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SOCS1 is a tumor suppressor in hepatocellular carcinoma (HCC). Recently, we showed that a loss of SOCS1 in hepatocytes promotes NRF2 activation. Here, we investigated how SOCS1 expression in HCC cells affected oxidative stress response and modulated the cellular proteome. Murine Hepa1-6 cells expressing SOCS1 (Hepa-SOCS1) or control vector (Hepa-Vector) were treated with cisplatin or tert-butyl hydroperoxide (t-BHP). The induction of NRF2 and its target genes, oxidative stress, lipid peroxidation, cell survival and cellular proteome profiles were evaluated. NRF2 induction was significantly reduced in Hepa-SOCS1 cells. The gene and protein expression of NRF2 targets were differentially induced in Hepa-Vector cells but markedly suppressed in Hepa-SOCS1 cells. Hepa-SOCS1 cells displayed an increased induction of reactive oxygen species but reduced lipid peroxidation. Nonetheless, Hepa-SOCS1 cells treated with cisplatin or t-BHP showed reduced survival. GCLC, poorly induced in Hepa-SOCS1 cells, showed a strong positive correlation with NFE2L2 and an inverse correlation with SOCS1 in the TCGA-LIHC transcriptomic data. A proteomic analysis of Hepa-Vector and Hepa-SOCS1 cells revealed that SOCS1 differentially modulated many proteins involved in diverse molecular pathways, including mitochondrial ROS generation and ROS detoxification, through peroxiredoxin and thioredoxin systems. Our findings indicate that maintaining sensitivity to oxidative stress is an important tumor suppression mechanism of SOCS1 in HCC.
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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.
Assuntos
Linfócitos T CD8-Positivos , Histonas , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/farmacologia , Acetilação , Histonas/metabolismo , Corpos Cetônicos , Animais , CamundongosRESUMO
Both clinical and experimental data suggest that podocyte injury is involved in the onset and progression of diabetic kidney disease (DKD). Although the mechanisms underlying the development of podocyte loss are not completely understood, critical structural proteins such as podocin play a major role in podocyte survival and function. We have reported that the protein tyrosine phosphatase SHP-1 expression increased in podocytes of diabetic mice and glomeruli of patients with diabetes. However, the in vivo contribution of SHP-1 in podocytes is unknown. Conditional podocyte-specific SHP-1-deficient mice (Podo-SHP-1-/-) were generated to evaluate the impact of SHP-1 deletion at four weeks of age (early) prior to the onset of diabetes and after 20 weeks (late) of diabetes (DM; Ins2+/C96Y) on kidney function (albuminuria and glomerular filtration rate) and kidney pathology. Ablation of the SHP-1 gene specifically in podocytes prevented and even reversed the elevated albumin/creatinine ratio, glomerular filtration rate progression, mesangial cell expansion, glomerular hypertrophy, glomerular basement membrane thickening and podocyte foot process effacement induced by diabetes. Moreover, podocyte-specific deletion of SHP-1 at an early and late stage prevented diabetes-induced expression of collagen IV, fibronectin, transforming growth factor-ß, transforming protein RhoA, and serine/threonine kinase ROCK1, whereas it restored nephrin, podocin and cation channel TRPC6 expression. Mass spectrometry analysis revealed that SHP-1 reduced SUMO2 post-translational modification of podocin while podocyte-specific deletion of SHP-1 preserved slit diaphragm protein complexes in the diabetic context. Thus, our data uncovered a new role of SHP-1 in the regulation of cytoskeleton dynamics and slit diaphragm protein expression/stability, and its inhibition preserved podocyte function preventing DKD progression.
