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Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism.
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Metabolômica , Proteoma , Proteômica , Proteoma/metabolismo , Metabolômica/métodos , Proteômica/métodos , Humanos , Animais , Glutationa/metabolismo , Metaboloma , Transaminases/metabolismoRESUMO
The mechanisms underlying the pathophysiology of endometriosis, characterized by the presence of endometrium-like tissue outside the uterus, remain poorly understood. This study aimed to identify cell type-specific gene expression changes in superficial peritoneal endometriotic lesions and elucidate the crosstalk among the stroma, epithelium, and macrophages compared to patient-matched eutopic endometrium. Surprisingly, comparison between lesions and eutopic endometrium revealed transcriptional similarities, indicating minimal alterations in the sub-epithelial stroma and epithelium of lesions. Spatial transcriptomics highlighted increased signaling between the lesion epithelium and macrophages, emphasizing the role of the epithelium in driving lesion inflammation. We propose that the superficial endometriotic lesion epithelium orchestrates inflammatory signaling and promotes a pro-repair phenotype in macrophages, providing a new role for Complement 3 in lesion pathobiology. This study underscores the significance of considering spatial context and cellular interactions in uncovering mechanisms governing disease in endometriotic lesions.
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PURPOSE: The importance of cellular context to the synergy of DNA damage response (DDR)-targeted agents is important for tumors with mutations in DDR pathways, but less well-established for tumors driven by oncogenic transcription factors. In this study, we exploit the widespread transcriptional dysregulation of the EWS-FLI1 transcription factor to identify an effective DDR-targeted combination therapy for Ewing sarcoma. EXPERIMENTAL DESIGN: We used matrix drug screening to evaluate synergy between a DNA-PK inhibitor (M9831) or an ATR inhibitor (berzosertib) and chemotherapy. The combination of berzosertib and cisplatin was selected for broad synergy, mechanistically evaluated for Ewing sarcoma selectivity, and optimized for in vivo schedule. RESULTS: Berzosertib combined with cisplatin demonstrates profound synergy in multiple Ewing sarcoma cell lines at clinically achievable concentrations. The synergy is due to loss of expression of the ATR downstream target CHEK1, loss of cell-cycle check-points, and mitotic catastrophe. Consistent with the goals of the project, EWS-FLI1 drives the expression of CHEK1 and five other ATR pathway members. The loss of CHEK1 expression is not due to transcriptional repression and instead caused by degradation coupled with suppression of protein translation. The profound synergy is realized in vivo with a novel optimized schedule of this combination in subsets of Ewing sarcoma models, leading to durable complete responses in 50% of animals bearing two different Ewing sarcoma xenografts. CONCLUSIONS: These data exploit EWS-FLI1 driven alterations in cell context to broaden the therapeutic window of berzosertib and cisplatin to establish a promising combination therapy and a novel in vivo schedule. See related commentary by Ohmura and Grünewald, p. 3358.
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Proteínas Mutadas de Ataxia Telangiectasia , Cisplatino , Sinergismo Farmacológico , Proteína Proto-Oncogênica c-fli-1 , Proteína EWS de Ligação a RNA , Sarcoma de Ewing , Ensaios Antitumorais Modelo de Xenoenxerto , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/patologia , Sarcoma de Ewing/genética , Humanos , Cisplatino/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Camundongos , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Linhagem Celular Tumoral , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Dano ao DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isoxazóis , PirazinasRESUMO
BACKGROUND: Perinatal depression (including antenatal-, postnatal-, and depression that spans both timepoints) is a prevalent disorder with high morbidity that affects both mother and child. Even though the full biological blueprints of perinatal depression remain incomplete, multiple studies indicate that, at least for antenatal depression, the disorder has an inflammatory component likely linked to a dysregulation of the enzymatic kynurenine pathway. The production of neuroactive metabolites in this pathway, including quinolinic acid (QUIN), is upregulated in the placenta due to the multiple immunological roles of the metabolites during pregnancy. Since neuroactive metabolites produced by the pathway also may affect mood by directly affecting glutamate neurotransmission, we sought to investigate whether the placental expression of kynurenine pathway enzymes controlling QUIN production was associated with both peripheral inflammation and depressive symptoms during pregnancy. METHODS: 68 placentas obtained at birth were analyzed using qPCR to determine the expression of kynurenine pathway enzymes. Cytokines and metabolites were quantified in plasma using high-sensitivity electroluminescence and ultra-performance liquid chromatography, respectively. Maternal depressive symptoms were assessed using the Edinburgh Postnatal Depression Scale (EPDS) throughout pregnancy and the post-partum. Associations between these factors were assessed using robust linear regression with ranked enzymes. RESULTS: Low placental quinolinate phosphoribosyl transferase (QPRT), the enzyme responsible for degrading QUIN, was associated with higher IL-6 and higher QUIN/kynurenic acid ratios at the 3rd trimester. Moreover, women with severe depressive symptoms in the 3rd trimester had significantly lower placental expression of both QPRT and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD); impaired activity of these two enzymes leads to QUIN accumulation. CONCLUSION: Overall, our data support that a compromised placental environment, featuring low expression of critical kynurenine pathway enzymes is associated with increased levels of plasma cytokines and the dysregulated kynurenine metabolite pattern observed in depressed women during pregnancy.
