RESUMO
Perturbation of the excitation/inhibition (E/I) balance leads to neurodevelopmental diseases including to autism spectrum disorders, intellectual disability, and epilepsy. Loss-of-function mutations in the DYRK1A gene, located on human chromosome 21 (Hsa21,) lead to an intellectual disability syndrome associated with microcephaly, epilepsy, and autistic troubles. Overexpression of DYRK1A, on the other hand, has been linked with learning and memory defects observed in people with Down syndrome (DS). Dyrk1a is expressed in both glutamatergic and GABAergic neurons, but its impact on each neuronal population has not yet been elucidated. Here we investigated the impact of Dyrk1a gene copy number variation in glutamatergic neurons using a conditional knockout allele of Dyrk1a crossed with the Tg(Camk2-Cre)4Gsc transgenic mouse. We explored this genetic modification in homozygotes, heterozygotes and combined with the Dp(16Lipi-Zbtb21)1Yey trisomic mouse model to unravel the consequence of Dyrk1a dosage from 0 to 3, to understand its role in normal physiology, and in MRD7 and DS. Overall, Dyrk1a dosage in postnatal glutamatergic neurons did not impact locomotor activity, working memory or epileptic susceptibility, but revealed that Dyrk1a is involved in long-term explicit memory. Molecular analyses pointed at a deregulation of transcriptional activity through immediate early genes and a role of DYRK1A at the glutamatergic post-synapse by deregulating and interacting with key post-synaptic proteins implicated in mechanism leading to long-term enhanced synaptic plasticity. Altogether, our work gives important information to understand the action of DYRK1A inhibitors and have a better therapeutic approach.
Assuntos
Transtorno Autístico/genética , Transtornos Cognitivos/genética , Síndrome de Down/genética , Dosagem de Genes , Ácido Glutâmico/metabolismo , Deficiência Intelectual/genética , Neurônios/metabolismo , Distúrbios da Fala/genética , Animais , Encéfalo/patologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Transtornos Cognitivos/complicações , Modelos Animais de Doenças , Síndrome de Down/complicações , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Proteômica/métodos , Transmissão Sináptica/genética , Transcrição GênicaRESUMO
In multiple sclerosis (MS), Th17 cells are critical drivers of autoimmune central nervous system (CNS) inflammation and demyelination. Th17 cells exhibit functional heterogeneity fostering both pathogenic and nonpathogenic, tissue-protective functions. Still, the factors that control Th17 pathogenicity remain incompletely defined. Here, using experimental autoimmune encephalomyelitis, an established mouse MS model, we report that therapeutic administration of activin-A ameliorates disease severity and alleviates CNS immunopathology and demyelination, associated with decreased activation of Th17 cells. In fact, activin-A signaling through activin-like kinase-4 receptor represses pathogenic transcriptional programs in Th17-polarized cells, while it enhances antiinflammatory gene modules. Whole-genome profiling and in vivo functional studies revealed that activation of the ATP-depleting CD39 and CD73 ectonucleotidases is essential for activin-A-induced suppression of the pathogenic signature and the encephalitogenic functions of Th17 cells. Mechanistically, the aryl hydrocarbon receptor, along with STAT3 and c-Maf, are recruited to promoter elements on Entpd1 and Nt5e (encoding CD39 and CD73, respectively) and other antiinflammatory genes, and control their expression in Th17 cells in response to activin-A. Notably, we show that activin-A negatively regulates the metabolic sensor, hypoxia-inducible factor-1α, and key inflammatory proteins linked to pathogenic Th17 cell states. Of translational relevance, we demonstrate that activin-A is induced in the CNS of individuals with MS and restrains human Th17 cell responses. These findings uncover activin-A as a critical controller of Th17 cell pathogenicity that can be targeted for the suppression of autoimmune CNS inflammation.
