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Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that SDC4 knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression.
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Neoplasias Gástricas , Sindecano-4 , Humanos , Heparitina Sulfato/metabolismo , Invasividad Neoplásica , Neoplasias Gástricas/genética , Sindecano-4/genética , Sindecano-4/metabolismoRESUMEN
The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery-suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these-the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex-as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.
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Adenocarcinoma , Antineoplásicos , Neoplasias del Colon , Humanos , Proteoma/metabolismo , Adenocarcinoma/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico , Chaperonas Moleculares , Antineoplásicos/farmacología , Espectrometría de Masas , Cromatografía en GelRESUMEN
Herbaspirillum seropedicae is a ß-proteobacterium that establishes as an endophyte in various plants. These bacteria can consume diverse carbon sources, including hexoses and pentoses like D-xylose. D-xylose catabolic pathways have been described in some microorganisms, but databases of genes involved in these routes are limited. This is of special interest in biotechnology, considering that D-xylose is the second most abundant sugar in nature and some microorganisms, including H. seropedicae, are able to accumulate poly-3-hydroxybutyrate when consuming this pentose as a carbon source. In this work, we present a study of D-xylose catabolic pathways in H. seropedicae strain Z69 using RNA-seq analysis and subsequent analysis of phenotypes determined in targeted mutants in corresponding identified genes. G5B88_22805 gene, designated xylB, encodes a NAD+-dependent D-xylose dehydrogenase. Mutant Z69∆xylB was still able to grow on D-xylose, although at a reduced rate. This appears to be due to the expression of an L-arabinose dehydrogenase, encoded by the araB gene (G5B88_05250), that can use D-xylose as a substrate. According to our results, H. seropedicae Z69 uses non-phosphorylative pathways to catabolize D-xylose. The lower portion of metabolism involves co-expression of two routes: the Weimberg pathway that produces α-ketoglutarate and a novel pathway recently described that synthesizes pyruvate and glycolate. This novel pathway appears to contribute to D-xylose metabolism, since a mutant in the last step, Z69∆mhpD, was able to grow on this pentose only after an extended lag phase (40-50 h). KEY POINTS: ⢠xylB gene (G5B88_22805) encodes a NAD+-dependent D-xylose dehydrogenase. ⢠araB gene (G5B88_05250) encodes a L-arabinose dehydrogenase able to recognize D-xylose. ⢠A novel route involving mhpD gene is preferred for D-xylose catabolism.
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Biotecnología , Xilosa , HerbaspirillumRESUMEN
Chemoresistance is a serious limitation of cancer treatment. Until recently, almost all the work done to study this limitation has been restricted to tumour cells. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.
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Daño del ADN , Resistencia a Antineoplásicos/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Citocinas/biosíntesis , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos/genética , Células Endoteliales/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/deficiencia , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Humanos , Ratones , FN-kappa B/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Neoplasias/radioterapia , Fosforilación/efectos de los fármacosRESUMEN
Focal adhesion kinase (FAK) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. In vitro activation of the FAK kinase domain triggers autophosphorylation of Y397, Src activation, and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK Y397 mutations affect FAK kinase-dead (KD) phenotypes in tumour angiogenesis in vivo is unknown. We developed three Pdgfb-iCreert -driven endothelial cell (EC)-specific, tamoxifen-inducible homozygous mutant mouse lines: FAK wild-type (WT), FAK KD, and FAK double mutant (DM), i.e. KD with a putatively phosphomimetic Y397E mutation. These ECCre+;FAKWT/WT , ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAKWT/WT and ECCre-;FAKWT/WT control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice, as compared with Cre - littermates. Despite no change in the percentage of perfused vessels or pericyte coverage in either genotype, tumour hypoxia was elevated