Eosinophilic esophagitis (EoE) is a chronic non-IgE-mediated allergic disease of the esophagus. An unbiased proteomics approach was performed to investigate pathophysiological changes in esophageal ...epithelium. Additionally, an RNAseq-based transcriptomic analysis in paired samples was also carried out.
Total proteins were purified from esophageal endoscopic biopsies in a cohort of adult EoE patients (n = 25) and healthy esophagus controls (n = 10). Differentially accumulated (DA) proteins in EoE patients compared to control tissues were characterized to identify altered biological processes and signaling pathways. Results were also compared with a quantitative proteome dataset of the human esophageal mucosa. Next, results were contrasted with those obtained after RNAseq analysis in paired samples. Finally, we matched up protein expression with two EoE-specific mRNA panels (EDP and Eso-EoE panel).
A total of 1667 proteins were identified, of which 363 were DA in EoE. RNA sequencing in paired samples identified 1993 differentially expressed (DE) genes. Total RNA and protein levels positively correlated, especially in DE mRNA-proteins pairs. Pathway analysis of these proteins in EoE showed alterations in immune and inflammatory responses for the upregulated proteins, and in epithelial differentiation, cornification and keratinization in those downregulated. Interestingly, a set of DA proteins, including eosinophil-related and secreted proteins, were not detected at the mRNA level. Protein expression positively correlated with EDP and Eso-EoE, and corresponded with the most abundant proteins of the human esophageal proteome.
We unraveled for the first time key proteomic features involved in EoE pathogenesis. An integrative analysis of transcriptomic and proteomic datasets provides a deeper insight than transcriptomic alone into understanding complex disease mechanisms.
Acute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune ...cells. The anti-diabetic drug Metformin inhibits acute and chronic inflammation through mechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppression by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immunosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macrophages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression.
Embryonic stem cell (ESC) differentiation and somatic cell reprogramming are biological processes governed by antagonistic expression or repression of a largely common set of genes. Accurate ...regulation of gene expression is thus essential for both processes, and alterations in RNA processing are predicted to negatively affect both. We show that truncation of the DIDO gene alters RNA splicing and transcription termination in ESC and mouse embryo fibroblasts (MEF), which affects genes involved in both differentiation and reprogramming. We combined transcriptomic, protein interaction, and cellular studies to identify the underlying molecular mechanism. We found that DIDO3 interacts with the helicase DHX9, which is involved in R-loop processing and transcription termination, and that DIDO3-exon16 deletion increases nuclear R-loop content and causes DNA replication stress. Overall, these defects result in failure of ESC to differentiate and of MEF to be reprogrammed. MEF immortalization restored impaired reprogramming capacity. We conclude that DIDO3 has essential functions in ESC differentiation and somatic cell reprogramming by supporting accurate RNA metabolism, with its exon16-encoded domain playing the main role.
Accumulation of lipid-laden macrophages within the arterial neointima is a critical step in atherosclerotic plaque formation. Here, we show that reduced levels of the cellular plasticity factor ZEB1 ...in macrophages increase atherosclerotic plaque formation and the chance of cardiovascular events. Compared to control counterparts (Zeb1
/Apoe
), male mice with Zeb1 ablation in their myeloid cells (Zeb1
/Apoe
) have larger atherosclerotic plaques and higher lipid accumulation in their macrophages due to delayed lipid traffic and deficient cholesterol efflux. Zeb1
/Apoe
mice display more pronounced systemic metabolic alterations than Zeb1
/Apoe
mice, with higher serum levels of low-density lipoproteins and inflammatory cytokines and larger ectopic fat deposits. Higher lipid accumulation in Zeb1
macrophages is reverted by the exogenous expression of Zeb1 through macrophage-targeted nanoparticles. In vivo administration of these nanoparticles reduces atherosclerotic plaque formation in Zeb1
/Apoe
mice. Finally, low ZEB1 expression in human endarterectomies is associated with plaque rupture and cardiovascular events. These results set ZEB1 in macrophages as a potential target in the treatment of atherosclerosis.
Chronic lymphocytic leukemia (CLL) is the most frequent, and still incurable, form of leukemia in the Western World. It is widely accepted that cancer results from an evolutionary process shaped by ...the acquisition of driver mutations which confer selective growth advantage to cells that harbor them. Clear examples are missense mutations in classic RAS genes (KRAS, HRAS and NRAS) that underlie the development of approximately 13% of human cancers. Although autonomous B cell antigen receptor (BCR) signaling is involved and mutations in many tumor suppressor genes and oncogenes have been identified, an oncogenic driver gene has not still been identified for CLL.
