RESUMEN
The IGF2 mRNA-binding protein 1 (IGF2BP1) is a non-catalytic post-transcriptional enhancer of tumor growth upregulated and associated with adverse prognosis in solid cancers. However, conserved effector pathway(s) and the feasibility of targeting IGF2BP1 in cancer remained elusive. We reveal that IGF2BP1 is a post-transcriptional enhancer of the E2F-driven hallmark in solid cancers. IGF2BP1 promotes G1/S cell cycle transition by stabilizing mRNAs encoding positive regulators of this checkpoint like E2F1. This IGF2BP1-driven shortening of the G1 cell cycle phase relies on 3'UTR-, miRNA- and m6A-dependent regulation and suggests enhancement of cell cycle progression by m6A-modifications across cancers. In addition to E2F transcription factors, IGF2BP1 also stabilizes E2F-driven transcripts directly indicating post-transcriptional 'super'-enhancer role of the protein in E2F-driven gene expression in cancer. The small molecule BTYNB disrupts this enhancer function by impairing IGF2BP1-RNA association. Consistently, BTYNB interferes with E2F-driven gene expression and tumor growth in experimental mouse tumor models.
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Factores de Transcripción E2F/genética , Neoplasias/genética , Proteínas de Unión al ARN/genética , Regiones no Traducidas 3'/genética , Animales , Línea Celular Tumoral , Factor de Transcripción E2F1/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Neoplasias/patología , Proteínas de Unión al ARN/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
OBJECTIVE: This report summarizes findings from a 2020 survey of US child and adolescent psychiatry training programs that explored the impact of the COVID-19 pandemic on pediatric telepsychiatry training. The authors hypothesized that telepsychiatry training significantly increased during the pandemic, in part due to legal and regulatory waivers during the COVID-19 public health emergency. METHODS: In August 2020, an anonymous, 28-question online survey was emailed to all (138) accredited child psychiatry fellowships on the Accreditation Council for Graduate Medical Education website. Forty-nine programs responded (36%). This analysis focuses on three of the 28 questions relevant to the hypotheses: characteristics of the program's training in telepsychiatry; perceived impediments to clinical training; and perceived impediments to didactic training pre-COVID onset vs. post-COVID onset, respectively. Total scores were created to investigate differences in training programs and impediments to including telepsychiatry pre- and post-COVID onset. Paired sample t-tests were used to compare means pre- and post-COVID onset. RESULTS: Results provided support for significant differences between training components related to telepsychiatry pre- and post-COVID onset, with participants reporting more training components post-COVID onset (M = 5.69) than pre-COVID onset (M = 1.80); t(48) = 9.33, p < .001. Participants also reported significantly fewer barriers to providing clinical experiences in pediatric telepsychiatry post-COVID onset (M = 2.65) than pre-COVID onset (M = 4.90); t(48) = - 4.20, p < .001. CONCLUSIONS: During the COVID-19 pandemic, pediatric telepsychiatry training in child psychiatry fellowships increased significantly. Perceived barriers to providing clinical, but not didactic, training decreased significantly.
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COVID-19 , Psiquiatría , Telemedicina , Adolescente , Niño , Humanos , Becas , Psiquiatría del Adolescente , Psiquiatría/educación , PandemiasRESUMEN
The oncofetal IGF2 mRNA binding proteins (IGF2BPs) are upregulated in most cancers but their paralogue-specific roles in tumor cells remain poorly understood. In a panel of five cancer-derived cell lines, IGF2BP1 shows highly conserved oncogenic potential. Consistently, the deletion of IGF2BP1 impairs the growth and metastasis of ovarian cancer-derived cells in nude mice. Gene expression analyses in ovarian cancer-derived cells reveal that the knockdown of IGF2BPs is associated with the downregulation of mRNAs that are prone to miRNA regulation. All three IGF2BPs preferentially associate upstream of miRNA binding sites (MBSs) in the 3'UTR of mRNAs. The downregulation of mRNAs co-regulated by miRNAs and IGF2BP1 is abrogated at low miRNA abundance or when miRNAs are depleted. IGF2BP1 associates with these target mRNAs in RISC-free complexes and its deletion enhances their association with AGO2. The knockdown of most miRNA-regulated target mRNAs of IGF2BP1 impairs tumor cell properties. In four primary cancers, elevated synthesis of these target mRNAs is largely associated with upregulated IGF2BP1 mRNA levels. In ovarian cancer, the enhanced expression of IGF2BP1 and most of its miRNA-controlled target mRNAs is associated with poor prognosis. In conclusion, these findings indicate that IGF2BP1 enhances an aggressive tumor cell phenotype by antagonizing miRNA-impaired gene expression.