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Depressão , Inflamação , Cinurenina , Placenta , Ácido Quinolínico , Humanos , Feminino , Gravidez , Cinurenina/metabolismo , Cinurenina/sangue , Placenta/metabolismo , Adulto , Inflamação/metabolismo , Depressão/metabolismo , Ácido Quinolínico/metabolismo , Ácido Quinolínico/sangue , Citocinas/metabolismo , Complicações na Gravidez/metabolismo , Carboxiliases/metabolismo , PentosiltransferasesRESUMO
DNA mutations are necessary drivers of cancer, yet only a small subset of mutated cells go on to cause the disease. To date, the mechanisms that determine which rare subset of cells transform and initiate tumorigenesis remain unclear. Here, we take advantage of a unique model of intrinsic developmental heterogeneity (Trim28+/D9) and demonstrate that stochastic early life epigenetic variation can trigger distinct cancer-susceptibility 'states' in adulthood. We show that these developmentally primed states are characterized by differential methylation patterns at typically silenced heterochromatin, and that these epigenetic signatures are detectable as early as 10 days of age. The differentially methylated loci are enriched for genes with known oncogenic potential. These same genes are frequently mutated in human cancers, and their dysregulation correlates with poor prognosis. These results provide proof-of-concept that intrinsic developmental heterogeneity can prime individual, life-long cancer risk.
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Proper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the activity of the pathway in the skeleton, cause high bone mass in human subjects and mouse models. Increased bone mass is often accompanied by severe headaches from increased intracranial pressure, which can lead to fatality and loss of vision or hearing due to the entrapment of cranial nerves. In addition, progressive forehead bossing and mandibular overgrowth occur in almost all subjects. Treatments that would provide symptomatic relief in these subjects are limited. Porcupine-mediated palmitoylation is necessary for Wnt secretion and binding to the frizzled receptor. Chemical inhibition of porcupine is a highly selective method of Wnt signaling inhibition. We treated three different mouse models of high bone mass caused by aberrant Wnt signaling, including homozygosity for loss-of-function in Sost, which models sclerosteosis, and two strains of mice carrying different point mutations in Lrp5 (equivalent to human G171V and A214V), at 3 months of age with porcupine inhibitors for 5-6 weeks. Treatment significantly reduced both trabecular and cortical bone mass in all three models. This demonstrates that porcupine inhibition is potentially therapeutic for symptomatic relief in subjects who suffer from these disorders and further establishes that the continued production of Wnts is necessary for sustaining high bone mass in these models.
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Mutação com Ganho de Função , Hiperostose , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal , Secreções Corporais , Modelos Animais de Doenças , Hiperostose/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , MutaçãoRESUMO
Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. Here, we identified ketone bodies (KBs)-including ß-hydroxybutyrate (ßOHB) and acetoacetate (AcAc)-as essential fuels supporting CD8+ T cell metabolism and effector function. ßOHB directly increased CD8+ T effector (Teff) cell cytokine production and cytolytic activity, and KB oxidation (ketolysis) was required for Teff cell responses to bacterial infection and tumor challenge. CD8+ Teff cells preferentially used KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boosted the respiratory capacity and TCA cycle-dependent metabolic pathways that fuel CD8+ T cell function. Mechanistically, ßOHB was a major substrate for acetyl-CoA production in CD8+ T cells and regulated effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses.
