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Background Delirium is a syndrome of acute brain failure that represents a change from an individual's baseline cognitive functioning characterized by deficits in attention and multiple aspects of cognition that fluctuate in severity over time. The symptomatic management of delirium's behavioral manifestations remains difficult. The alpha-2 agonists, dexmedetomidine and clonidine, are efficacious, but their potential cardiovascular adverse effects limit their utilization. Guanfacine is an oral alpha-2 agonist with a lower potential for such adverse outcomes; however, its use in delirium has not been studied. Methods A retrospective descriptive analysis of guanfacine for managing hyperactive or mixed delirium at Tampa General Hospital from January 2020 to October 2020 was conducted. The primary outcome was the time reduction in acute sedative administration. Secondary outcomes included renewed participation in physical therapy or occupational therapy (PT/OT), decreased opioid use, and an incidence of cardiovascular adverse effects. Results One hundred forty-nine patients were identified as having received guanfacine for managing delirium during the study period. All experienced a reduction in acute sedative use after the initiation of guanfacine. In 93 patients receiving PT/OT and no longer participating due to behavioral agitation, 74% had a documented renewal of services within four days. Of 112 patients on opioids, 70% experienced a 25% reduction in opioid administration within four days. No patients experienced consecutive episodes of hypotension that required a change in their clinical care. Two patients experienced a single episode of consecutive bradycardia that led to the discontinuation of guanfacine. Conclusions Based on our retrospective study, guanfacine is a well-tolerated medication for the management of delirium. Even in medically and critically ill patients, cardiovascular adverse events were rare with guanfacine. Patients treated with guanfacine experienced decreased acute sedative use for behavioral agitation. Additionally, patients treated with guanfacine received fewer opioids and were better able to participate in PT/OT. Future studies with prospective, randomized, placebo-controlled designs are warranted to evaluate this promising intervention for delirium further.
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Exposure to early-life stress (ELS) can persistently modify neuronal circuits and functions, and contribute to the expression of misfolded and aggregated proteins that are hallmarks of several neurodegenerative diseases. The healthy brain is able to clear dysfunctional proteins through the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP). Accumulating evidence indicates that impairment of these pathways contributes to enhanced protein aggregation and neurodegeneration. While stress is a known precipitant of neurological decline, few specific mechanistic links underlying this relationship have been identified. We hypothesized that neonatal maternal separation (MatSep), a well-established model of ELS, has the ability to alter the levels of UPS and ALP components in the brain, and thus has the potential to disrupt proteostasis. The expression of proteostasis-associated protein markers was evaluated by immunoblotting in the hippocampus and cortex of adult Wistar rats that were previously subjected to MatSep. We observed multiple sex- and MatSep-specific changes in the expression of proteins in the ALP, mitophagy, and UPS pathways, particularly in the hippocampus of adult animals. In contrast, MatSep had limited influence on proteostasis marker expression in the cortex of adult animals. Our results indicate that MatSep can selectively modify the intracellular protein degradation machinery in ways that may impact the development and progression of neurodegenerative disease.
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Childhood brain tumors have suspected prenatal origins. To identify vulnerable developmental states, we generated a single-cell transcriptome atlas of >65,000 cells from embryonal pons and forebrain, two major tumor locations. We derived signatures for 191 distinct cell populations and defined the regional cellular diversity and differentiation dynamics. Projection of bulk tumor transcriptomes onto this dataset shows that WNT medulloblastomas match the rhombic lip-derived mossy fiber neuronal lineage and embryonal tumors with multilayered rosettes fully recapitulate a neuronal lineage, while group 2a/b atypical teratoid/rhabdoid tumors may originate outside the neuroectoderm. Importantly, single-cell tumor profiles reveal highly defined cell hierarchies that mirror transcriptional programs of the corresponding normal lineages. Our findings identify impaired differentiation of specific neural progenitors as a common mechanism underlying these pediatric cancers and provide a rational framework for future modeling and therapeutic interventions.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Encéfalo/patologia , Linhagem Celular Tumoral , Humanos , Lactente , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/patologia , Fibras Nervosas/patologia , Fibras Nervosas/fisiologia , Prosencéfalo/citologia , Prosencéfalo/embriologia , Tumor Rabdoide/genética , Tumor Rabdoide/patologia , Análise de Célula ÚnicaAssuntos
Ligante Coestimulador de Linfócitos T Induzíveis/deficiência , Imunodeficiência Combinada Severa/genética , Adulto , Substituição de Aminoácidos , Apresentação de Antígeno , Quimiotaxia de Leucócito , Complexo de Golgi/química , Humanos , Imunogenicidade da Vacina , Ligante Coestimulador de Linfócitos T Induzíveis/química , Ligante Coestimulador de Linfócitos T Induzíveis/genética , Ligante Coestimulador de Linfócitos T Induzíveis/imunologia , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Infecções/etiologia , Ativação Linfocitária , Masculino , Mutação de Sentido Incorreto , Fenótipo , Transporte Proteico , Recidiva , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/imunologiaRESUMO
Primary immunodeficiencies represent naturally occurring experimental models to decipher human immunobiology. We report a patient with combined immunodeficiency, marked by recurrent respiratory tract and DNA-based viral infections, hypogammaglobulinemia, and panlymphopenia. He also developed moderate neutropenia but without prototypical pyogenic infections. Using whole-exome sequencing, we identified a homozygous mutation in the inducible T cell costimulator ligand gene (ICOSLG; c.657C>G; p.N219K). Whereas WT ICOSL is expressed at the cell surface, the ICOSLN219K mutation abrogates surface localization: mutant protein is retained in the endoplasmic reticulum/Golgi apparatus, which is predicted to result from deleterious conformational and biochemical changes. ICOSLN219K diminished B cell costimulation of T cells, providing a compelling basis for the observed defect in antibody and memory B cell generation. Interestingly, ICOSLN219K also impaired migration of lymphocytes and neutrophils across endothelial cells, which normally express ICOSL. These defects likely contributed to the altered adaptive immunity and neutropenia observed in the patient, respectively. Our study identifies human ICOSLG deficiency as a novel cause of a combined immunodeficiency.