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Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Neoplasias/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/fisiología , Animales , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Eliminación de Gen , Humanos , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , Neoplasias/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Fenotipo , Estabilidad del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
The tumor-suppressive let-7 microRNA family targets various oncogene-encoding mRNAs. We identify the let-7 targets HMGA2, LIN28B and IGF2BP1 to form a let-7 antagonizing self-promoting oncogenic triangle. Surprisingly, 3'-end processing of IGF2BP1 mRNAs is unaltered in aggressive cancers and tumor-derived cells although IGF2BP1 synthesis was proposed to escape let-7 attack by APA-dependent (alternative polyadenylation) 3' UTR shortening. However, the expression of the triangle factors is inversely correlated with let-7 levels and promoted by LIN28B impairing let-7 biogenesis. Moreover, IGF2BP1 enhances the expression of all triangle factors by recruiting the respective mRNAs in mRNPs lacking AGO proteins and let-7 miRNAs. This indicates that the downregulation of let-7, largely facilitated by LIN28B upregulation, and the protection of let-7 target mRNAs by IGF2BP1-directed shielding in mRNPs synergize in enhancing the expression of triangle factors. The oncogenic potential of this triangle was confirmed in ovarian cancer (OC)-derived ES-2 cells transduced with let-7 targeting decoys. In these, the depletion of HMGA2 only diminishes tumor cell growth under permissive conditions. The depletion of LIN28B and more prominently IGF2BP1 severely impairs tumor cell viability, self-renewal and 2D as well as 3D migration. In conclusion, this suggests the targeting of the HMGA2-LIN28B-IGF2BP1 triangle as a promising strategy in cancer treatment.
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Regulación Neoplásica de la Expresión Génica , Proteína HMGA2/genética , MicroARNs/metabolismo , Proteínas de Unión al ARN/genética , Línea Celular Tumoral , Movimiento Celular , Femenino , Células HEK293 , Proteína HMGA2/antagonistas & inhibidores , Proteína HMGA2/metabolismo , Humanos , MicroARNs/antagonistas & inhibidores , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/fisiopatología , Isoformas de ARN/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismoRESUMEN
Inappropriately activated mineralocorticoid receptor (MR) is a risk factor for vascular remodeling with unclear molecular mechanism. Recent findings suggest that post-transcriptional regulation by micro-RNAs (miRs) may be involved. Our aim was to search for MR-dependent miRs in vascular smooth muscle cells (VSMCs) and to explore the underlying molecular mechanism and the pathologic relevance. We detected that aldosteroneviathe MR reduces miR-29bin vivoin murine aorta and in human primary and cultured VSMCs (ED50= 0.07 nM) but not in endothelial cells [quantitative PCR (qPCR), luciferase assays]. This effect was mediated by an increased decay of miR-29b in the cytoplasm with unchanged miR-29 family member or primary-miR levels. Decreased miR-29b led to an increase in extracellular matrix measured by ELISA and qPCR and enhanced VSMC migration in single cell-tracking experiments. Additionally, cell proliferation and the apoptosis/necrosis ratio (caspase/lactate dehydrogenase assay) was modulated by miR-29b. Enhanced VSMC migration by aldosterone required miR-29b regulation. Control experiments were performed with scrambled RNA and empty plasmids, by comparing aldosterone-stimulated with vehicle-incubated cells. Overall, our findings provide novel insights into the molecular mechanism of aldosterone-mediated vascular pathogenesis by identifying miR-29b as a pathophysiologic relevant target of activated MR in VSMCs and by highlighting the importance of miR processing for miR regulation.-Bretschneider, M., Busch, B., Mueller, D., Nolze, A., Schreier, B., Gekle, M., Grossmann, C. Activated mineralocorticoid receptor regulates micro-RNA-29b in vascular smooth muscle cells.
