RESUMEN
The standard treatment approach for stage II/III rectal cancer is neoadjuvant chemoradiation therapy (nCRT) followed by surgery. In recent years, new treatment approaches have led to higher rates of complete tumor eradication combined with organ-preservation strategies. However, better tools are still needed to personalize therapy for the individual patient. In this prospective observational study, we analyzed colon-derived cell-free (cf)DNA (c-cfDNA) using a tissue-specific DNA methylation signature, and its association with therapy outcomes. Analyzing plasma samples (n = 303) collected during nCRT from 37 patients with locally advanced rectal cancer (LARC), we identified colon-specific methylation markers that discriminated healthy individuals from patients with untreated LARC (area under the curve, 0.81; 95% confidence interval, 0.70-0.92; P < .0001). Baseline c-cfDNA predicted tumor response, with increased levels linked to larger residual cancer. c-cfDNA measured after the first week of therapy identified patients with maximal response and complete cancer eradication, who had significantly lower c-cfDNA compared with those who had residual disease (8.6 vs 57.7 average copies/ml, respectively; P = .013). Increased c-cfDNA after 1 week of therapy was also associated with disease recurrence. Methylation-based liquid biopsy can predict nCRT outcomes and facilitate patient selection for escalation and de-escalation strategies.
Asunto(s)
Ácidos Nucleicos Libres de Células , Neoplasias del Recto , Humanos , Ácidos Nucleicos Libres de Células/genética , Recurrencia Local de Neoplasia , Quimioradioterapia , Neoplasias del Recto/genética , Neoplasias del Recto/terapia , Neoplasias del Recto/patología , Recto/patología , Terapia Neoadyuvante , Resultado del Tratamiento , Estudios RetrospectivosRESUMEN
Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.
Asunto(s)
Antígenos Bacterianos/farmacología , Toxinas Bacterianas/farmacología , Edema/metabolismo , Endosomas/efectos de los fármacos , Uniones Intercelulares/efectos de los fármacos , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Cadherinas/metabolismo , Línea Celular , Endosomas/metabolismo , Uniones Intercelulares/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Development of effective therapies against SARS-CoV-2 infections relies on mechanistic knowledge of virus-host interface. Abundant physical interactions between viral and host proteins have been identified, but few have been functionally characterized. Harnessing the power of fly genetics, we develop a comprehensive Drosophila COVID-19 resource (DCR) consisting of publicly available strains for conditional tissue-specific expression of all SARS-CoV-2 encoded proteins, UAS-human cDNA transgenic lines encoding established host-viral interacting factors, and GAL4 insertion lines disrupting fly homologs of SARS-CoV-2 human interacting proteins. We demonstrate the utility of the DCR to functionally assess SARS-CoV-2 genes and candidate human binding partners. We show that NSP8 engages in strong genetic interactions with several human candidates, most prominently with the ATE1 arginyltransferase to induce actin arginylation and cytoskeletal disorganization, and that two ATE1 inhibitors can reverse NSP8 phenotypes. The DCR enables parallel global-scale functional analysis of SARS-CoV-2 components in a prime genetic model system.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Animales , SARS-CoV-2/genética , Drosophila , Actinas , Animales Modificados GenéticamenteRESUMEN
Clostridium difficile infections (CDIs) cause severe and occasionally life-threatening diarrhea. Hyper-virulent strains produce CDT, a toxin that ADP-ribosylates actin monomers and inhibits actin polymerization. We created transgenic Drosophila lines expressing the catalytic subunit CDTa to investigate its interaction with host signaling pathways in vivo. When expressed in the midgut, CDTa reduces body weight and fecal output and compromises survival, suggesting severe impairment of digestive functions. At the cellular level, CDTa induces F-actin network collapse, elimination of the intestinal brush border, and disruption of intercellular junctions. We confirm toxin-dependent re-distribution of Rab11 to enterocytes' apical surface and observe suppression of CDTa phenotypes by a Dominant-Negative form of Rab11 or RNAi of the dedicated Rab11GEF Crag (DENND4). We also report that Calmodulin (Cam) is required to mediate CDTa activity. In parallel, chemical inhibition of the Cam/Calcineurin pathway by Cyclosporin A or FK506 also reduces CDTa phenotypes, potentially opening new avenues for treating CDIs.