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Linfócitos T CD8-Positivos , Histonas , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/farmacologia , Acetilação , Histonas/metabolismo , Corpos Cetônicos , Animais , CamundongosRESUMO
Here, we provide an in-depth analysis of the usefulness of single-sample metabolite/RNA extraction for multi-'omics readout. Using pulverized frozen livers of mice injected with lymphocytic choriomeningitis virus (LCMV) or vehicle (Veh), we isolated RNA prior (RNA) or following metabolite extraction (MetRNA). RNA sequencing (RNAseq) data were evaluated for differential expression analysis and dispersion, and differential metabolite abundance was determined. Both RNA and MetRNA clustered together by principal component analysis, indicating that inter-individual differences were the largest source of variance. Over 85% of LCMV versus Veh differentially expressed genes were shared between extraction methods, with the remaining 15% evenly and randomly divided between groups. Differentially expressed genes unique to the extraction method were attributed to randomness around the 0.05 FDR cut-off and stochastic changes in variance and mean expression. In addition, analysis using the mean absolute difference showed no difference in the dispersion of transcripts between extraction methods. Altogether, our data show that prior metabolite extraction preserves RNAseq data quality, which enables us to confidently perform integrated pathway enrichment analysis on metabolomics and RNAseq data from a single sample. This analysis revealed pyrimidine metabolism as the most LCMV-impacted pathway. Combined analysis of genes and metabolites in the pathway exposed a pattern in the degradation of pyrimidine nucleotides leading to uracil generation. In support of this, uracil was among the most differentially abundant metabolites in serum upon LCMV infection. Our data suggest that hepatic uracil export is a novel phenotypic feature of acute infection and highlight the usefulness of our integrated single-sample multi-'omics approach.
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Metabolômica , Viroses , Animais , Camundongos , Análise de Sequência de RNA , Fígado , RNARESUMO
PURPOSE: On the basis of preclinical evidence of epigenetic contribution to sensitivity and resistance to immune checkpoint inhibitors (ICI), we hypothesized that guadecitabine (hypomethylating agent) and atezolizumab [anti-programmed cell death ligand 1 (PD-L1)] together would potentiate a clinical response in patients with metastatic urothelial carcinoma (UC) unresponsive to initial immune checkpoint blockade therapy. PATIENTS AND METHODS: We designed a single arm phase II study (NCT03179943) with a safety run-in to identify the recommended phase II dose of the combination therapy of guadecitabine and atezolizumab. Patients with recurrent/advanced UC who had previously progressed on ICI therapy with programmed cell death protein 1 or PD-L1 targeting agents were eligible. Preplanned correlative analysis was performed to characterize peripheral immune dynamics and global DNA methylation, transcriptome, and immune infiltration dynamics of patient tumors. RESULTS: Safety run-in enrolled 6 patients and phase II enrolled 15 patients before the trial was closed for futility. No dose-limiting toxicity was observed. Four patients, with best response of stable disease (SD), exhibited extended tumor control (8-11 months) and survival (>14 months). Correlative analysis revealed lack of DNA demethylation in tumors after 2 cycles of treatment. Increased peripheral immune activation and immune infiltration in tumors after treatment correlated with progression-free survival and SD. Furthermore, high IL6 and IL8 levels in the patients' plasma was associated with short survival. CONCLUSIONS: No RECIST responses were observed after combination therapy in this trial. Although we could not detect the anticipated tumor-intrinsic effects of guadecitabine, the addition of hypomethylating agent to ICI therapy induced immune activation in a few patients, which associated with longer patient survival.