in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice. Furthermore, although ECCre+;FAKKD/KD mice showed reduced blood vessel leakage, ECCre+;FAKDM/DM and ECCre-;FAKDM/DM mice showed no difference in leakage. Mechanistically, fibronectin-stimulated Y397 autophosphorylation was reduced in Cre+;FAKKD/KD ECs as compared with Cre+;FAKWT/WT cells, with no change in phosphorylation of the known Src targets FAK-Y577, FAK-Y861, FAK-Y925, paxillin-Y118, p130Cas-Y410. Cre+;FAKDM/DM ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE-cadherin-pY658 levels in Cre+;FAKKD/KD ECs were rescued in Cre+FAKDM/DM ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAKDM/DM mice. We show that EC-specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAKDM/DM mice restored the KD-dependent tumour vascular leakage observed in ECCre+;FAKKD/KD mice in vivo. This study opens new fields in in vivo FAK signalling. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Permeabilidad Capilar/genética , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Melanoma/enzimología , Animales , Antineoplásicos Hormonales/farmacología , Permeabilidad Capilar/efectos de los fármacos , División Celular/genética , Hipoxia de la Célula/genética , Línea Celular Tumoral , Endotelio Vascular/enzimología , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/deficiencia , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Homocigoto , Melanoma/irrigación sanguínea , Melanoma/genética , Ratones , Mutación/genética , Trasplante de Neoplasias , Neovascularización Patológica/enzimología , Neovascularización Patológica/genética , Tamoxifeno/farmacologíaRESUMEN
Telomeres protect the natural ends of chromosomes from being repaired as deleterious DNA breaks. In fission yeast, absence of Taz1 (homologue of human TRF1 and TRF2) renders telomeres vulnerable to DNA repair. During the G1 phase, when non-homologous end joining (NHEJ) is upregulated, taz1Δ cells undergo telomere fusions with consequent loss of viability. Here, we show that disruption of the fission yeast MRN (Rad23(MRE11)-Rad50-Nbs1) complex prevents NHEJ at telomeres and, as a result, rescues taz1Δ lethality in G1. Neither Tel1(ATM) activation nor 5'-end resection was required for telomere fusion. Nuclease activity of Rad32(MRE11) was also dispensable for NHEJ. Mutants unable to coordinate metal ions required for nuclease activity were proficient in NHEJ repair. In contrast, Rad32(MRE11) mutations that affect binding and/or positioning of DNA ends leaving the nuclease function largely unaffected also impaired NHEJ at telomeres and restored the viability of taz1Δ in G1. Consistently, MRN structural integrity but not nuclease function is also required for NHEJ of independent DNA ends in a novel split-molecule plasmid assay. Thus, MRN acts to tether unlinked DNA ends, allowing for efficient NHEJ.
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Reparación del ADN por Unión de Extremidades/fisiología , Fase G1/fisiología , Complejos Multiproteicos/metabolismo , Telómero/metabolismo , Supervivencia Celular/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Cartilla de ADN/genética , Electroforesis en Gel de Campo Pulsado , Exodesoxirribonucleasas/metabolismo , Reacción en Cadena de la Polimerasa , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Unión a Telómeros/deficienciaRESUMEN
BACKGROUND/OBJECTIVES: The vascular heterogeneity of pancreatic ductal adenocarcinoma (PDAC) has never been characterised. We analysed the heterogeneous vascular density of human PDAC along with its prognostic correlation. METHODS: Tissue Microarrays of 87 patients with different pancreatico-biliary pathologies were analysed in an automated manner (Ariol™) after CD31 staining to assess vascular density in juxta-tumoral and panstromal compartments. In vitro and ex vivo assays were carried out to assess the role of PSC. RESULTS: PDAC has a distinct vascular density and distribution of vessels compared to cholangiocarcinoma. The PDAC juxta-tumoral stroma was hypovascular and the normal adjacent rim was hypervascular compared to the panstromal compartment. These features adversely affected patient prognosis, suggesting a model for spatio-temporal PDAC evolution. Mice aortic rings and 3D organotypic cultures demonstrated pro- and anti-angiogenic signalling from activated PSC and cancer cells respectively. ATRA-induced quiescence suppressed the pro-angiogenic activity of PSC. CONCLUSION: Human PDAC has variable vascularity at microscopic level suggesting that novel stromal directed therapies would need to be determined by pathological characteristics.