Assuntos
5'-Nucleotidase/metabolismo , Ativinas/farmacologia , Antígenos CD/metabolismo , Apirase/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/prevenção & controle , Esclerose Múltipla/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Células Th17/metabolismoRESUMO
Intrahepatic cholestasis of pregnancy (ICP) causes increased transfer of maternal bile acids to the fetus and an increased incidence of sudden fetal death. Treatment includes ursodeoxycholic acid (UDCA), but it is not clear if UDCA protects the fetus. This study explores the placental transport of the bile acid taurocholate (TC) by the organic anion-transporting polypeptide, (OATP)4A1, its effects on the placental proteome and vascular function, and how these are modified by UDCA. Various methodological approaches including placental villous fragments and Xenopus laevis oocytes were used to investigate UDCA transport. Placental perfusions and myography investigated the effect of TC on vasculature. The effects of acute TC exposure on placental tissue were investigated using quantitative proteomics. UDCA inhibited OATP4A1 activity in placental villous fragments and oocytes. TC induced vasoconstriction in placental and rat vasculature, which was attenuated by UDCA. Quantitative proteomic analysis of villous fragments showed direct effects of TC on multiple placental pathways, including oxidative stress and autophagy. The effects of TC on the placental proteome and vasculature demonstrate how bile acids may cause fetal distress in ICP. UDCA inhibition of OATP4A1 suggests it will protect the mother and fetus against the vascular effects of TC by inhibiting its cellular uptake. UDCA may protect the fetus in ICP by inhibiting OATP4A1-mediated bile acid transfer and TC-induced placental vasoconstriction. Understanding the physiologic mechanisms of UDCA may allow better therapeutic interventions to be designed specifically for the fetus in the future.-Lofthouse, E. M., Torrens, C., Manousopoulou, A., Nahar, M., Cleal, J. K., O'Kelly, I. M., Sengers, B. G., Garbis, S. D., Lewis, R. M. Ursodeoxycholic acid inhibits uptake and vasoconstrictor effects of taurocholate in human placenta.
Assuntos
Placenta , Ácido Taurocólico/metabolismo , Ácido Ursodesoxicólico/farmacologia , Vasoconstrição/efeitos dos fármacos , Animais , Morte Celular Autofágica/efeitos dos fármacos , Feminino , Humanos , Masculino , Transportadores de Ânions Orgânicos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Placenta/irrigação sanguínea , Placenta/metabolismo , Gravidez , Ratos , Ratos Wistar , Xenopus laevisRESUMO
Approximately 800,000 leukemia and lymphoma cases are diagnosed worldwide each year. Burkitt's lymphoma (BL) and chronic lymphocytic leukemia (CLL) are examples of contrasting B-cell cancers; BL is a highly aggressive lymphoid tumor, frequently affecting children, whereas CLL typically presents as an indolent, slow-progressing leukemia affecting the elderly. The B-cell-specific overexpression of the myc and TCL1 oncogenes in mice induce spontaneous malignancies modeling BL and CLL, respectively. Quantitative mass spectrometry proteomics and isobaric labeling were employed to examine the biology underpinning contrasting Eµ-myc and Eµ-TCL1 B-cell tumors. Additionally, the plasma proteome was evaluated using subproteome enrichment to interrogate biomarker emergence and the systemic effects of tumor burden. Over 10,000 proteins were identified (q<0.01) of which 8270 cellular and 2095 plasma proteins were quantitatively profiled. A common B-cell tumor signature of 695 overexpressed proteins highlighted ribosome biogenesis, cell-cycle promotion and chromosome segregation. Eµ-myc tumors overexpressed several methylating enzymes and underexpressed many cytoskeletal components. Eµ-TCL1 tumors specifically overexpressed ER stress response proteins and signaling components in addition to both subunits of the interleukin-5 (IL5) receptor. IL5 treatment promoted Eµ-TCL1 tumor proliferation, suggesting an amplification of IL5-induced AKT signaling by TCL1. Tumor plasma contained a substantial tumor lysis signature, most prominent in Eµ-myc plasma, whereas Eµ-TCL1 plasma contained signatures of immune-response, inflammation and microenvironment interactions, with putative biomarkers in early-stage cancer. These findings provide a detailed characterization of contrasting B-cell tumor models, identifying common and specific tumor mechanisms. Integrated plasma proteomics allowed the dissection of a systemic response and a tumor lysis signature present in early- and late-stage cancers, respectively. Overall, this study suggests common B-cell cancer signatures exist and illustrates the potential of the further evaluation of B-cell cancer subtypes by integrative proteomics.