Conditional knock-in mice were generated to overexpress wild type RRAS2 and prove its driver role. RT-qPCR analysis of a human CLL sample cohort was carried out to measure RRAS2 transcriptional expression. Sanger DNA sequencing was used to identify a SNP in the 3'UTR region of RRAS2 in human CLL samples. RNAseq of murine CLL was carried out to identify activated pathways, molecular mechanisms and to pinpoint somatic mutations accompanying RRAS2 overexpression. Flow cytometry was used for phenotypic characterization and shRNA techniques to knockdown RRAS2 expression in human CLL.
RRAS2 mRNA is found overexpressed in its wild type form in 82% of the human CLL samples analyzed (n = 178, mean and median = 5-fold) as well as in the explored metadata. A single nucleotide polymorphism (rs8570) in the 3'UTR of the RRAS2 mRNA has been identified in CLL patients, linking higher expression of RRAS2 with more aggressive disease. Deliberate overexpression of wild type RRAS2 in mice, but not an oncogenic Q72L mutation in the coding sequence, provokes the development of CLL. Overexpression of wild type RRAS2 in mice is accompanied by a strong convergent selection of somatic mutations in genes that have been identified in human CLL. R-RAS2 protein is physically bound to the BCR and mediates BCR signals in CLL.
The results indicate that overexpression of wild type RRAS2 is behind the development of CLL.
Cancer stem cells (CSC) exhibit high tumorigenic capacity in several tumor models. We have now determined an extended phenotype for cervical cancer stem cells. Our results showed increased CK-17, ...p63+, AII+, CD49f+ expression in these cells, together with higher Aldehyde dehydrogenase (ALDHbright)activity in Cervical CSC (CCSC) enriched in cervospheres. An increase in stem cell markers, represented by OCT-4, Nanog, and β-catenin proteins, was also observed, indicating that under our culture conditions, CCSC are enriched in cervospheres, as compared to monolayer cultures. In addition, we were able to show that an increased ALDHbright activity correlated with higher tumorigenic activity. Flow cytometry and immunflorescence assays demonstrated that CCSC in cervosphere cultures contain a sub-population of cells that contain Annexin II, a Human papillomavirus (HPV) co-receptor. Taken together, under our conditions there is an increase in the number of CCSC in cervosphere cultures which exhibit the following phenotype: CK-17, p63+, AII+, CD49f+ and high ALDH activity, which in turn correlates with higher tumorigenicity. The presence of Annexin II and CD49f in CCSC opens the possibility that normal cervical stem cells could be the initial target of infection by high risk HPV.
Abstract Background Recently, we have identified a dysregulated protein signature in the esophageal epithelium of eosinophilic esophagitis (EoE) patients including proteins associated with ...inflammation and epithelial barrier function; however, the effect of proton pump inhibitor (PPI) treatment on this signature is unknown. Herein, we used a proteomic approach to investigate: (1) whether PPI treatment alters the esophageal epithelium protein profile observed in EoE patients and (2) whether the protein signature at baseline predicts PPI response. Methods We evaluated the protein signature of esophageal biopsies using a cohort of adult EoE ( n = 25) patients and healthy controls (C) ( n = 10). In EoE patients, esophageal biopsies were taken before (pre) and after (post) an 8‐week PPI treatment, determining the histologic response. Eosinophil count PostPPI was used to classify the patients: ≥15 eosinophils/hpf as non‐responders (non‐responder) and < 15 eosinophils/hpf as responders (R). Protein signature was determined and differentially accumulated proteins were characterized to identify altered biological processes and signaling pathways. Results Comparative analysis of differentially accumulated proteins between groups revealed common signatures between three groups of patients with inflammation (responder‐PrePPI, non‐responder‐PrePPI, and non‐responder‐PostPPI) and without inflammation (controls and responder‐PostPPI). PPI therapy almost reversed the EoE specific esophageal protein signature, which is enriched in pathways associated with inflammation and epithelial barrier function, in responder‐PostPPI. Furthermore, we identified a set of candidate proteins to differentiate responder‐PrePPI and non‐responder‐PrePPI EoE patients before treatment. Conclusion These findings provide evidence that PPI therapy reverses the alterations in esophageal inflammatory and epithelial proteins characterizing EoE, thereby providing new insights into the mechanism of PPI clinical response. Interestingly, our results also suggest that PPI response could be predicted at baseline in EoE.