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MicroARNs/genética , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Receptores de Mineralocorticoides/genética , Aldosterona/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Apoptosis/genética , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/genética , Células Cultivadas , Colágeno/metabolismo , Fibronectinas/metabolismo , Humanos , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Mineralocorticoides/agonistas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
MicroRNAs (miRNAs) control gene expression at the post-transcriptional level. However, the identification of miRNAs regulating the fate of a specific messenger RNA remains limited due to the imperfect complementarity of miRNAs and targeted transcripts. Here, we describe miTRAP (miRNA trapping by RNA in vitro affinity purification), an advanced protocol of previously reported MS2-tethering approaches. MiTRAP allows the rapid identification of miRNAs targeting an in vitro transcribed RNA in cell lysates. Selective co-purification of regulatory miRNAs was confirmed for the MYC- as well as ZEB2-3'UTR, two well-established miRNA targets in vivo. Combined with miRNA-sequencing, miTRAP identified in addition to miRNAs reported to control MYC expression, 18 novel candidates including not in silico predictable miRNAs. The evaluation of 10 novel candidate miRNAs confirmed 3'UTR-dependent regulation of MYC expression as well as putative non-canonical targeting sites for the not in silico predictable candidates. In conclusion, miTRAP provides a rapid, cost-effective and easy-to-handle protocol allowing the identification of regulatory miRNAs for RNAs of choice in a cellular context of interest. Most notably, miTRAP not only identifies in silico predictable but also unpredictable miRNAs regulating the expression of a specific target RNA.
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Regiones no Traducidas 3' , MicroARNs/aislamiento & purificación , Línea Celular , Regulación de la Expresión Génica , Genes myc , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , MicroARNs/metabolismo , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
Recently, bryophytes, which diverged from the ancestor of seed plants more than 400 million years ago, came into focus in photosynthesis research as they can provide valuable insights into the evolution of photosynthetic complexes during the adaptation to terrestrial life. This study isolated intact photosystem I (PSI) with its associated light-harvesting complex (LHCI) from the moss Physcomitrella patens and characterized its structure, polypeptide composition, and light-harvesting function using electron microscopy, mass spectrometry, biochemical, and physiological methods. It became evident that Physcomitrella possesses a strikingly high number of isoforms for the different PSI core subunits as well as LHCI proteins. It was demonstrated that all these different subunit isoforms are expressed at the protein level and are incorporated into functional PSI-LHCI complexes. Furthermore, in contrast to previous reports, it was demonstrated that Physcomitrella assembles a light-harvesting complex consisting of four light-harvesting proteins forming a higher-plant-like PSI superstructure.
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Bryopsida/metabolismo , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/metabolismo , Bryopsida/química , Bryopsida/genética , Bryopsida/efectos de la radiación , Luz , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Fotosíntesis , Complejo de Proteína del Fotosistema I/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
The use and development of post-genomic tools naturally depends on large-scale genome sequencing projects. The usefulness of post-genomic applications is dependent on the accuracy of genome annotations, for which the correct identification of intron-exon borders in complex genomes of eukaryotic organisms is often an error-prone task. Although automated algorithms for predicting intron-exon structures are available, supporting exon evidence is necessary to achieve comprehensive genome annotation. Besides cDNA and EST support, peptides identified via MS/MS can be used as extrinsic evidence in a proteogenomic approach. We describe an improved version of the Genomic Peptide Finder (GPF), which aligns de novo predicted amino acid sequences to the genomic DNA sequence of an organism while correcting for peptide sequencing errors and accounting for the possibility of splicing. We have coupled GPF and the gene finding program AUGUSTUS in a way that provides automatic structural annotations of the Chlamydomonas reinhardtii genome, using highly unbiased GPF evidence. A comparison of the AUGUSTUS gene set incorporating GPF evidence to the standard JGI FM4 (Filtered Models 4) gene set reveals 932 GPF peptides that are not contained in the Filtered Models 4 gene set. Furthermore, the GPF evidence improved the AUGUSTUS gene models by altering 65 gene models and adding three previously unidentified genes.