Assuntos
Biomarcadores Tumorais/análise , Linfoma de Burkitt/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Linfoma de Burkitt/genética , Estresse do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Espectrometria de Massas/métodos , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: Early-onset breast cancer (EOBC) affects about one in 300 women aged 40 years or younger and is associated with worse outcomes than later onset breast cancer. This study explored novel serum proteins as surrogate markers of prognosis in patients with EOBC. METHODS: Serum samples from EOBC patients (stages 1-3) were analysed using agnostic high-precision quantitative proteomics. Patients received anthracycline-based chemotherapy. The discovery cohort (n = 399) either had more than 5-year disease-free survival (DFS) (good outcome group, n = 203) or DFS of less than 2 years (poor outcome group, n = 196). Expressed proteins were assessed for differential expression between the two groups. Bioinformatics pathway and network analysis in combination with literature research were used to determine clinically relevant proteins. ELISA analysis against an independent sample set from the Prospective study of Outcomes in Sporadic versus Hereditary breast cancer (POSH) cohort (n = 181) was used to validate expression levels of the selected target. Linear and generalized linear modelling was applied to determine the effect of target markers, body mass index (BMI), lymph node involvement (LN), oestrogen receptor (ER), progesterone receptor and human epidermal growth factor receptor 2 status on patients' outcome. RESULTS: A total of 5346 unique proteins were analysed (peptide FDR p ≤ 0.05). Of these, 812 were differentially expressed in the good vs poor outcome groups and showed significant enrichment for the insulin signalling (p = 0.01) and the glycolysis/gluconeogenesis (p = 0.01) pathways. These proteins further correlated with interaction networks involving glucose and fatty acid metabolism. A consistent nodal protein to these metabolic networks was resistin (upregulated in the good outcome group, p = 0.009). ELISA validation demonstrated resistin to be upregulated in the good outcome group (p = 0.04), irrespective of BMI and ER status. LN involvement was the only covariate with a significant association with resistin measurements (p = 0.004). An ancillary in-silico observation was the induction of the inflammatory response, leucocyte infiltration, lymphocyte migration and recruitment of phagocytes (p < 0.0001, z-score > 2). Survival analysis showed that resistin overexpression was associated with improved DFS. CONCLUSIONS: Higher circulating resistin correlated with node-negative patients and longer DFS independent of BMI and ER status in women with EOBC. Overexpression of serum resistin in EOBC may be a surrogate indicator of improved prognosis.
Assuntos
Proteínas Sanguíneas/genética , Neoplasias da Mama/sangue , Proteômica , Resistina/sangue , Adulto , Biomarcadores Tumorais/sangue , Índice de Massa Corporal , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Resistência à Insulina , Linfonodos/patologia , Células Neoplásicas Circulantes/patologia , Prognóstico , Receptores de Estrogênio/genética , Receptores de Progesterona/genéticaRESUMO
BACKGROUND: Cancer-associated fibroblasts (CAFs) form the major stromal component of the tumour microenvironment (TME). The present study aimed to examine the proteomic profiles of CAFs vs. normal fibroblasts (NOFs) from patients with oesophageal adenocarcinoma to gain insight into their pro-oncogenic phenotype. METHODS: CAFs/NOFs from four patients were sub-cultured and analysed using quantitative proteomics. Differentially expressed proteins (DEPs) were subjected to bioinformatics and compared with published proteomics and transcriptomics datasets. RESULTS: Principal component analysis of all profiled proteins showed that CAFs had high heterogeneity and clustered separately from NOFs. Bioinformatics interrogation of the DEPs demonstrated inhibition of adhesion of epithelial cells, adhesion of connective tissue cells and cell death of fibroblast cell lines in CAFs vs. NOFs (p < 0.0001). KEGG pathway analysis showed a significant enrichment of the insulin-signalling pathway (p = 0.03). Gene ontology terms related with myofibroblast phenotype, metabolism, cell adhesion/migration, hypoxia/oxidative stress, angiogenesis, immune/inflammatory response were enriched in CAFs vs. NOFs. Nestin, a stem-cell marker up-regulated in CAFs vs. NOFs, was confirmed to be expressed in the TME with immunohistochemistry. CONCLUSIONS: The identified pathways and participating proteins may provide novel insight on the tumour-promoting properties of CAFs and unravel novel adjuvant therapeutic targets in the TME.
Assuntos
Adenocarcinoma/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Neoplasias Esofágicas/metabolismo , Fibroblastos/metabolismo , Proteoma/análise , Adenocarcinoma/patologia , Fibroblastos Associados a Câncer/patologia , Células Cultivadas , Conjuntos de Dados como Assunto , Neoplasias Esofágicas/patologia , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Cultura Primária de Células , Proteoma/metabolismo , Proteômica/métodos , Transcriptoma , Microambiente Tumoral/fisiologiaRESUMO
Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory properties, so this finding indicates it may be involved in metabolic remodeling and also serve as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly those two candidates were not up-regulated in pregnancy or exercise induced CH, indicating PKM2 and eEF1 were pathological CH specific markers. We anticipate that the methodologies described here will be valuable for other researchers studying protein synthesis in primary cells.