RESUMEN
Tumor-specific, circulating cell-free DNA in liquid biopsies is a promising source of biomarkers for minimally invasive serial monitoring of treatment responses in cancer management. We will review the current understanding of the origin of circulating cell-free DNA and different forms of DNA release (including various types of cell death and active secretion processes) and clearance routes. The dynamics of extracellular DNA in blood during therapy and the role of circulating DNA in pathophysiological processes (tumor-associated inflammation, NETosis, and pre-metastatic niche development) provide insights into the mechanisms that contribute to tumor development and metastases formation. Better knowledge of circulating tumor-specific cell-free DNA could facilitate the development of new therapeutic and diagnostic options for cancer management.
Asunto(s)
Biomarcadores de Tumor , Ácidos Nucleicos Libres de Células , ADN Tumoral Circulante , ADN de Neoplasias , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/metabolismo , Humanos , Biopsia Líquida/métodos , Neoplasias/sangre , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Especificidad de ÓrganosRESUMEN
Responses of teachers and children in Northern Ireland and observations in other areas of the world beset by the daily threat of violence and death are reported. Respondents showed a deep concern about the role of schools in relation to children who have grown up with warfare in the streets. The concept of school-as-sanctuary is discussed in light of its effects on the processes of teaching, learning, and the emotional development of students.
Asunto(s)
Trastornos de la Conducta Infantil/psicología , Trastornos Reactivos del Niño/psicología , Enseñanza , Actuación (Psicología) , Adaptación Psicológica , Trastorno de Personalidad Antisocial/psicología , Actitud , Niño , Humanos , Discapacidades para el Aprendizaje/psicología , Irlanda del Norte , Desarrollo de la Personalidad , Política , ViolenciaRESUMEN
In maturing T lineage cells, the helix-loop-helix protein E47 has been shown to enforce a critical proliferation and developmental checkpoint commonly referred to as beta selection. To examine how E47 regulates cellular expansion and developmental progression, we have used an E2A-deficient lymphoma cell line and DNA microarray analysis to identify immediate E47 target genes. Hierarchical cluster analysis of gene expression patterns revealed that E47 coordinately regulates the expression of genes involved in cell survival, cell cycle progression, lipid metabolism, stress response, and lymphoid maturation. These include Plcgamma2, Cdk6, CD25, Tox, Gadd45a, Gadd45b, Gfi1, Gfi1b, Socs1, Socs3, Id2, Eto2, and Xbp1. We propose a regulatory network linking Janus kinase (JAK)/signal transducer and activator of transcription (STAT)-mediated signaling, E47, and suppressor of cytokine signaling (SOCS) proteins in a common pathway. Finally, we suggest that the aberrant activation of Cdk6 in E47-deficient T lineage cells contributes to the development of lymphoid malignancy.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Linfoma/genética , Linfocitos T/inmunología , Factores de Transcripción TCF/fisiología , Ciclo Celular/genética , Línea Celular Tumoral , Linaje de la Célula/genética , Proliferación Celular , Supervivencia Celular/genética , Citocinas/metabolismo , Humanos , Janus Quinasa 1 , Metabolismo de los Lípidos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Tirosina Quinasas/genética , Receptores de Interleucina-2/genética , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/genética , Linfocitos T/citología , Linfocitos T/metabolismo , Factores de Transcripción TCF/genética , Proteína 1 Similar al Factor de Transcripción 7RESUMEN
The response of thymocytes to pre-T cell receptor (pre-TCR) signaling includes proliferation and gene rearrangement, two cellular processes that are incompatible. The control of proliferation by pre-TCR signals depends on the activities of the transcription factors RORgammat, Egr3, E12, and E47. Here, we describe a regulatory network in which interplay between these factors ensures transient proliferation that is temporally distinct from gene rearrangement. RORgammat expression was elevated after pre-TCR signaling, and RORgammat promoted gene rearrangement in CD4+, CD8+ cells by inhibiting cell division, promoting survival via Bcl-X(L), and inducing Rag2. Egr3 was transiently induced by pre-TCR signals and promoted a distinct proliferative phase by reducing E protein-dependent RORgammat expression and interacting with RORgammat to prevent induction of target genes. After Egr3 subsided, the expression and function of RORgammat increased. Thus, transient induction of Egr3 delays the effects of RORgammat and enables pre-TCR signaling to induce both proliferation and gene rearrangement.