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Antineoplásicos , Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Antineoplásicos/uso terapêutico , Carcinoma de Células de Transição/tratamento farmacológico , Carcinoma de Células de Transição/secundário , Antígeno B7-H1 , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Recidiva Local de Neoplasia/tratamento farmacológicoRESUMO
Uterine fibroids (leiomyomas) affect Black women disproportionately compared with women of other races and ethnicities in terms of prevalence, incidence, and severity of symptoms. The causes of this racial disparity are essentially unknown. We hypothesized that myometria of Black women are more susceptible to developing fibroids, and we examined the transcriptomic and DNA methylation profiles of myometria and fibroids from Black and White women for comparison. Myometrial samples cluster by race in both their transcriptome and DNA methylation profiles, whereas fibroid samples only cluster by race in the latter. More differentially expressed genes (DEGs) were detected in the Black and White myometrial sample comparison than in the fibroid comparison. Leiomyoma gene set expression analysis identified 4 clusters of DEGs, including a cluster of 24 genes with higher expression in myometrial samples from Black women. One of the DEGs in this group, von Willibrands factor (VWF), was significantly hypomethylated in both myometrial samples from Black women and in all fibroids at 2 CpG probes that are near a putative enhancer site and that are correlated with VWF expression levels. These results suggest that the molecular basis for the disparity in fibroid disease between Black and White women could be found in the myometria before fibroid development and not in the fibroids themselves.
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Leiomioma , Neoplasias Uterinas , Feminino , Humanos , Neoplasias Uterinas/genética , Neoplasias Uterinas/epidemiologia , Neoplasias Uterinas/metabolismo , Transcriptoma , Epigenoma , Fator de von Willebrand/genética , Leiomioma/genética , Leiomioma/epidemiologia , Leiomioma/metabolismoRESUMO
The DNA methylation status of the X-chromosome in cancer cells is often overlooked because of computational difficulties. Most of the CpG islands on the X-chromosome are mono-allelically methylated in normal female cells and only present as a single copy in male cells. We treated two colorectal cancer cell lines from a male (HCT116) and a female (RKO) with increasing doses of a DNA methyltransferase 1 (DNMT1)-specific inhibitor (GSK3685032/GSK5032) over several months to remove as much non-essential CpG methylation as possible. Profiling of the remaining DNA methylome revealed an unexpected, enriched retention of DNA methylation on the X-chromosome. Strikingly, the identified retained X-chromosome DNA methylation patterns accurately predicted de novo DNA hypermethylation in colon cancer patient methylomes in the TCGA COAD/READ cohort. These results suggest that a re-examination of tumors for X-linked DNA methylation changes may enable greater understanding of the importance of epigenetic silencing of cancer related genes.
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Metilação de DNA , Neoplasias , Ilhas de CpG , DNA , Feminino , Humanos , Masculino , Neoplasias/genética , Cromossomo XRESUMO
Mechanisms of Alzheimer's disease (AD) and its putative prodromal stage, amnestic mild cognitive impairment (aMCI), involve the dysregulation of multiple candidate molecular pathways that drive selective cellular vulnerability in cognitive brain regions. However, the spatiotemporal overlap of markers for pathway dysregulation in different brain regions and cell types presents a challenge for pinpointing causal versus epiphenomenal changes characterizing disease progression. To approach this problem, we performed Weighted Gene Co-expression Network Analysis and STRING interactome analysis of gene expression patterns quantified in frontal cortex samples (Brodmann area 10) from subjects who died with a clinical diagnosis of no cognitive impairment, aMCI, or mild/moderate AD. Frontal cortex was chosen due to the relatively protracted involvement of this region in AD, which might reveal pathways associated with disease onset. A co-expressed network correlating with clinical diagnosis was functionally associated with insulin signaling, with insulin (INS) being the most highly connected gene within the network. Co-expressed networks correlating with neuropathological diagnostic criteria (e.g., NIA-Reagan Likelihood of AD) were associated with platelet-endothelium-leucocyte cell adhesion pathways and hypoxia-oxidative stress. Dysregulation of these functional pathways may represent incipient alterations impacting disease progression and the clinical presentation of aMCI and AD.