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Síndromes de Imunodeficiência , Ligante Coestimulador de Linfócitos T Induzíveis/deficiência , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Linfócitos B/imunologia , Linfócitos B/patologia , Linhagem Celular Transformada , Células Endoteliais/imunologia , Células Endoteliais/patologia , Feminino , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Memória Imunológica , Ligante Coestimulador de Linfócitos T Induzíveis/imunologia , Masculino , Linfócitos T/imunologia , Linfócitos T/patologia , Sequenciamento Completo do GenomaRESUMO
Therapies targeting epidermal growth factor receptor (EGFR) have variable and unpredictable responses in breast cancer. Screening triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), we identify a subset responsive to EGFR inhibition by gefitinib, which displays heterogeneous expression of wild-type EGFR. Deep single-cell RNA sequencing of 3,500 cells from an exceptional responder identified subpopulations displaying distinct biological features, where elevated EGFR expression was significantly enriched in a mesenchymal/stem-like cellular cluster. Sorted EGFRhi subpopulations exhibited enhanced stem-like features, including ALDH activity, sphere-forming efficiency, and tumorigenic and metastatic potential. EGFRhi cells gave rise to EGFRhi and EGFRlo cells in primary and metastatic tumors, demonstrating an EGFR-dependent expansion and hierarchical state transition. Similar tumorigenic EGFRhi subpopulations were identified in independent PDXs, where heterogeneous EGFR expression correlated with gefitinib sensitivity. This provides new understanding for an EGFR-dependent hierarchy in TNBC and for patient stratification for therapeutic intervention.
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Receptores ErbB/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Proteína BRCA1/metabolismo , Feminino , Gefitinibe , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia de Fluorescência , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , RNA Neoplásico/química , RNA Neoplásico/isolamento & purificação , RNA Neoplásico/metabolismo , Análise de Sequência de RNA , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Renin, a key component in the regulation of blood pressure in mammals, is produced by the rare and highly specialized juxtaglomerular cells of the kidney. Chronic stimulation of renin release results in a recruitment of new juxtaglomerular cells by the apparent conversion of adjacent smooth muscle cells along the afferent arterioles. Because juxtaglomerular cells rapidly dedifferentiate when removed from the kidney, their developmental origin and the mechanism that explains their phenotypic plasticity remain unclear. To overcome this limitation, we have performed RNA expression analysis on 4 human renin-producing tumors. The most highly expressed genes that were common between the reninomas were subsequently used for in situ hybridization in kidneys of 5-day-old mice, adult mice, and adult mice treated with captopril. From the top 100 genes, 10 encoding for ligands were selected for further analysis. Medium of human embryonic kidney 293 cells transfected with the mouse cDNA encoding these ligands was applied to (pro)renin-synthesizing As4.1 cells. Among the ligands, only platelet-derived growth factor B reduced the medium and cellular (pro)renin levels, as well as As4.1 renin gene expression. In addition, platelet-derived growth factor B-exposed As4.1 cells displayed a more elongated and aligned shape with no alteration in viability. This was accompanied by a downregulated expression of α-smooth muscle actin and an upregulated expression of interleukin-6, suggesting a phenotypic shift from myoendocrine to inflammatory. Our results add 36 new genes to the list that characterize renin-producing cells and reveal a novel role for platelet-derived growth factor B as a regulator of renin-synthesizing cells.