High doses of ethylenebisdithiocarbamate (EBDC) are used in banana production, and unused pesticide mixture (solution) is often disposed of improperly. This can result in soil and water contamination ...and present an undue risk to rural communities and the environment. An alternative to reduce the environmental impacts caused by pesticide residues is the biobeds treatment. It is necessary to establish if the composition of the proposed biomixtures supports microbial activity to degrade pesticides in biobeds. This research aimed to evaluate the EBDC effect on the distribution and abundance of microbial populations in polluted biomixtures .
For this purpose, a biomixture based on banana stem, mulch, and Fluvisol soil (50:25:25% v/v) was prepared and polluted with 1,000 mg L
EBDC. The response variables kinetics were determined every 14 days for three months, such as pH, organic matter, moisture, cation exchange capacity, microbial colonies, and cell counts at three depths within the experimental units.
EBDC reduced the number of microbial colonies by 72%. Bacterial cells rapidly decreased by 69% and fungi 89% on the surface, while the decrease was gradual and steady at the middle and bottom of the biobed. The microbial populations stabilized at day 42, and the bacteria showed a total recovery on day 84, but the fungi slightly less. At the end of the experiment, the concentration of EBDC in the biomixture was 1.3-4.1 mg L
. A correlation was found between fungal count (colonies and cells) with EBDC concentration. A replacement of the biomixture is suggested if the bacterial population becomes less than 40 × 10
CFU mL
and the fungal population less than 8 × 10
CFU mL
or if the direct cell count becomes lower than 50 × 10
cells mL
in bacteria and 8 × 10
cells mL
in fungi.
The biomixture based on banana stem supports the microbial activity necessary for the degradation of the EBDC pesticide. It was found that fungi could be used as indicators of the pollutant degradation process in the biomixtures. Microbial counts were useful to establish the mobility and degradation time of the pesticide and the effectiveness of the biomixture. Based on the results, it is appropriate to include the quantification of microbial populations to assess the effectiveness of pesticide degradation and the maturity level of the biomixture.
The aim of the study was to characterize the circulating immunome of patients with EoE before and after proton pump inhibitor (PPI) treatment in order to identify potential non-invasive biomarkers of ...treatment response.
PBMCs from 19 healthy controls and 24 EoE patients were studied using a 39-plex spectral cytometry panel. The plasmacytoid dendritic cell (pDC) population was differentially characterized by spectral cytometry analysis and immunofluorescence assays in esophageal biopsies from 7 healthy controls and 13 EoE patients.
Interestingly, EoE patients at baseline had lower levels of circulating pDC compared with controls. Before treatment, patients with EoE who responded to PPI therapy had higher levels of circulating pDC and classical monocytes, compared with non-responders. Moreover, following PPI therapy pDC levels were increased in all EoE patients, while normal levels were only restored in PPI-responding patients. Finally, circulating pDC levels inversely correlated with peak eosinophil count and pDC count in esophageal biopsies. The number of tissue pDCs significantly increased during active EoE, being even higher in non-responder patients when compared to responder patients pre-PPI. pDC levels decreased after PPI intake, being further restored almost to control levels in responder patients post-PPI.
We hereby describe a unique immune fingerprint of EoE patients at diagnosis. Moreover, circulating pDC may be also used as a novel non-invasive biomarker to predict subsequent response to PPI treatment.
Esta obra presenta una serie de investigaciones acerca de las prácticas y métodos de documentación sobre ocho lenguas fuegopatagónicas: selk'nam, haush, alakaluf / kawesqar, yagan, aonekko ’a’ien, ...teushen, günün a yajüch y mapuzungun realizadas en la región en el último tercio del siglo xix y hasta mediados del xx en un contexto de expropiación territorial y genocidio de los pueblos preexistentes. Además, pone a disposición los documentos inéditos de estas lenguas que han permanecido albergados en repositorios de Argentina, Chile, Alemania e Italia. La publicación procura visibilizar el trabajo de las y los coproductores de los pueblos originarios en la recolección de información y métodos de documentación de sus lenguas así como en las condiciones materiales en que se desarrollaron. Sin dudas, se valoriza un corpus manuscrito poco conocido, que en la época fue compartido por diversos estudiosos, objetualizado y extraído de sus comunidades hablantes, y así contribuye a consolidar un posicionamiento ético en el trabajo de investigación interdisciplinario acerca de las lenguas de los pueblos originarios de la región fuegopatagónica.
PDF