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Diabetes Mellitus Experimental , Nefropatias Diabéticas , Podócitos , Animais , Camundongos , Diabetes Mellitus Experimental/induzido quimicamente , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/prevenção & controle , Nefropatias Diabéticas/metabolismo , Podócitos/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Quinases Associadas a rho/metabolismo , SumoilaçãoRESUMO
Aggressive tumors evade cytotoxic T lymphocytes by suppressing MHC class-I (MHC-I) expression that also compromises tumor responsiveness to immunotherapy. MHC-I defects strongly correlate to defective expression of NLRC5, the transcriptional activator of MHC-I and antigen processing genes. In poorly immunogenic B16 melanoma cells, restoring NLRC5 expression induces MHC-I and elicits antitumor immunity, raising the possibility of using NLRC5 for tumor immunotherapy. As the clinical application of NLRC5 is constrained by its large size, we examined whether a smaller NLRC5-CIITA fusion protein, dubbed NLRC5-superactivator (NLRC5-SA) as it retains the ability to induce MHC-I, could be used for tumor growth control. We show that stable NLRC5-SA expression in mouse and human cancer cells upregulates MHC-I expression. B16 melanoma and EL4 lymphoma tumors expressing NLRC5-SA are controlled as efficiently as those expressing full-length NLRC5 (NLRC5-FL). Comparison of MHC-I-associated peptides (MAPs) eluted from EL4 cells expressing NLRC5-FL or NLRC5-SA and analyzed by mass spectrometry revealed that both NLRC5 constructs expanded the MAP repertoire, which showed considerable overlap but also included a substantial proportion of distinct peptides. Thus, we propose that NLRC5-SA, with its ability to increase tumor immunogenicity and promote tumor growth control, could overcome the limitations of NLRC5-FL for translational immunotherapy applications.
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Regulação da Expressão Gênica , Melanoma Experimental , Humanos , Animais , Camundongos , Melanoma Experimental/genética , Melanoma Experimental/terapia , Genes MHC Classe I , Antígenos de Histocompatibilidade Classe I , Apresentação de Antígeno , Peptídeos e Proteínas de Sinalização Intracelular/genéticaRESUMO
BACKGROUND: Radioresistance of HNSCCs remains a major challenge for effective tumor control. Combined radiotherapy (RT) and immunotherapy (IT) treatment improved survival for a subset of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To overcome radioresistance and improve treatment outcomes, an understanding of factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs. In this study, we investigated how radiation modulates Treg infiltration in tumors through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity. METHODS: Human and mouse HNSCC cell lines with different immune phenotypes were irradiated at doses of 2 or 10 Gy. Conditioned media, RNA and protein were collected for assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cells were implanted into the buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for analysis of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Mass-spectrometry was performed to analyze proteomic changes in the tumor microenvironment after anti-CCL20 treatment. RESULTS: Cal27 and MOC2 HNSCCs had a gene signature associated with Treg infiltration, whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed TME. In vitro, tumor irradiation at 10 Gy significantly induced CCL20 in Cal27 and MOC2 cells relative to control. The increase in CCL20 was associated with increased Treg migration. Neutralization of CCL20 reversed radiation-induced migration of Treg cells in vitro and decreased intratumoral Tregs in vivo. Furthermore, inhibition of CCL20 resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. This effect was further enhanced after combination with RT compared to either treatment alone. CONCLUSION: Our results suggest that radiation promotes CCL20 secretion by tumor cells which is responsible for the attraction of Tregs. Inhibition of the CCR6-CCL20 axis prevents infiltration of Tregs in tumors and suppresses tumor growth resulting in improved response to radiation.
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Neoplasias de Cabeça e Pescoço , Linfócitos T Reguladores , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Proteômica , Neoplasias de Cabeça e Pescoço/radioterapia , Neoplasias de Cabeça e Pescoço/metabolismo , Microambiente Tumoral , Receptores CCR6/genética , Receptores CCR6/metabolismoRESUMO
Radio-chemotherapy with 5-flu orouracil (5-FU) is the standard of care treatment for patients with colorectal cancer, but it is only effective for a third of them. Despite our understanding of the mechanism of action of 5-FU, drug resistance remains a significant limitation to the clinical use of 5-FU, as both intrinsic and acquired chemoresistance represents the major obstacles for the success of 5-FU-based chemotherapy. In order to identify the mechanism of acquired resistance, 5-FU chemoresistance was induced in CRC cell lines by passaging cells with increasing concentrations of 5-FU. To study global molecular changes, quantitative proteomics and transcriptomics analyses were performed on these cell lines, comparing the resistant cells as well as the effect of chemo and radiotherapy. Interestingly, a very high proportion of downregulated genes were annotated as transcription factors coding for Krüppel-associated box (KRAB) domain-containing zinc-finger proteins (KZFPs), the largest family of transcriptional repressors. Among nearly 350 KRAB-ZFPs, almost a quarter were downregulated after the induction of a 5-FU-resistance including a common one between the three CRC cell lines, ZNF649, whose role is still unknown. To confirm the observations of the proteomic and transcriptomic approaches, the abundance of 20 different KZFPs and control mRNAs was validated by RT-qPCR. In fact, several KZFPs were no longer detectable using qPCR in cell lines resistant to 5-FU, and the KZFPs that were downregulated only in one or two cell lines showed similar pattern of expression as measured by the omics approaches. This proteomic, transcriptomic and genomic analysis of intrinsic and acquired resistance highlights a possible new mechanism involved in the cellular adaptation to 5-FU and therefore identifies potential new therapeutic targets to overcome this resistance.