Assuntos
Insulina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Fenilefrina/farmacologia , Proteoma/efeitos dos fármacos , Proteômica/métodos , Animais , Células Cultivadas , Cromatografia Líquida , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Espectrometria de Massas , Miócitos Cardíacos/metabolismo , Proteoma/genética , Ratos , Ratos Sprague-DawleyRESUMO
Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Córtex Cerebral/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Córtex Cerebral/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Overweight adults are at increased risk for cardiovascular disease and vitamin D deficiency, whereas an important feature to vitamin D physiology is its sex dependence. The aim of this study was to examine whether vitamin D status improvement exerts a sexually dimorphic effect on serum proteins associated with cardiovascular risk among overweight adults. MATERIALS AND METHODS: Unprocessed serum from age- and BMI-matched men (n = 26) and premenopausal women (n = 24) with vitamin D deficiency and after they achieved sufficiency through a 12-month nutritional intervention was analysed using our previously published depletion-free quantitative proteomics method. Key findings were verified with ELISA. Differentially expressed proteins were subjected to in silico bioinformatics assessment using principal component analysis, hierarchical clustering and Metacore™ pathway analysis. All mass spectrometry proteomic data are available via ProteomeXchange (identifier: PXD003663). RESULTS: A total of 282 proteins were differentially expressed after the intervention between men and women (P-value ≤ 0·05), in which the blood coagulation pathway was significantly enriched. In agreement with the proteomics findings, ELISA measurements showed vitamin K-dependent protein C, von Willebrand factor, fibrinogen gamma chain and multimerin-1 proteins, of relevance to blood coagulation, to be differentially affected (P-value ≤ 0·05) between sexes after vitamin D status correction. CONCLUSIONS: This study identified novel protein-level molecular indicators on the sexually dimorphic effect of vitamin D status correction associated with blood coagulation among overweight adults. These sex-mediated vitamin D effects should be factored in the design and interpretation of vitamin D observational and interventional studies testing cardiometabolic outcomes.
Assuntos
Proteínas Sanguíneas/metabolismo , Fibrinogênio/metabolismo , Sobrepeso/metabolismo , Proteína C/metabolismo , Deficiência de Vitamina D/metabolismo , Vitamina D/análogos & derivados , Fator de von Willebrand/metabolismo , Adulto , Coagulação Sanguínea , Estudos de Casos e Controles , Simulação por Computador , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Espectrometria de Massas , Sobrepeso/complicações , Análise de Componente Principal , Proteômica , Fatores Sexuais , Resultado do Tratamento , Vitamina D/metabolismo , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/dietoterapia , Adulto JovemRESUMO
BACKGROUND: Obesity is a risk factor for both vitamin D deficiency and cardiovascular disease. A link between vitamin D status optimisation and improved cardiometabolic profile among adults with obesity could inform public health initiatives. METHODS: PubMed, Embase and Web of Science were searched for interventional studies examining the effects of vitamin D status improvement on cardiovascular risk factors (anthropometric measures, lipid profile, blood pressure, glucose tolerance) among nondiabetic adults with obesity. RESULTS: Seventeen publications reporting results from 11 different studies were included. Number of participants ranged from 34 to 1179 subjects. Duration was between 6 weeks and 4 years. Vitamin D was administered as a supplement in ten studies (1000 IU daily to 120 000 IU fortnightly). In one study, participants were advised to increase sunlight exposure and dietary vitamin D intake. The random and fixed-effects meta-analysis showed that vitamin D significantly increased systolic blood pressure and LDL-C levels. The fixed-effects model also indicated a significant decrease in triglyceride levels, which was not evident using the random-effects model. Caution should be given to these results given the small number of studies used and the high heterogeneity between studies for the two latter outcomes. Additionally, a subset of eligible studies with compatible data presentation was included in the meta-analysis. CONCLUSION: This systematic review highlights a paucity of interventional studies examining the effects of vitamin D status improvement on cardiovascular risk factors among otherwise healthy adults with obesity. Large-scale studies at pharmacologically relevant doses and with sufficient duration are warranted.