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Doença de Alzheimer , Disfunção Cognitiva , Insulinas , Humanos , Doença de Alzheimer/patologia , Mapeamento Encefálico , Imageamento por Ressonância Magnética , Disfunção Cognitiva/patologia , Encéfalo , Lobo Frontal , Progressão da DoençaRESUMO
Enzalutamide resistance has been observed in approximately 50% of patients with prostate cancer (PCa) bone metastases. Therefore, there is an urgent need to investigate the mechanisms and develop strategies to overcome resistance. We observed enzalutamide resistance in bone lesion development induced by PCa cells in mouse models. We found that the bone microenvironment was indispensable for enzalutamide resistance because enzalutamide significantly inhibited the growth of subcutaneous C4-2B tumors and the proliferation of C4-2B cells isolated from the bone lesions, and the resistance was recapitulated only when C4-2B cells were co-cultured with osteoblasts. In revealing how osteoblasts contribute to enzalutamide resistance, we found that enzalutamide decreased TGFBR2 protein expression in osteoblasts, which was supported by clinical data. This decrease was possibly through PTH1R-mediated endocytosis. We showed that PTH1R blockade rescued enzalutamide-mediated decrease in TGFBR2 levels and enzalutamide responses in C4-2B cells that were co-cultured with osteoblasts. This is the first study to reveal the contribution of the bone microenvironment to enzalutamide resistance and identify PTH1R as a feasible target to overcome the resistance in PCa bone metastases.
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Benzamidas/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Nitrilas/farmacologia , Feniltioidantoína/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Masculino , Camundongos , Metástase Neoplásica , Osteoblastos/efeitos dos fármacos , Próstata/efeitos dos fármacos , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteólise/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
OBJECTIVE: Brain metastasis is the most common intracranial neoplasm. Although anatomical spatial distributions of brain metastasis may vary according to primary cancer subtype, these patterns are not understood and may have major implications for treatment. METHODS: To test the hypothesis that the spatial distribution of brain metastasis varies according to cancer origin in nonrandom patterns, the authors leveraged spatial 3D coordinate data derived from stereotactic Gamma Knife radiosurgery procedures performed to treat 2106 brain metastases arising from 5 common cancer types (melanoma, lung, breast, renal, and colorectal). Two predictive topographic models (regional brain metastasis echelon model [RBMEM] and brain region susceptibility model [BRSM]) were developed and independently validated. RESULTS: RBMEM assessed the hierarchical distribution of brain metastasis to specific brain regions relative to other primary cancers and showed that distinct regions were relatively susceptible to metastasis, as follows: bilateral temporal/parietal and left frontal lobes were susceptible to lung cancer; right frontal and occipital lobes to melanoma; cerebellum to breast cancer; and brainstem to renal cell carcinoma. BRSM provided probability estimates for each cancer subtype, independent of other subtypes, to metastasize to brain regions, as follows: lung cancer had a propensity to metastasize to bilateral temporal lobes; breast cancer to right cerebellar hemisphere; melanoma to left temporal lobe; renal cell carcinoma to brainstem; and colon cancer to right cerebellar hemisphere. Patient topographic data further revealed that brain metastasis demonstrated distinct spatial patterns when stratified by patient age and tumor volume. CONCLUSIONS: These data support the hypothesis that there is a nonuniform spatial distribution of brain metastasis to preferential brain regions that varies according to cancer subtype in patients treated with Gamma Knife radiosurgery. These topographic patterns may be indicative of the abilities of various cancers to adapt to regional neural microenvironments, facilitate colonization, and establish metastasis. Although the brain microenvironment likely modulates selective seeding of metastasis, it remains unknown how the anatomical spatial distribution of brain metastasis varies according to primary cancer subtype and contributes to diagnosis. For the first time, the authors have presented two predictive models to show that brain metastasis, depending on its origin, in fact demonstrates distinct geographic spread within the central nervous system. These findings could be used as a predictive diagnostic tool and could also potentially result in future translational and therapeutic work to disrupt growth of brain metastasis on the basis of anatomical region.