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Adenocarcinoma/patología , Vasos Sanguíneos/patología , Carcinoma Ductal Pancreático/patología , Células Estrelladas Pancreáticas/patología , Animales , Células Cultivadas , Colangiocarcinoma/patología , Humanos , Ratones , Análisis por Micromatrices , Microcirculación/efectos de los fármacos , Neovascularización Patológica/patología , Técnicas de Cultivo de Órganos , Pronóstico , Análisis de Supervivencia , Tretinoina/uso terapéutico , Microambiente TumoralRESUMEN
Pampa Rocha (PR) is a breed of pig that emerged in eastern Uruguay during the 18th century. They represent an important resource for non-intensive production using purebred or crossbred animals. However, productive activities have been oriented towards intensive production using commercial breeds, abandoning, except by some academic and educational institutions, the promotion of this creole breed. Thus, a population of few animals is still maintained, which could be in danger of disappearing. This work focuses on the fecal microbiota of these animals, which is related to the animal genetic background but also to their grazing capacity and resistance to weather. The structure and diversity of bacterial communities in the intestines of four PR adult females and of other breeds, including crosses, reared under non-grazing conditions, were analyzed and compared. Results obtained indicate that PR fecal microbiota is clearly different from those of other animals analyzed. Some sequences, corresponding to particular groups apparently related to the consumption of fiber, were strongly associated with PR pigs.
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Ethanol production by the D-xylose fermentation of lignocellulosic biomass would augment environmental sustainability by increasing the yield of biofuel obtained per cultivated area. A set of recombinant strains derived from the industrial strain Saccharomyces cerevisiae CAT-1 was developed for this purpose. First, two recombinant strains were obtained by the chromosomal insertion of genes involved in the assimilation and transport of D-xylose (Gal2-N376F). Strain CAT-1-XRT was developed with heterologous genes for D-xylose metabolism from the oxo-reductive pathway of Scheffersomyces stipitis (XYL1-K270R, XYL2); and strain CAT-1-XIT, with D-xylose isomerase (xylA gene, XI) from Streptomyces coelicolor. Moreover, both recombinant strains contained extra copies of homologous genes for xylulose kinase (XK) and transaldolase (TAL1). Furthermore, plasmid (pRS42K::XI) was constructed with xylA from Piromyces sp. transferred to CAT-1, CAT-1-XRT, and CAT-1-XIT, followed by an evolution protocol. After 10 subcultures, CAT-1-XIT (pRS42K::XI) consumed 74% of D-xylose, producing 12.6 g/L ethanol (0.31 g ethanol/g D-xylose). The results of this study show that CAT-1-XIT (pRS42K::XI) is a promising recombinant strain for the efficient utilization of D-xylose to produce ethanol from lignocellulosic materials.
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Antarctic environments can sustain a great diversity of well-adapted microorganisms known as psychrophiles or psychrotrophs. The potential of these microorganisms as a resource of enzymes able to maintain their activity and stability at low temperature for technological applications has stimulated interest in exploration and isolation of microbes from this extreme environment. Enzymes produced by these organisms have a considerable potential for technological applications because they are known to have higher enzymatic activities at lower temperatures than their mesophilic and thermophilic counterparts. A total of 518 Antarctic microorganisms, were isolated during Antarctic expeditions organized by the Instituto Antártico Uruguayo. Samples of particules suspended in air, ice, sea and freshwater, soil, sediment, bird and marine animal faeces, dead animals, algae, plants, rocks and microbial mats were collected from different sites in maritime Antarctica. We report enzymatic activities present in 161 microorganisms (120 bacteria, 31 yeasts and 10 filamentous fungi) isolated from these locations. Enzymatic performance was evaluated at 4 and 20°C. Most of yeasts and bacteria grew better at 20°C than at 4°C, however the opposite was observed with the fungi. Amylase, lipase and protease activities were frequently found in bacterial strains. Yeasts and fungal isolates typically exhibited lipase, celullase and gelatinase activities. Bacterial isolates with highest enzymatic activities were identified by 16S rDNA sequence analysis as Pseudomonas spp., Psychrobacter sp., Arthrobacter spp., Bacillus sp. and Carnobacterium sp. Yeasts and fungal strains, with multiple enzymatic activities, belonged to Cryptococcus victoriae, Trichosporon pullulans and Geomyces pannorum.