Assuntos
Doenças Cardiovasculares/etiologia , Obesidade/complicações , Deficiência de Vitamina D/etiologia , Adulto , Idoso , Glicemia/metabolismo , Pressão Sanguínea/fisiologia , Cálcio/administração & dosagem , Suplementos Nutricionais , Feminino , Humanos , Metabolismo dos Lipídeos/fisiologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto , Fatores de Risco , Luz Solar , Vitamina D/administração & dosagem , Deficiência de Vitamina D/dietoterapia , Deficiência de Vitamina D/prevenção & controle , Vitaminas/administração & dosagemRESUMO
BACKGROUNDNovel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections.METHODSWe applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modeling and network correlation analyses identified 118 differentially expressed proteins, significant through 3 complementary analytical pipelines. Candidate biomarkers were subsequently analyzed in 2 validation cohorts of differing ethnicity using antibody-based proximity extension assays.RESULTSTB-specific host biomarkers were confirmed. A 6-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14, and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts.CONCLUSIONThis biomarker panel exceeds the World Health Organization Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.FUNDINGMedical Research Council (MRC) (MR/R001065/1, MR/S024220/1, MR/P023754/1, and MR/W025728/1); the MRC and the UK Foreign Commonwealth and Development Office; the UK National Institute for Health Research (NIHR); the Wellcome Trust (094000, 203135, and CC2112); Starter Grant for Clinical Lecturers (Academy of Medical Sciences UK); the British Infection Association; the Program for Advanced Research Capacities for AIDS in Peru at Universidad Peruana Cayetano Heredia (D43TW00976301) from the Fogarty International Center at the US NIH; the UK Technology Strategy Board/Innovate UK (101556); the Francis Crick Institute, which receives funding from UKRI-MRC (CC2112); Cancer Research UK (CC2112); and the NIHR Biomedical Research Centre of Imperial College NHS.
Assuntos
Biomarcadores , Proteômica , Tuberculose Pulmonar , Humanos , Biomarcadores/sangue , Proteômica/métodos , Masculino , Feminino , Adulto , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/sangue , Mycobacterium tuberculosis , Pessoa de Meia-Idade , Peru/epidemiologia , África do Sul/epidemiologia , Estudos de Casos e Controles , Sensibilidade e EspecificidadeRESUMO
Elimination of the latent HIV cell reservoir may be possible, if the molecular identity of latently infected cells were fully elucidated. We conducted comprehensive molecular profiling, at the protein and RNA levels, of primary T cells latently infected with HIV in vitro. Isobaric labelling quantitative proteomics and RNA sequencing identified 1453 proteins and 618 genes, altered in latently infected cells compared to mock-infected controls (p < 0.05). Biomarker selection was based on results from integrated data analysis. Relative enrichment for latently infected cells was monitored using flow cytometric sorting and the HIV integrant assay. Antibodies against selected proteins, encoded by CEACAM1 and PLXNB2, enabled enrichment of latently infected cells from cell mixtures by 3-10 fold (5.8 average, p < 0.001), comparable to levels obtained with biomarkers reported previously. Individual biomarkers are likely linked to subsets of latently infected cells, and an extended antibody panel will be required to inclusively target the latent HIV reservoir.
Assuntos
Infecções por HIV , HIV-1 , Linfócitos T CD4-Positivos , HIV-1/genética , Humanos , Proteômica , Transcriptoma , Ativação Viral , Latência ViralRESUMO
The chemotherapy resistance of esophageal adenocarcinomas (EACs) is underpinned by cancer cell extrinsic mechanisms of the tumor microenvironment (TME). We demonstrate that, by targeting the tumor-promoting functions of the predominant TME cell type, cancer-associated fibroblasts (CAFs) with phosphodiesterase type 5 inhibitors (PDE5i), we can enhance the efficacy of standard-of-care chemotherapy. In ex vivo conditions, PDE5i prevent the transdifferentiation of normal fibroblasts to CAF and abolish the tumor-promoting function of established EAC CAFs. Using shotgun proteomics and single-cell RNA-seq, we reveal PDE5i-specific regulation of pathways related to fibroblast activation and tumor promotion. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient and in vivo PDX-based model systems. These findings demonstrate that CAFs drive chemotherapy resistance in EACs and can be targeted by repurposing PDE5i, a safe and well-tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction.