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Neoplasias Colorretais , Fluoruracila , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Proteômica , Dedos de Zinco/genéticaRESUMO
PURPOSE: Recent studies have identified transanastomotic tubes (TATs) as a risk factor for the development of anastomotic strictures after repair of esophageal atresia with tracheoesophageal fistula (EATEF). We further investigated these findings in a multicenter study. METHODS: We conducted a retrospective cohort study at three university-affiliated hospitals in the province of Quebec. All patients with types C and D EATEF who underwent primary repair between January 1993 and August 2018 were included. Anastomotic stricture was defined as clinical symptoms of stricture with confirmation on esophagram or endoscopy. Multivariate logistic regression and the Wilcoxon Rank-Sum test were used to evaluate the primary outcome of stricture within one year of surgery and secondary outcome of duration of postoperative total parenteral nutrition (TPN). RESULTS: 244 patients were included, of which 234 (96%) were type C and 10 (4%) were type D. The anastomotic stricture rate at 1 year was 30%. TATs were utilized in 61% of patients. Thirty-six percent of patients with TATs developed a stricture within one year, as compared to 19% of patients without TATs (p = 0.005). TATs were associated with stricture on univariate analysis (OR 2.49, p = 0.004, 95% CI: 1.37-4.69). On multivariate analysis, after adjusting for gestational age, birth weight, leak, long gap, anastomotic tension, and daily acid suppression, patients with TATs had 2.72 times higher odds of developing a stricture as compared to patients without TATs (p = 0.006, 95% CI: 1.35-5.74). The median duration of TPN was 9 days in both groups (p = 0.139, IQR 6-14 in patients with TATs versus IQR 7-16 in patients without). CONCLUSION: Transanastomotic tubes are associated with a significantly higher risk of postoperative stricture following repair of esophageal atresia with tracheoesophageal fistula and do not shorten the duration of total parenteral nutrition. LEVEL OF EVIDENCE: III.
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Atresia Esofágica , Estenose Esofágica , Fístula Traqueoesofágica , Anastomose Cirúrgica/efeitos adversos , Fístula Anastomótica , Constrição Patológica/cirurgia , Atresia Esofágica/cirurgia , Estenose Esofágica/etiologia , Estenose Esofágica/cirurgia , Humanos , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Quebeque/epidemiologia , Estudos Retrospectivos , Fístula Traqueoesofágica/etiologia , Fístula Traqueoesofágica/cirurgia , Resultado do TratamentoRESUMO
Suppressor of Cytokine Signaling 1 (SOCS1) functions as a tumor suppressor in hepatocellular carcinoma and many other types of cancers. SOCS1 mediates its functions by inhibiting tyrosine kinases, promoting ubiquitination and proteasomal degradation of signal transducing proteins, and by modulating transcription factors. Here, we studied the impact of SOCS1 on the hepatocyte proteome using Stable Isotopic Labelling of Amino acids in Cell culture (SILAC)-based mass spectrometry on the Hepa1-6 murine HCC cell line stably expressing wildtype SOCS1 or a mutant SOCS1 with impaired SH2 domain. As SOCS1 regulates the hepatocyte growth factor (HGF) receptor, the MET receptor tyrosine kinase (RTK), the SILAC-labelled cells were stimulated or not with HGF. Following mass spectrometry analysis, differentially modulated proteins were identified, quantified and analyzed for pathway enrichment. Of the 3440 proteins identified in Hepa-SOCS1 cells at steady state, 181 proteins were significantly modulated compared to control cells. The SH2 domain mutation and HGF increased the number of differentially modulated proteins. Protein interaction network analysis revealed enrichment of SOCS1-modulated proteins within multiprotein complexes such as ubiquitin conjugating enzymes, proteasome, mRNA spliceosome, mRNA exosome and mitochondrial ribosome. Notably, the expression of UBE2D ubiquitin conjugating enzyme, which is implicated in the control of growth factor receptor tyrosine kinase signaling, was found to be regulated by SOCS1. These findings suggest that SOCS1, induced by cytokines, growth factors and diverse other stimuli, has the potential to dynamically modulate of large macromolecular regulatory complexes to help maintain cellular homeostasis.