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Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Neoplasias do Sistema Nervoso Central/patologia , Neoplasias/patologia , Adulto , Fatores Etários , Idoso , Algoritmos , Mapeamento Encefálico , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias do Sistema Nervoso Central/diagnóstico por imagem , Feminino , Humanos , Imageamento Tridimensional , Masculino , Pessoa de Meia-Idade , Modelos Neurológicos , Metástase Neoplásica , Neoplasias/diagnóstico por imagem , Procedimentos Neurocirúrgicos , Valor Preditivo dos Testes , Radiocirurgia , Estudos RetrospectivosRESUMO
Hyperglycemia affects over 400 million individuals worldwide. The detrimental health effects are well studied at the tissue level, but the in vivo effects at the organelle level are poorly understood. To establish such an in vivo model, we used mice lacking TXNIP, a negative regulator of glucose uptake. Examining mitochondrial function in brown adipose tissue, we find that TXNIP KO mice have a lower content of polyunsaturated fatty acids (PUFAs) in their membrane lipids, which affects mitochondrial integrity and electron transport chain efficiency and ultimately results in lower mitochondrial heat output. This phenotype can be rescued by a ketogenic diet, confirming the usefulness of this model and highlighting one facet of early cellular damage caused by excess glucose influx.
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Tecido Adiposo Marrom/metabolismo , Carboidratos da Dieta/efeitos adversos , Mitocôndrias/metabolismo , Tecido Adiposo Marrom/ultraestrutura , Animais , Transporte Biológico/genética , Proteínas de Transporte/metabolismo , Dieta Cetogênica , Ácidos Graxos Insaturados/metabolismo , Regulação da Expressão Gênica , Lipidômica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/ultraestrutura , Termogênese/genética , Tiorredoxinas/metabolismoRESUMO
The repurposing of drugs developed to treat type 2 diabetes for the treatment of Parkinson's disease (PD) was encouraged by the beneficial effect exerted by the glucagon-like peptide 1 (GLP-1) analogue exenatide in a phase 2 clinical trial. The effects of GLP-1 analogues have been investigated extensively using rodent toxin models of PD. However, many of the toxin-based models used lack robust α-synuclein (α-syn) pathology, akin to the Lewy bodies and neurites seen in PD. One prior study has reported a protective effect of a GLP-1 analogue on midbrain dopamine neurons following injection of α-syn preformed fibrils (PFF) into the striatum. Here, we used olfactory bulb injections of PFF as a model of prodromal PD and monitored the effect of a long-acting GLP-1 analogue on the propagation of α-syn pathology in the olfactory system. Thirteen weeks after PFF injection, mice treated with long-acting the GLP-1 analogue had a significant increase in pathological α-syn in brain regions connected to the olfactory bulb, accompanied by signs of microglia activation. Our results suggest that the nature of the neuronal insult and intrinsic properties of the targeted neuronal population markedly influence the effect of GLP-1 analogues.
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Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/metabolismo , Sintomas Prodrômicos , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade , Animais , Modelos Animais de Doenças , Feminino , Peptídeo 1 Semelhante ao Glucagon/administração & dosagem , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , alfa-Sinucleína/administração & dosagemRESUMO
Genomic imprinting occurs before fertilization, impacts every cell of the developing child, and may be sensitive to environmental perturbations. The noncoding RNA, nc886 (also called VTRNA2-1) is the only known example of the â¼100 human genes imprinted by DNA methylation, that shows polymorphic imprinting in the population. The nc886 gene is part of an â¼1.6-kb differentially methylated region (DMR) that is methylated in the oocyte and silenced on the maternal allele in about 75% of humans worldwide. Here, we show that the presence or absence of imprinting at the nc886 DMR in an individual is consistent across different tissues, confirming that the imprint is established before cellular differentiation and is maintained into adulthood. We investigated the relationships between the frequency of imprinting in newborns and maternal age, alcohol consumption and cigarette smoking before conception in more than 1,100 mother/child pairs from South Africa. The probability of imprinting in newborns was increased in older mothers and decreased in mothers who drank alcohol before conception. On the other hand, cigarette smoking had no apparent relationship with the frequency of imprinting. These data show an epigenetic change during oocyte maturation which is potentially subject to environmental influence. Much focus has been placed on avoiding alcohol consumption during pregnancy, but our data suggest that drinking before conception may affect the epigenome of the newborn.