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Bacterias/enzimología , Bacterias/aislamiento & purificación , Microbiología Ambiental , Hongos/enzimología , Hongos/aislamiento & purificación , Amilasas/análisis , Regiones Antárticas , Bacterias/clasificación , Bacterias/genética , Celulasa/análisis , Análisis por Conglomerados , Frío , ADN Bacteriano/química , ADN Bacteriano/genética , ADN de Hongos/química , ADN de Hongos/genética , Hongos/clasificación , Hongos/genética , Gelatinasas/análisis , Lipasa/análisis , Filogenia , Análisis de Secuencia de ADNRESUMEN
Colorectal cancer (CRC) is the third most common cancer in the world and represents the third most deadly tumor worldwide. About 15-25% of patients present metastasis in the moment of diagnosis, the liver being the most common site of metastization. Therefore, the development of new therapeutic agents is needed, to improve the patients' prognosis. Amino acids transporters, LAT1 and ASCT2, are described as upregulated in CRC, being associated with a poor prognosis. Extracellular vesicles have emerged as key players in cell-to-cell communication due to their ability to transfer biomolecules between cells, with a phenotypic impact on the recipient cells. Thus, this study analyzes the presence of LAT1 and ASCT2 mRNAs in CRC-EVs and evaluates their role in phenotype modulation in a panel of four recipient cell lines (HCA-7, HEPG-2, SK-HEP-1, HKC-8). We found that HCT 116-EVs carry LAT1, ASCT2 and other oncogenic mRNAs being taken up by recipient cells. Moreover, the HCT 116-EVs' internalization was associated with the increase of LAT1 mRNA in SK-HEP-1 cells. We also observed that HCT 116-EVs induce a higher cell migration capacity and proliferation of SK-HEP-1 and HKC-8 cells. The present study supports the LAT1-EVs' mRNA involvement in cell phenotype modulation, conferring advantages in cell migration and proliferation.
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(1) Background: Pancreatic ductal adenocarcinoma (PDAC) is expected to be the second-leading cause of cancer deaths by 2030. Imaging techniques are the standard for monitoring the therapy response in PDAC, but these techniques have considerable limits, including delayed disease progression detection and difficulty in distinguishing benign from malignant lesions. Extracellular vesicle (EV) liquid biopsy is an emerging diagnosis modality. Nonetheless, the majority of research for EV-based diagnosis relies on point analyses of EVs at specified times, while longitudinal EV population studies before and during therapeutic interventions remain largely unexplored. (2) Methods: We analyzed plasma EV protein composition at diagnosis and throughout PDAC therapy. (3) Results: We found that IgG is linked with the diagnosis of PDAC and the patient's response to therapy, and that the IgG+ EV population increases with disease progression and reduces with treatment response. Importantly, this covers PDAC patients devoid of the standard PDAC seric marker CA19.9 expression. We also observed that IgG is bound to EVs via the tumor antigen MAGE B1, and that this is independent of the patient's inflammatory condition and IgG seric levels. (4) Conclusions: We here propose that a population analysis of IgG+ EVs in PDAC plasma represents a novel method to supplement the monitoring of the PDAC treatment response.