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Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Proteômica , Proteínas Proto-Oncogênicas c-met/biossíntese , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Linhagem Celular , Camundongos , Proteínas Proto-Oncogênicas c-met/genética , Proteína 1 Supressora da Sinalização de Citocina/genética , Enzimas de Conjugação de Ubiquitina/genética , Domínios de Homologia de srcRESUMO
HNF4α is a nuclear receptor produced as 12 isoforms from two promoters by alternative splicing. To characterize the transcriptional capacities of all 12 HNF4α isoforms, stable lines expressing each isoform were generated. The entire transcriptome associated with each isoform was analyzed as well as their respective interacting proteome. Major differences were noted in the transcriptional function of these isoforms. The α1 and α2 isoforms were the strongest regulators of gene expression whereas the α3 isoform exhibited significantly reduced activity. The α4, α5, and α6 isoforms, which use an alternative first exon, were characterized for the first time, and showed a greatly reduced transcriptional potential with an inability to recognize the consensus response element of HNF4α. Several transcription factors and coregulators were identified as potential specific partners for certain HNF4α isoforms. An analysis integrating the vast amount of omics data enabled the identification of transcriptional regulatory mechanisms specific to certain HNF4α isoforms, hence demonstrating the importance of considering all isoforms given their seemingly diverse functions.
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Fator 4 Nuclear de Hepatócito/metabolismo , Transcrição Gênica , Linhagem Celular Tumoral , DNA/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Fluorescência Verde/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Humanos , Ligação Proteica , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
Hepatocyte nuclear factor 4α (HNF4α) is a transcription factor that acts as a master regulator of genes for several endoderm-derived tissues, including the intestine, in which it plays a central role during development and tumorigenesis. To better define the mechanisms by which HNF4α can influence these processes, we identified proteins interacting with HNF4α using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics with either immunoprecipitation of green fluorescent protein (GFP) or with proximity-dependent purification by the biotin ligase BirA (BioID), both fused to HNF4α. Surprisingly, these analyses identified a significant enrichment of proteins characterized with a role in DNA repair, a so far unidentified biological feature of this transcription factor. Several of these proteins including PARP1, RAD50, and DNA-PKcs were confirmed to interact with HNF4α in colorectal cancer cell lines. Following DNA damage, HNF4α was able to increase cell viability in colorectal cancer cells. Overall, these observations identify a potential role for this transcription factor during the DNA damage response.
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DNA replication during S phase involves thousands of replication forks that must be coordinated to ensure that every DNA section is replicated only once. The minichromosome maintenance proteins, MCM2 to MCM7, form a heteromeric DNA helicase required for both the initiation and elongation of DNA replication. Although only two DNA helicase activities are necessary to establish a bidirectional replication fork from each replication origin, a large excess of MCM complexes is amassed and distributed along the chromatin. The function of the additional MCM complexes is not well understood, as most are displaced from the DNA during the S-phase, apparently without playing an active role in DNA replication. DNA damage response (DDR) kinases activated by stalled forks prevent the replication machinery from being activated, indicating a tight relationship between DDR and DNA replication. To investigate the role of MCM proteins in the cellular response to DNA damage, we used shRNA targeting MCM2 or MCM3 to determine the impact of a reduction in MCM complex. The alteration of MCM proteins induced a change in the activation of key factors of the DDR in response to Etoposide treatment. Etoposide-induced DNA damage affected the phosphorylation of γ-H2AX, CHK1 and CHK2 without affecting cell viability. Using assays measuring homologous recombination (HR) and non-homologous end-joining (NHEJ), we identified a decrease in both HR and NHEJ associated with a decrease in MCM complex.