Assuntos
Adenocarcinoma , Fibroblastos Associados a Câncer , Neoplasias Esofágicas , Adenocarcinoma/tratamento farmacológico , Fibroblastos Associados a Câncer/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Humanos , Masculino , Inibidores da Fosfodiesterase 5/farmacologia , Microambiente TumoralRESUMO
Pregnancy 25-hydroxyvitamin D [25(OH)D] concentrations are associated with maternal and fetal health outcomes. Using physiological human placental perfusion and villous explants, we investigate the role of the placenta in regulating the relationships between maternal 25(OH)D and fetal physiology. We demonstrate active placental uptake of 25(OH)D3 by endocytosis, placental metabolism of 25(OH)D3 into 24,25-dihydroxyvitamin D3 and active 1,25-dihydroxyvitamin D [1,25(OH)2D3], with subsequent release of these metabolites into both the maternal and fetal circulations. Active placental transport of 25(OH)D3 and synthesis of 1,25(OH)2D3 demonstrate that fetal supply is dependent on placental function rather than simply the availability of maternal 25(OH)D3. We demonstrate that 25(OH)D3 exposure induces rapid effects on the placental transcriptome and proteome. These map to multiple pathways central to placental function and thereby fetal development, independent of vitamin D transfer. Our data suggest that the underlying epigenetic landscape helps dictate the transcriptional response to vitamin D treatment. This is the first quantitative study demonstrating vitamin D transfer and metabolism by the human placenta, with widespread effects on the placenta itself. These data demonstrate a complex interplay between vitamin D and the placenta and will inform future interventions using vitamin D to support fetal development and maternal adaptations to pregnancy.
Assuntos
Placenta , Vitamina D , Calcifediol/metabolismo , Feminino , Feto/metabolismo , Humanos , Placenta/metabolismo , Gravidez , Vitamina D/metabolismo , Vitaminas/metabolismoRESUMO
INTRODUCTION: Placental oxidative stress features in pregnancy pathologies but in clinical trials antioxidant supplementation has not improved outcomes. N-acetylcysteine (NAC) stimulates glutathione production and is proposed as a therapeutic agent in pregnancy. However, key elements of N-acetylcysteine biology, including its cellular uptake mechanism, remains unclear. This study explores how the cystine/glutamate transporter xCT may mediate N-acetylcysteine uptake and how N-acetylcysteine alters placental redox status. METHODS: The involvement of xCT in NAC uptake by the human placenta was studied in perfused placenta and Xenopus oocytes. The effect of short-term N-acetylcysteine exposure on the placental villous proteome was determined using LC-MS. The effect of N-acetylcysteine on Maxi-chloride channel activity was investigated in perfused placenta, villous fragments and cell culture. RESULTS: Maternoplacental N-acetylcysteine administration stimulated intracellular glutamate efflux suggesting a role of the exchange transporter xCT, which was localised to the microvillous membrane of the placental syncytiotrophoblast. Placental exposure to a bolus of N-acetylcysteine inhibited subsequent activation of the redox sensitive Maxi-chloride channel independently of glutathione synthesis. Stable isotope quantitative proteomics of placental villi treated with N-acetylcysteine demonstrated changes in pathways associated with oxidative stress, apoptosis and the acute phase response. DISCUSSION: This study suggests that xCT mediates N-acetylcysteine uptake into the placenta and that N-acetylcysteine treatment of placental tissue alters the placental proteome while regulating the redox sensitive Maxi-chloride channel. Interestingly N-acetylcysteine had antioxidant effects independent of the glutathione pathway. Effective placental antioxidant therapy in pregnancy may require maintaining the balance between normalising redox status without inhibiting physiological redox signalling.