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Carcinoma Ductal Pancreático , Vesículas Extracelulares , Neoplasias Pancreáticas , Antígenos de Neoplasias , Biomarcadores de Tumor , Carcinoma Ductal Pancreático/terapia , Progresión de la Enfermedad , Vesículas Extracelulares/patología , Humanos , Inmunoglobulina G , Neoplasias Pancreáticas/diagnóstico , Neoplasias PancreáticasRESUMEN
(1) Background: Extracellular vesicles (EVs) have emerged as crucial players in the communication between cells in both physiological and pathological scenarios. The functions of EVs are strongly determined by their molecular content, which includes all bioactive molecules, such as proteins, lipids, RNA, and, as more recently described, double-stranded DNA. It has been shown that in oncological settings DNA associated with EVs (EV-DNA) is representative of the genome of parental cells and that it reflects the mutational status of the tumor, gaining much attention as a promising source of biomarker mutant DNA. However, one of the challenges in studies of EV-DNA is the lack of standardization of protocols for the DNA extraction from EVs, as well as ways to assess quality control, which hinders its future implementation in clinics. (2) Methods: We performed a comprehensive comparison of commonly used approaches for EV-DNA extraction by assessing DNA quantity, quality, and suitability for downstream analyses. (3) Results: We here established strategic points to consider for EV-DNA preparation for mutational analyses, including qPCR and NGS. (4) Conclusions: We put in place a workflow that can be applied for the detection of clinically relevant mutations in the EV-DNA of cancer patients.
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Integrin alpha3beta1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of alpha3beta1 can affect angiogenesis either positively or negatively, either a direct in vivo role for alpha3beta1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of alpha3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where alpha3 is deleted specifically in endothelial cells (ec-alpha3-/-). Here we show that ec-alpha3-/- mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that alpha3beta1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial alpha3beta1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.
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Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Hipoxia/metabolismo , Integrina alfa3beta1/metabolismo , Neovascularización Patológica/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Western Blotting , Células Endoteliales/patología , Endotelio Vascular/patología , Femenino , Citometría de Flujo , Hipoxia/genética , Hipoxia/patología , Inmunohistoquímica , Integrina alfa3beta1/genética , Masculino , Ratones , Ratones Noqueados , Trasplante de Neoplasias , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Reacción en Cadena de la Polimerasa , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The objective of this study is to identify and analyze integrons and antibiotic resistance genes (ARGs) in samples collected from diverse sites in terrestrial Antarctica. Integrons were studied using two independent methods. One involved the construction and analysis of intI gene amplicon libraries. In addition, we sequenced 17 metagenomes of microbial mats and soil by high-throughput sequencing and analyzed these data using the IntegronFinder program. As expected, the metagenomic analysis allowed for the identification of novel predicted intI integrases and gene cassettes (GCs), which mostly encode unknown functions. However, some intI genes are similar to sequences previously identified by amplicon library analysis in soil samples collected from non-Antarctic sites. ARGs were analyzed in the metagenomes using ABRIcate with CARD database and verified if these genes could be classified as GCs by IntegronFinder. We identified 53 ARGs in 15 metagenomes, but only four were classified as GCs, one in MTG12 metagenome (Continental Antarctica), encoding an aminoglycoside-modifying enzyme (AAC(6´)acetyltransferase) and the other three in CS1 metagenome (Maritime Antarctica). One of these genes encodes a class D ß-lactamase (blaOXA-205) and the other two are located in the same contig. One is part of a gene encoding the first 76 amino acids of aminoglycoside adenyltransferase (aadA6), and the other is a qacG2 gene.
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Bacterias/genética , Farmacorresistencia Microbiana/genética , Genes Bacterianos , Integrasas/genética , Integrones/genética , Metagenoma , Regiones Antárticas , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenómica/métodos , Filogenia , Microbiología del SueloRESUMEN
Extracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. EVs-based liquid biopsies usually require EVs isolation before content analysis, which frequently increases sample volume requirements. We here present a Flow Cytometry (FC) strategy that does not require isolation or concentration of EVs prior to staining. By doing so, it enables population analysis of EVs in samples characterized by challenging small volumes, while reducing overall sample processing time. To illustrate its application, we performed longitudinal non-lethal population analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By quantifying the proportion of vesicular particles in purified and non-purified biological samples, this method also serves as a precious tool to quality control isolates of EVs purified by different protocols. Our FC strategy has an unexplored clinical potential to analyze EVs in biofluids with intrinsically limited volumes and to multiply the number of different analytes in EVs that can be studied from a single collection of biofluid.