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Quebras de DNA de Cadeia Dupla , Proteínas de Manutenção de Minicromossomo/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades , Etoposídeo/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Histonas/metabolismo , Recombinação Homóloga , Humanos , Espectrometria de Massas , Componente 2 do Complexo de Manutenção de Minicromossomo/antagonistas & inibidores , Componente 2 do Complexo de Manutenção de Minicromossomo/genética , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 3 do Complexo de Manutenção de Minicromossomo/antagonistas & inibidores , Componente 3 do Complexo de Manutenção de Minicromossomo/genética , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/antagonistas & inibidores , Proteínas de Manutenção de Minicromossomo/genética , Fosfopeptídeos/análise , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
NudCD1, also known as CML66 or OVA66, is a protein initially identified as overexpressed in patients with chronic myelogenous leukemia. The mRNA of NudCD1 is expressed in heart and testis of normal tissues, and is overexpressed in several cancers. Previous studies have shown that the expression level of the protein correlates with tumoral phenotype, possibly interacting upstream of the Insulin Growth Factor - 1 Receptor (IGF-1R). The gene encoding the NudCD1 protein consists of 12 exons that can be alternative spliced, leading to the expression of three different isoforms. These isoforms possess a common region of 492 amino acids in their C-terminus region and have an isoform specific N-terminus. To determine the distinct function of each isoforms, we have localised the isoforms within the cells using immunofluorescence microscopy and used a quantitative proteomics approach (SILAC) to identify specific protein interaction partners for each isoforms. Localization studies showed a different subcellular distribution for the different isoforms, with the first isoform being nuclear, while the other two isoforms have distinct cytoplasmic and nuclear location. We found that the different NudCD1 isoforms have unique interacting partners, with the first isoform binding to a putative RNA helicase named DHX15 involved in mRNA splicing.
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Processamento Alternativo/genética , Antígenos de Neoplasias/genética , Mapas de Interação de Proteínas , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Anotação de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas , Transporte Proteico , RNA Helicases/metabolismoRESUMO
PURPOSE: Gastrojejunostomy tubes (GJTs) enable enteral nutrition in infants/children with feeding intolerance. However, complications may be increased in small infants. We evaluated our single-institution GJT complication rate and systematically reviewed existing literature. METHODS: With REB approval, a retrospective single-institution analysis of GJT placements between 2009 and 2015 was performed. For the systematic review, MOOSE guidelines were followed. RESULTS: At our institution, 48 children underwent 154/159 successful insertions primarily for gastroesophageal reflux (n=27; 55%) and aspiration (n=11; 23%). Median age at first GJT insertion was 2.2years (0.2-18). Thirty-five (73%) had an index insertion when ≤10kg. GJTs caused 2 perforations and 1 death. The systematic review assessed 48 articles representing 2726 procedures. Overall perforation rate was estimated as 2.1% (n=36 studies, 23/1092, 95% CI: 1.0-3.2). Perforation rates in children <10kg versus ≥10kg were estimated as 3.1%/procedure (95% CI: 1.1%-5.0%) and 0.1%/procedure (95% CI: 0%-0.3%), respectively. The relative risk of perforation was 9.4 (95% CI: 2.8-31.3). Overall mortality was estimated as 0.9%/patient (n=39 studies; 95% CI: 0.2-1.6%). Most perforations (19/23; 83%) occurred ≤30days of attempted tube placement. CONCLUSION: Gastrojejunostomy tubes are associated with significant complications and frequently require revision/replacement. Insertion in patients <10kg is associated with increased perforation risk. Caution is warranted in this subgroup. LEVEL OF EVIDENCE: Level II.
