDA model and discovered that metastatic organoids exhibit an accelerated global proteome turnover compared to primary tumor organoids. Globally, most turnover changes are not reflected at the level of protein abundance. Interestingly, the group of proteins that show the highest turnover increase in metastatic PDA compared to tumor is involved in mitochondrial respiration. This indicates that metastatic PDA may adopt alternative respiratory chain functionality that is controlled by the rate at which proteins are turned over. Collectively, our analysis of proteome turnover in PDA organoids offers insights into the mechanisms underlying PDA metastasis.


Assuntos
Carcinoma Ductal Pancreático , Organoides , Neoplasias Pancreáticas , Proteoma , Organoides/metabolismo , Organoides/patologia , Proteoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Humanos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Marcação por Isótopo , Proteômica/métodos
16.
J Exp Med ; 221(8)2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38869500

RESUMO

UNC93B1 is a transmembrane domain protein mediating the signaling of endosomal Toll-like receptors (TLRs). We report five families harboring rare missense substitutions (I317M, G325C, L330R, R466S, and R525P) in UNC93B1 causing systemic lupus erythematosus (SLE) or chilblain lupus (CBL) as either autosomal dominant or autosomal recessive traits. As for a D34A mutation causing murine lupus, we recorded a gain of TLR7 and, to a lesser extent, TLR8 activity with the I317M (in vitro) and G325C (in vitro and ex vivo) variants in the context of SLE. Contrastingly, in three families segregating CBL, the L330R, R466S, and R525P variants were isomorphic with respect to TLR7 activity in vitro and, for R525P, ex vivo. Rather, these variants demonstrated a gain of TLR8 activity. We observed enhanced interaction of the G325C, L330R, and R466S variants with TLR8, but not the R525P substitution, indicating different disease mechanisms. Overall, these observations suggest that UNC93B1 mutations cause monogenic SLE or CBL due to differentially enhanced TLR7 and TLR8 signaling.


Assuntos
Pérnio , Lúpus Eritematoso Sistêmico , Receptor 7 Toll-Like , Feminino , Humanos , Masculino , Pérnio/genética , Mutação com Ganho de Função , Células HEK293 , Lúpus Eritematoso Cutâneo/genética , Lúpus Eritematoso Cutâneo/patologia , Lúpus Eritematoso Sistêmico/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação de Sentido Incorreto , Linhagem , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/genética , Receptor 8 Toll-Like/metabolismo , Pré-Escolar , Criança , Adulto Jovem , Adulto
17.
Nat Genet ; 36(5): 462-70, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15107855

RESUMO

BBS4 is one of several proteins that cause Bardet-Biedl syndrome (BBS), a multisystemic disorder of genetic and clinical complexity. Here we show that BBS4 localizes to the centriolar satellites of centrosomes and basal bodies of primary cilia, where it functions as an adaptor of the p150(glued) subunit of the dynein transport machinery to recruit PCM1 (pericentriolar material 1 protein) and its associated cargo to the satellites. Silencing of BBS4 induces PCM1 mislocalization and concomitant deanchoring of centrosomal microtubules, arrest in cell division and apoptotic cell death. Expression of two truncated forms of BBS4 that are similar to those found in some individuals with BBS had a similar effect on PCM1 and microtubules. Our findings indicate that defective targeting or anchoring of pericentriolar proteins and microtubule disorganization contribute to the BBS phenotype and provide new insights into possible causes of familial obesity, diabetes and retinal degeneration.


Assuntos
Síndrome de Bardet-Biedl/metabolismo , Ciclo Celular , Centrossomo/metabolismo , Microtúbulos/metabolismo , Proteínas/metabolismo , Animais , Apoptose , Autoantígenos , Síndrome de Bardet-Biedl/patologia , Células COS , Proteínas de Ciclo Celular/metabolismo , Centrossomo/patologia , Chlorocebus aethiops , Dineínas/metabolismo , Inativação Gênica , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Proteínas Associadas aos Microtúbulos , Fragmentos de Peptídeos/imunologia , Fenótipo , Ligação Proteica , Subunidades Proteicas , Transporte Proteico , Proteínas/antagonistas & inibidores , Proteínas/genética , RNA Interferente Pequeno/farmacologia , Coelhos , Saccharomyces cerevisiae , Técnicas do Sistema de Duplo-Híbrido
18.
Nat Genet ; 36(9): 989-93, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15314642