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The overexpression of the protein tyrosine kinase, Focal adhesion kinase (FAK), in endothelial cells has implicated its requirement in angiogenesis and tumour growth, but how pericyte FAK regulates tumour angiogenesis is unknown. We show that pericyte FAK regulates tumour growth and angiogenesis in multiple mouse models of melanoma, lung carcinoma and pancreatic B-cell insulinoma and provide evidence that loss of pericyte FAK enhances Gas6-stimulated phosphorylation of the receptor tyrosine kinase, Axl with an upregulation of Cyr61, driving enhanced tumour growth. We further show that pericyte derived Cyr61 instructs tumour cells to elevate expression of the proangiogenic/protumourigenic transmembrane receptor Tissue Factor. Finally, in human melanoma we show that when 50% or more tumour blood vessels are pericyte-FAK negative, melanoma patients are stratified into those with increased tumour size, enhanced blood vessel density and metastasis. Overall our data uncover a previously unknown mechanism of tumour growth by pericytes that is controlled by pericyte FAK.
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Proteína 61 Rica en Cisteína/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neovascularización Patológica , Pericitos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Aorta Torácica/patología , Carcinoma Pulmonar de Lewis/metabolismo , Adhesión Celular , Proliferación Celular , Femenino , Quinasa 1 de Adhesión Focal/genética , Humanos , Linfocinas/metabolismo , Masculino , Melanoma/irrigación sanguínea , Melanoma/metabolismo , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/patología , Factor de Crecimiento Placentario/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-sis/metabolismo , Transducción de Señal , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismo , Tirosina Quinasa del Receptor AxlRESUMEN
The rhizobial DctA permease is essential for the development of effective nitrogen-fixing bacteroids, which was correlated with its requirement for growth on C(4)-dicarboxylates. A previously described dctA mutant of Rhizobium tropici CIAT899, strain GA1 (dctA), however, was unexpectedly still able to grow on succinate as a sole carbon source but less efficiently than CIAT899. Like other rhizobial dctA mutants, GA1 was unable to grow on fumarate or malate as a carbon source and induced the formation of ineffective nodules. We report an alternative succinate uptake system identified by Tn5 mutagenesis of strain GA1 that was required for the remaining ability to transport and utilize succinate. The alternative uptake system required a three-gene cluster that is highly characteristic of a dctABD locus. The predicted permease-encoding gene had high sequence similarity with open reading frames encoding putative 2-oxoglutarate permeases (KgtP) of Ralstonia solanacearum and Agrobacterium tumefaciens. This analysis was in agreement with the requirement for this gene for optimal growth on and induction by 2-oxoglutarate. The permease-encoding gene of the alternative system was also designated kgtP in R. tropici. The dctBD-like genes in this cluster were found to be required for kgtP expression and were designated kgtSR. Analysis of a kgtP::lacZ transcriptional fusion indicated that a kgtSR-dependent promoter of kgtP was specifically induced by 2-oxoglutarate. The expression of kgtPp was found in bacteroids of nodules formed with either CIAT899 or GA1 on roots of Phaseolus vulgaris. Results suggested that 2-oxoglutarate might be transported or conceivably exported in nodules induced by R. tropici on roots of P. vulgaris.
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Ácidos Cetoglutáricos/metabolismo , Phaseolus/microbiología , Rhizobium tropici/crecimiento & desarrollo , Rhizobium tropici/metabolismo , Nódulos de las Raíces de las Plantas/microbiología , Ácido Succínico/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Datos de Secuencia Molecular , Familia de Multigenes/genética , Familia de Multigenes/fisiología , Análisis de Secuencia de ADNRESUMEN
Gastrointestinal cancers are still responsible for high numbers of cancer-related deaths despite advances in therapy. Tumor-associated cells play a key role in tumor biology, by supporting or halting tumor development through the production of extracellular matrix, growth factors, cytokines, and extracellular vesicles. Here, we review the roles of these tumor-associated cells in the initiation, angiogenesis, immune modulation, and resistance to therapy of gastrointestinal cancers. We also discuss novel diagnostic and therapeutic strategies directed at tumor-associated cells and their potential benefits for the survival of these patients.