Assuntos
Acetilcisteína/farmacologia , Sistema y+ de Transporte de Aminoácidos/genética , Canais de Cloreto/antagonistas & inibidores , Placenta , Acetilcisteína/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Canais de Cloreto/metabolismo , Vilosidades Coriônicas/efeitos dos fármacos , Vilosidades Coriônicas/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Xenopus laevisRESUMO
OBJECTIVE: Fetal growth restriction is associated with increased postnatal cardiovascular morbidity. The alterations in heart physiology and structure caused by in utero nutrient deprivation have not been extensively studied. We aim to investigate the impact of maternal food restriction on the cardiac proteome of newborn rats with normal (non-fetal growth-restricted (FGR)) and reduced (FGR) birth weight. METHODS: On day 14 of gestation, 10 timed pregnant rats were randomized into two nutritional groups: (a) Standard laboratory diet and (b) 50% global food restriction. Pups born to food-restricted mothers were subdivided, based on birthweight, into fetal growth-restricted (FGR) and non-FGR, while pups born from normally nourished mothers were considered controls. Rat neonates were euthanized immediately after birth and the hearts of 11 randomly selected male offspring (n = 4 FGR, n = 4 non-FGR, n = 3 control group) were analyzed using quantitative proteomics. RESULTS: In total, 7422 proteins were quantified (q < 0.05). Of these, 1175 were differentially expressed in FGR and 231 in non-FGR offspring vs. control with 151 common differentially expressed proteins (DEPs) between the two groups. Bioinformatics analysis of DEPs in FGR vs. control showed decreased integrin and apelin cardiac fibroblast signaling, decreased muscle contraction and glycolysis, and over-representation of a protein network related to embryonic development, and cell death and survival. CONCLUSION: Our study illustrates the distinct proteomic profile of FGR and non-FGR offspring of food-restricted dams underlying the importance of both prenatal adversities and birth weight in cardiac physiology and development.
Assuntos
Animais Recém-Nascidos/metabolismo , Privação de Alimentos , Miocárdio/química , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Proteoma/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Peso ao Nascer , Feminino , Gravidez , Ratos , Ratos WistarRESUMO
BACKGROUND: Fetal growth restriction (FGR) has been associated with a higher risk of developing adverse perinatal outcomes and distinct neurodevelopmental and neurobehavioral disorders. The aim of the present study was to investigate the impact of prenatal food restriction on the brain proteome in both FGR and appropriately grown rats and to identify potential pathways connecting maternal malnutrition with altered brain development. METHODS: Ten time-dated pregnant Wistar rats were housed individually at their 12th day of gestation. On the 15th day of gestation, the rats were randomly divided into two groups, namely the food restricted one (n = 6) and the control group (n = 4). From days 15 to 21 the control group had unlimited access to food and the food restricted group was given half the amount of food that was on average consumed by the control group, based on measurements taken place the day before. On the 21st day of gestation, all rats delivered spontaneously and after birth all newborn pups of the food restricted group were weighed and matched as appropriately grown (non-FGR) or growth restricted (FGR) and brain tissues were immediately collected. A multiplex experiment was performed analyzing brain tissues from 4 FGR, 4 non-FGR, and 3 control male offspring. Differentially expressed proteins (DEPs) were subjected to bioinformatics analysis in order to identify over-represented processes. RESULTS: Proteomic analysis resulted in the profiling of 3,964 proteins. Gene ontology analysis of the common DEPs using DAVID (https://david.ncifcrf.gov/) showed significant enrichment for terms related to cellular morphology, learning, memory and positive regulation of NF-kappaB signaling. Ingenuity Pathway Analysis showed significant induction of inflammation in FGR pups, whereas significant induction of cell migration and cell spreading were observed in non-FGR pups. CONCLUSION: This study demonstrated that in both FGR and non-FGR neonates, a range of adaptive neurodevelopmental processes takes place, which may result in altered cellular morphology, chronic stress, poor memory and learning outcomes. Furthermore, this study highlighted that not only FGR, but also appropriately grown pups, which have been exposed to prenatal food deprivation may be at increased risk for impaired cognitive and developmental outcomes.
RESUMO
Age is the major risk factor in most carcinomas, yet little is known about how proteomes change with age in any human epithelium. We present comprehensive proteomes comprised of >9,000 total proteins and >15,000 phosphopeptides from normal primary human mammary epithelia at lineage resolution from ten women ranging in age from 19 to 68 years. Data were quality controlled and results were biologically validated with cell-based assays. Age-dependent protein signatures were identified using differential expression analyses and weighted protein co-expression network analyses. Upregulation of basal markers in luminal cells, including KRT14 and AXL, were a prominent consequence of aging. PEAK1 was identified as an age-dependent signaling kinase in luminal cells, which revealed a potential age-dependent vulnerability for targeted ablation. Correlation analyses between transcriptome and proteome revealed age-associated loss of proteostasis regulation. Age-dependent proteome changes in the breast epithelium identified heretofore unknown potential therapeutic targets for reducing breast cancer susceptibility.