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Consumo de Bebidas Alcoólicas/efeitos adversos , Metilação de DNA , Epigênese Genética , Impressão Genômica , Exposição Materna/efeitos adversos , Oócitos/metabolismo , RNA não Traduzido/genética , Alelos , Ilhas de CpG , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Idade Materna , GravidezRESUMO
Analysis of formalin-fixed paraffin-embedded (FFPE) tissue by immunohistochemistry (IHC) is commonplace in clinical and research laboratories. However, reports suggest that IHC results can be compromised by biospecimen preanalytical factors. The National Cancer Institute's Biospecimen Preanalytical Variables Program conducted a systematic study to examine the potential effects of delay to fixation (DTF) and time in fixative (TIF) on IHC using 24 cancer biomarkers. Differences in IHC staining, relative to controls with a DTF of 1 hr, were observed in FFPE kidney tumor specimens after a DTF of ≥2 hr. Reductions in H-score and/or staining intensity were observed for c-MET, p53, PAX2, PAX8, pAKT, and survivin, whereas increases were observed for RCC1, EGFR, and CD10. Prolonged TIF of 72 hr resulted in significantly reduced H-scores of CD44 and c-Met in kidney tumor specimens, compared with controls with 12-hr TIF. An elevated probability of altered staining intensity due to DTF was observed for nine antigens, whereas for prolonged TIF an elevated probability was observed for one antigen. Results reported here and elsewhere across tumor types and antigens support limiting DTF to ≤1 hr when possible and fixing tissues in formalin for 12-24 hr to avoid confounding effects of these preanalytical factors on IHC.
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Biomarcadores Tumorais/análise , Imuno-Histoquímica/métodos , Formaldeído , Humanos , Neoplasias Renais/patologia , Inclusão em Parafina , Fixação de TecidosRESUMO
Background: In some people with Parkinson's disease (PD), α-synuclein (αSyn) accumulation may begin in the enteric nervous system (ENS) decades before development of brain pathology and disease diagnosis. Objective: To determine how different types and severity of intestinal inflammation could trigger αSyn accumulation in the ENS and the subsequent development of αSyn brain pathology. Methods: We assessed the effects of modulating short- and long-term experimental colitis on αSyn accumulation in the gut of αSyn transgenic and wild type mice by immunostaining and gene expression analysis. To determine the long-term effect on the brain, we induced dextran sulfate sodium (DSS) colitis in young αSyn transgenic mice and aged them under normal conditions up to 9 or 21 months before tissue analyses. Results: A single strong or sustained mild DSS colitis triggered αSyn accumulation in the submucosal plexus of wild type and αSyn transgenic mice, while short-term mild DSS colitis or inflammation induced by lipopolysaccharide did not have such an effect. Genetic and pharmacological modulation of macrophage-associated pathways modulated the severity of enteric αSyn. Remarkably, experimental colitis at three months of age exacerbated the accumulation of aggregated phospho-Serine 129 αSyn in the midbrain (including the substantia nigra), in 21- but not 9-month-old αSyn transgenic mice. This increase in midbrain αSyn accumulation is accompanied by the loss of tyrosine hydroxylase-immunoreactive nigral neurons. Conclusions: Our data suggest that specific types and severity of intestinal inflammation, mediated by monocyte/macrophage signaling, could play a critical role in the initiation and progression of PD.
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Rhabdoid tumor (RT) is a pediatric cancer characterized by the inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. Although this deletion is the known oncogenic driver, there are limited effective therapeutic options for these patients. Here we use unbiased screening of cell line panels to identify a heightened sensitivity of rhabdoid tumor to mithramycin and the second-generation analogue EC8042. The sensitivity of MMA and EC8042 was superior to traditional DNA damaging agents and linked to the causative mutation of the tumor, SMARCB1 deletion. Mithramycin blocks SMARCB1-deficient SWI/SNF activity and displaces the complex from chromatin to cause an increase in H3K27me3. This triggers chromatin remodeling and enrichment of H3K27ac at chromHMM-defined promoters to restore cellular differentiation. These effects occurred at concentrations not associated with DNA damage and were not due to global chromatin remodeling or widespread gene expression changes. Importantly, a single 3-day infusion of EC8042 caused dramatic regressions of RT xenografts, recapitulated the increase in H3K27me3, and cellular differentiation described in vitro to completely cure three out of eight mice.