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Derivação Gástrica , Perfuração Intestinal/etiologia , Complicações Pós-Operatórias , Adolescente , Peso Corporal , Criança , Pré-Escolar , Feminino , Seguimentos , Derivação Gástrica/instrumentação , Derivação Gástrica/mortalidade , Humanos , Incidência , Lactente , Perfuração Intestinal/diagnóstico , Perfuração Intestinal/epidemiologia , Masculino , Avaliação de Resultados em Cuidados de Saúde , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Estudos Retrospectivos , Fatores de RiscoRESUMO
Organoids have the potential to bridge 3D cell culture to tissue physiology by providing a model resembling in vivo organs. Long-term growing organoids were first isolated from intestinal crypt cells and recreated the renewing intestinal epithelial niche. Since then, this technical breakthrough was applied to many other organs, including prostate, liver, kidney and pancreas. We describe here how to apply a SILAC-based quantitative proteomic approach to measure protein expression changes in intestinal organoids under different experimental conditions. We generated SILAC organoid media that allow organoids to grow and differentiate normally, and confirmed the incorporation of isotopically labelled amino acids. Furthermore, we used a treatment reported to affect organoid differentiation to demonstrate the reproducibility of the quantification using this approach and to validate the identification of proteins that correlate with the inhibition of cellular growth and development. With the combined use of quantitative mass spectrometry, SILAC and organoid culture, we validated this approach and showed that large-scale proteome variations can be measured in an "organ-like" system.
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Mucosa Intestinal/metabolismo , Organoides/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Aminoácidos/metabolismo , Animais , Western Blotting , Células Cultivadas , Cromatografia Líquida , Células Epiteliais/metabolismo , Intestinos/citologia , Marcação por Isótopo/métodos , Camundongos , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
Anastomotic strictures (ASs) complicate the postoperative course of roughly one-third of all patients with esophageal atresia with or without tracheoesophageal fistula. Its development is multifactorial, but is due in part to tension on the anastomosis, gastroesophageal reflux disease, and the presence of a leak in the early postoperative period. Efforts at reducing the rate of AS have been largely unsuccessful, although meticulous technique and aggressive acid suppression remain the cornerstones of perioperative care. Once an AS has been confirmed, the first-line treatment remains a course of esophageal dilatation. Adjuncts to dilatation are frequently required, including steroid injection or the topical application of mitomycin C. Currently, there is insufficient evidence to promote one at the expense of the other. Esophageal stenting has recently been added to the algorithm of treatment, although additional literature is required to confirm its safety and efficacy. Finally, stricture resection followed by primary esophageal anastomosis or, rarely, esophageal replacement with an interposition graft remain options for AS refractory to all other forms of treatment.
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Atresia Esofágica/cirurgia , Estenose Esofágica/etiologia , Esôfago/cirurgia , Stents , Anastomose Cirúrgica/efeitos adversos , Estenose Esofágica/cirurgia , Humanos , ReoperaçãoRESUMO
BACKGROUND AND OBJECTIVES: Many mathematical equations based on height have been developed to estimate the esophageal length (EL) in children. The aim of this study was to confirm whether the preexisting and most frequently used equation by Strobel et al is accurate in calculating the EL in our pediatric population. Our secondary goal was to evaluate whether a new formula could be developed using our nonsurgical and surgical populations' data for the correlation between patients' height and measured EL by esophageal manometry (EM). METHODS: From 2000 to 2009, 116 children between the ages of 3 and 18 years without previous esophageal surgery underwent EM (nâ=â31) at the Montreal Children's Hospital. During the same period, 55 EMs were performed on 34 children with a previous history of esophageal surgery. For both groups, we collected the following data: height, EL calculated by the Strobel formula, and EL measured by EM. RESULTS: The Strobel equation was inaccurate in predicting the EL. The calculated EL was 3.0â±â0.32 cm longer than the EM measurements (Pâ<â0.001). The height (H) of nonsurgical children was found to be highly predictive of the lower esophageal sphincter location (L), and the derived linear regression equation is Lâ=â0.216 (H)â+â7.13 [r²â=â0.85]. CONCLUSIONS: This study confirmed that the Strobel formula is not sufficiently accurate to predict EL in the pediatric population that is between 3 and 18 years old. A correlation exists between height and esophageal sphincter location position. If EM is unavailable, the use of a new mathematical equation like ours can be considered.