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Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Biología Computacional/métodos , Genómica/métodos , Proteómica/métodos , Algoritmos , Secuencia de Aminoácidos , Secuencia de Bases , Bases de Datos Genéticas , Exones/genética , Genoma de Planta/genética , Intrones/genética , Espectrometría de Masas , Datos de Secuencia Molecular , Péptidos/análisis , Péptidos/genética , Proteínas de Plantas/análisis , Proteínas de Plantas/genética , Sitios de Empalme de ARN/genética , Homología de Secuencia de Aminoácido , Programas InformáticosRESUMEN
Cilia are disassembled prior to cell division, which is proposed to regulate proper cell cycle progression. The signaling pathways that regulate cilia disassembly are not well-understood. Recent biochemical and genetic data demonstrate that protein phosphorylation plays important roles in cilia disassembly. Here, we analyzed the phosphoproteins in the membrane/matrix fraction of flagella undergoing shortening as well as flagella from steady state cells of Chlamydomonas. The phosphopeptides were enriched by a combination of IMAC and titanium dioxide chromatography with a strategy of sequential elution from IMAC (SIMAC) and analyzed by tandem mass spectrometry. A total of 224 phosphoproteins derived from 1296 spectral counts of phosphopeptides were identified. Among the identified phosphoproteins are flagellar motility proteins such as outer dynein arm, intraflagellar transport proteins as well as signaling molecules including protein kinases, phosphatases, G proteins, and ion channels. Eighty-nine of these phosphoproteins were only detected in shortening flagella, whereas 29 were solely in flagella of steady growing cells, indicating dramatic changes of protein phosphorylation during flagellar shortening. Our data indicates that protein phosphorylation is a key event in flagellar disassembly, and paves the way for further study of flagellar assembly and disassembly controlled by protein phosphorylation.
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Chlamydomonas reinhardtii/metabolismo , Flagelos/metabolismo , Fosfoproteínas/metabolismo , Secuencia de Aminoácidos , Western Blotting , Cromatografía/métodos , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosforilación , Transducción de SeñalRESUMEN
MYCN gene amplification and upregulated expression are major hallmarks in the progression of high-risk neuroblastoma. MYCN expression and function in modulating gene synthesis in neuroblastoma is controlled at virtually every level, including poorly understood regulation at the post-transcriptional level. MYCN modulates the expression of various microRNAs including the miR-17-92 cluster. MYCN mRNA expression itself is subjected to the control by miRNAs, most prominently the miR-17-92 cluster that balances MYCN expression by feed-back regulation. This homeostasis seems disturbed in neuroblastoma where MYCN upregulation coincides with severely increased expression of the miR-17-92 cluster. In the presented study, we applied high-throughput next generation sequencing to unravel the miRNome in a cohort of 97 neuroblastomas, representing all clinical stages. Aiming to reveal the MYCN-dependent miRNome, we evaluate miRNA expression in MYCN-amplified as well as none amplified tumor samples. In correlation with survival data analysis of differentially expressed miRNAs, we present various putative oncogenic as well as tumor suppressive miRNAs in neuroblastoma. Using microRNA trapping by RNA affinity purification, we provide a comprehensive view of MYCN-regulatory miRNAs in neuroblastoma-derived cells, confirming a pivotal role of the miR-17-92 cluster and moderate association by the let-7 miRNA family. Attempting to decipher how MYCN expression escapes elevated expression of inhibitory miRNAs, we present evidence that RNA-binding proteins like the IGF2 mRNA binding protein 1 reduce miRNA-directed downregulation of MYCN in neuroblastoma. Our findings emphasize the potency of post-transcriptional regulation of MYCN in neuroblastoma and unravel new avenues to pursue inhibition of this potent oncogene.
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Objectives: Our goal was to develop an open access nationally disseminated online curriculum for use in graduate and continuing medical education on the topic of pediatric telepsychiatry to enhance the uptake of telepsychiatry among child psychiatry training programs and improve access to mental health care for youth and families. Methods: Following Kern's 6-stage model of curriculum development, we identified a core problem, conducted a needs assessment, developed broad goals and measurable objectives in a competency-based model, and developed educational content and methods. The curriculum was reviewed by experts and feedback incorporated. Given the urgent need for such a curriculum due to the COVID-19 pandemic, the curriculum was immediately posted on the American Academy of Child and Adolescent Psychiatry and American Association of Directors of Psychiatric Residency Training websites. Further evaluation will be conducted over the next year. Results: The curriculum covers the six areas of core competence adapted for pediatric telepsychiatry and includes teaching content and resources, evaluation tools, and information about other resources. Conclusion: This online curriculum is available online and provides an important resource and set of standards for pediatric telepsychiatry training. Its online format allows for ongoing revision as the telepsychiatry landscape changes.