RESUMO

RAB, ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins belong to the Ras superfamily of small GTP-binding proteins and are essential for various membrane-associated intracellular trafficking processes. None of the approximately 50 known members of this family are linked to human disease. Using a bioinformatic screen for ciliary genes in combination with mutational analyses, we identified ARL6 as the gene underlying Bardet-Biedl syndrome type 3, a multisystemic disorder characterized by obesity, blindness, polydactyly, renal abnormalities and cognitive impairment. We uncovered four different homozygous substitutions in ARL6 in four unrelated families affected with Bardet-Biedl syndrome, two of which disrupt a threonine residue important for GTP binding and function of several related small GTP-binding proteins. Analysis of the Caenorhabditis elegans ARL6 homolog indicates that it is specifically expressed in ciliated cells, and that, in addition to the postulated cytoplasmic functions of ARL proteins, it undergoes intraflagellar transport. These findings implicate a small GTP-binding protein in ciliary transport and the pathogenesis of a pleiotropic disorder.


Assuntos
Fatores de Ribosilação do ADP/genética , Síndrome de Bardet-Biedl/genética , Genes ras , Proteínas de Membrana/genética , Mutação , Sequência de Bases , Cílios/metabolismo , Proteínas de Ligação ao GTP/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios/citologia , Linhagem
19.
Eur J Med Genet ; 66(3): 104696, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36639056

RESUMO

Neurodevelopmental disorder with visual defects and brain anomalies (NEDVIBA) is a recently described genetic condition caused by de novo missense HK1 variants. Phenotypic data is currently limited; only seven patients have been published to date. This descriptive case series of a further four patients with de novo missense HK1 variants, alongside integration of phenotypic data with the reported cases, aims to improve our understanding of the associated phenotype. We provide further evidence that de novo HK1 variants located within the regulatory-terminal domain and alpha helix are associated with neurological problems and visual problems. We highlight for the first time an association with a raised cerebrospinal fluid lactate and specific abnormalities to the basal ganglia on brain magnetic resonance imaging, as well as associated respiratory issues and swallowing/feeding difficulties. We propose that this distinctive neurodevelopmental phenotype could arise through disruption of the regulatory glucose-6-phosphate binding site and subsequent gain of function of HK1 within the brain.


Assuntos
Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Humanos , Encéfalo/diagnóstico por imagem , Heterozigoto , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento/genética , Fenótipo
20.
Am J Med Genet A ; 158A(11): 2733-42, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23023959

RESUMO

Meier-Gorlin syndrome (MGS) is a rare autosomal recessive disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recently, mutations in the ORC1, ORC4, ORC6, CDT1, and CDC6 genes, encoding components of the pre-replication complex, have been identified. This complex is essential for DNA replication and therefore mutations are expected to impair cell proliferation and consequently could globally reduce growth. However, detailed growth characteristics of MGS patients have not been reported, and so this is addressed here through study of 45 MGS patients, the largest cohort worldwide. Here, we report that growth velocity (length) is impaired in MGS during pregnancy and first year of life, but, thereafter, height increases in paralleled normal reference centiles, resulting in a mean adult height of -4.5 standard deviations (SD). Height is dependent on ethnic background and underlying molecular cause, with ORC1 and ORC4 mutations causing more severe short stature and microcephaly. Growth hormone therapy (n = 9) was generally ineffective, though in two patients with significantly reduced IGF1 levels, growth was substantially improved by GH treatment, with 2SD and 3.8 SD improvement in height. Growth parameters for monitoring growth in future MGS patients are provided and as well we highlight that growth is disproportionately affected in certain structures, with growth related minor genital abnormalities (42%) and mammary hypoplasia (100%) frequently present, in addition to established effects on ears and patellar growth.


Assuntos
Gráficos de Crescimento , Transtornos do Crescimento/diagnóstico , Micrognatismo/diagnóstico , Desenvolvimento Sexual , Proteínas de Ciclo Celular/genética , Pré-Escolar , Estudos de Coortes , Microtia Congênita , Orelha/anormalidades , Feminino , Transtornos do Crescimento/tratamento farmacológico , Transtornos do Crescimento/genética , Hormônio do Crescimento Humano/sangue , Hormônio do Crescimento Humano/uso terapêutico , Humanos , Lactente , Masculino , Micrognatismo/tratamento farmacológico , Micrognatismo/genética , Mutação , Complexo de Reconhecimento de Origem/genética , Patela/anormalidades , Desenvolvimento Sexual/genética , Anormalidades Urogenitais
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