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Monitoramento do pH Esofágico , Esôfago , Refluxo Gastroesofágico , Manometria/métodos , Conceitos Matemáticos , Adolescente , Estatura , Catéteres , Criança , Pré-Escolar , Eletrodos , Esfíncter Esofágico Inferior , Feminino , Refluxo Gastroesofágico/diagnóstico , Humanos , Concentração de Íons de Hidrogênio , Modelos Lineares , Masculino , Reprodutibilidade dos TestesAssuntos
Acalasia Esofágica/diagnóstico , Junção Esofagogástrica/fisiopatologia , Adolescente , Criança , Diagnóstico Diferencial , Acalasia Esofágica/fisiopatologia , Acalasia Esofágica/cirurgia , Junção Esofagogástrica/cirurgia , Fundoplicatura/métodos , Humanos , Laparoscopia , Masculino , Manometria , Pressão , Tomografia Computadorizada por Raios XRESUMO
RNA-based compounds are promising agents to inactivate viruses. New specific hepatitis delta virus (HDV)-derived ribozymes are natural molecules that can be engineered to specifically target a viral RNA. We have designed specific on-off adaptor (SOFA)-HDV ribozymes targeting the tat and rev sequences of the human immunodeficiency virus type 1 (HIV-1) RNA. We show that the SOFA-HDV ribozymes cleave their RNA target in vitro. They inhibit the Tat-mediated transactivation of HIV-1 from 62% to 86% in different assays. In vivo, the amount of HIV RNA was decreased by 60 and 86% with two distinct ribozymes, which indicates that the inhibition of HIV production is directly correlated to the decline in spliced and unspliced viral RNAs. These SOFAHDV- ribozymes inhibited the expression and the viral production of four HIV-1 strains, indicating an extended potential to act on multiple HIV variants. In HEK 293T and HeLa cells transfected with pNL4-3 and the SOFA-HDV-ribozymes, the reduced RNA levels consequently decreased the Gag protein expression in the cell and virus production in the supernatant. When transfected before HIV-1 infection, the ribozymes prevented the incoming virus from being expressed. The ribozymes inhibited HIV production up to 90% when transfected in combination with the HIV protease inhibitor Atazanavir. Our results strongly suggest that SOFA-HDV ribozymes have a great potential to target HIV-1 and to be used as therapeutic agents in combination therapy.
Assuntos
HIV-1/enzimologia , RNA Catalítico/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Replicação Viral , Sulfato de Atazanavir , Sequência de Bases , Produtos do Gene gag/biossíntese , Produtos do Gene gag/genética , Produtos do Gene rev/genética , Produtos do Gene rev/metabolismo , Células HEK293 , Infecções por HIV/genética , HIV-1/genética , Células HeLa , Vírus Delta da Hepatite/enzimologia , Vírus Delta da Hepatite/genética , Humanos , Oligopeptídeos/farmacologia , Piridinas/farmacologia , Splicing de RNA , RNA Catalítico/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
OBJECTIVES: Esophageal atresia with or without tracheo-esophageal fistula is a frequent malformation that occurs in about 1 of 3000 live births. It can be associated with other congenital malformations. The aim of this study was to measure the frequency of heterotopic pancreas (HP) in children with esophageal atresia (EA) and to evaluate possible linkage with other malformations. MATERIALS AND METHODS: All patients with EA were prospectively followed since 2005 at Hôpital Sainte-Justine and since 2006 at the Montreal Children's Hospital. We compared 91 patients who underwent gastroscopy during that period with 182 control patients who submitted to gastroscopy for other indications. The presence or the absence of HP and its localization were noted in both groups. The following data were also collected on patients with EA: sex, gestational age, EA type, and other malformations. RESULTS: Seventeen (18.7%) of the 91 patients with EA had gastric HP compared with 1 (0.5%) in the control group (OR 42, 95% confidence interval 7-249, P < 0.001). There were no differences between patients with or without HP regarding sex, prematurity, EA type, and the presence or absence of other congenital abnormalities. CONCLUSIONS: This study demonstrates, for the first time, that gastric HP is associated with EA irrespective of other malformations.