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Psiquiatría del Adolescente/educación , COVID-19 , Psiquiatría Infantil/educación , Curriculum/tendencias , Educación Médica Continua , Educación de Postgrado en Medicina , Acceso a la Información , Adolescente , COVID-19/epidemiología , COVID-19/prevención & control , Niño , Educación/métodos , Educación/organización & administración , Educación Médica Continua/métodos , Educación Médica Continua/organización & administración , Educación de Postgrado en Medicina/métodos , Educación de Postgrado en Medicina/organización & administración , Accesibilidad a los Servicios de Salud , Humanos , Servicios de Salud Mental/normas , Servicios de Salud Mental/tendencias , Innovación Organizacional , Objetivos Organizacionales , SARS-CoV-2 , Telemedicina/métodosRESUMEN
The oncofetal IGF2 mRNA-binding protein 1 (IGF2BP1) promotes tumor progression in a variety of solid tumors and its expression is associated with adverse prognosis. The main role proposed for IGF2BP1 in cancer cells is the stabilization of mRNAs encoding pro-oncogenic factors. Several IGF2BP1-RNA association studies, however, revealed a plethora of putative IGF2BP1-RNA targets. Thus, at present the main conserved target RNAs and pathways controlled by IGF2BP1 in cancer remain elusive. In this study, we present a set of genes and cancer hallmark pathways showing a conserved pattern of deregulation in dependence of IGF2BP1 expression in cancer cell lines. By the integrative analysis of these findings with publicly available cancer transcriptome and IGF2BP1-RNA association data, we compiled a set of prime candidate target mRNAs. These analyses confirm a pivotal role of IGF2BP1 in controlling cancer cell cycle progression and reveal novel cancer hallmark pathways influenced by IGF2BP1. For three novel target mRNAs identified by these studies, namely AURKA, HDLBP and YWHAZ, we confirm IGF2BP1 mRNA stabilization. In sum our findings confirm and expand previous findings on the pivotal role of IGF2BP1 in promoting oncogenic gene expression by stabilizing target mRNAs in a mainly 3'UTR, m6A-, miRNA-, and potentially AU-rich element dependent manner.
RESUMEN
A catalytic manganese (Mn) cluster is required for the oxidation of water in the oxygen-evolving complex (OEC) of photosystem II (PSII) in plants. Despite this essential role of Mn in generating the electrons driving photosynthesis, limited information is available on how Mn deficiency affects PSII functionality. We have here used parameters derived from measurements of fluorescence induction kinetics (OJIP transients), non-photochemical quenching (NPQ) and PSII subunit composition to investigate how latent Mn deficiency changes the photochemistry in two barley genotypes differing in Mn efficiency. Mn deficiency caused dramatic reductions in the quantum yield of PSII and led to the appearance of two new inflection points, the K step and the D dip, in the OJIP fluorescence transients, indicating severe damage to the OEC. In addition, Mn deficiency decreased the ability to induce NPQ in the light, rendering the plants incapable of dissipating excess energy in a controlled way. Thus, the Mn deficient plants became severely affected in their ability to recover from high light-induced photoinhibition, especially under strong Mn deficiency. Interestingly, the Mn-efficient genotype was able to maintain a higher NPQ than the Mn-inefficient genotype when exposed to mild Mn deficiency. However, during severe Mn deficiency, there were no differences between the two genotypes, suggesting a general loss of the ability to disassemble and repair PSII. The pronounced defects of PSII activity were supported by a dramatic decrease in the abundance of the OEC protein subunits, PsbP and PsbQ in response to Mn deficiency for both genotypes. We conclude that regulation of photosynthetic performance by means of maintaining and inducing NPQ mechanisms contribute to genotypic differences in the Mn efficiency of barley genotypes growing under conditions with mild Mn deficiency.