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Perfilação da Expressão Gênica , Sistema Justaglomerular/citologia , Nefropatias/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Renina/biossíntese , Análise de Variância , Animais , Células Cultivadas , Modelos Animais de Doenças , Expressão Gênica , Humanos , Hibridização In Situ , Sistema Justaglomerular/metabolismo , Nefropatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Renina/genética , Transdução de SinaisRESUMO
The eukaryotic translation initiation factor eIF4E acts in the nuclear export and translation of a subset of mRNAs. Both of these functions contribute to its oncogenic potential. While the biochemical mechanisms that underlie translation are relatively well understood, the molecular basis for eIF4E's role in mRNA export remains largely unexplored. To date, over 3000 transcripts, many encoding oncoproteins, were identified as potential nuclear eIF4E export targets. These target RNAs typically contain a â¼50-nucleotide eIF4E sensitivity element (4ESE) in the 3' UTR and a 7-methylguanosine cap on the 5' end. While eIF4E associates with the cap, an unknown factor recognizes the 4ESE element. We previously identified cofactors that functionally interacted with eIF4E in mammalian cell nuclei including the leucine-rich pentatricopeptide repeat protein LRPPRC and the export receptor CRM1/XPO1. LRPPRC simultaneously interacts with both eIF4E bound to the 5' mRNA cap and the 4ESE element in the 3' UTR. In this way, LRPPRC serves as a specificity factor to recruit 4ESE-containing RNAs within the nucleus. Further, we show that CRM1 directly binds LRPPRC likely acting as the export receptor for the LRPPRC-eIF4E-4ESE RNA complex. We also found that Importin 8, the nuclear importer for cap-free eIF4E, imports RNA-free LRPPRC, potentially providing both coordinated nuclear recycling of the export machinery and an important surveillance mechanism to prevent futile export cycles. Our studies provide the first biochemical framework for the eIF4E-dependent mRNA export pathway.
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Fator de Iniciação 4E em Eucariotos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Animais , Núcleo Celular/metabolismo , Fator de Iniciação 4E em Eucariotos/química , Humanos , Carioferinas/metabolismo , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Proteínas de Neoplasias/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte de RNA , RNA Mensageiro/química , Receptores Citoplasmáticos e Nucleares/metabolismo , beta Carioferinas/metabolismo , Proteína Exportina 1RESUMO
Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing specific repertoires of genetic material. In mammals, EVs can mediate the horizontal transfer of various cargos and signaling molecules, notably miRNA and mRNA species. Whether this form of intercellular communication prevails in other metazoans remains unclear. Here, we report the first parallel comparative morphologic and transcriptomic characterization of EVs from Drosophila and human cellular models. Electronic microscopy revealed that human and Drosophila cells release similar EVs with diameters ranging from 30 to 200 nm, which contain complex populations of transcripts. RNA-seq identified abundant ribosomal RNAs, related pseudogenes and retrotransposons in human and Drosophila EVs. Vault RNAs and Y RNAs abounded in human samples, whereas small nucleolar RNAs involved in pseudouridylation were most prevalent in Drosophila EVs. Numerous mRNAs were identified, largely consisting of exonic sequences displaying full-length read coverage and enriched for translation and electronic transport chain functions. By analogy with human systems, these sizeable similarities suggest that EVs could potentially enable RNA-mediated intercellular communication in Drosophila.
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Drosophila/metabolismo , Vesículas Extracelulares/metabolismo , Transcriptoma , Animais , Drosophila/genética , Vesículas Extracelulares/genética , Vesículas Extracelulares/ultraestrutura , Humanos , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Especificidade da EspécieRESUMO
BACKGROUND: Genome-wide expression profiling is increasingly being used to identify transcriptional changes induced by drugs and environmental stressors. In this context, the Toxicogenomics Project-Genomics Assisted Toxicity Evaluation system (TG-GATEs) project generated transcriptional profiles from rat liver samples and human/rat cultured primary hepatocytes exposed to more than 100 different chemicals. OBJECTIVES: To assess the capacity of the cell culture models to recapitulate pathways induced by chemicals in vivo, we leveraged the TG-GATEs data set to compare the early transcriptional responses observed in the liver of rats treated with a large set of chemicals with those of cultured rat and human primary hepatocytes challenged with the same compounds in vitro. METHODS: We developed a new pathway-based computational pipeline that efficiently combines gene set enrichment analysis (GSEA) using pathways from the Reactome database with biclustering to identify common modules of pathways that are modulated by several chemicals in vivo and in vitro across species. RESULTS: We found that some chemicals induced conserved patterns of early transcriptional responses in in vitro and in vivo settings, and across human and rat genomes. These responses involved pathways of cell survival, inflammation, xenobiotic metabolism, oxidative stress, and apoptosis. Moreover, our results support the transforming growth factor beta receptor (TGF-ßR) signaling pathway as a candidate biomarker associated with exposure to environmental toxicants in primary human hepatocytes. CONCLUSIONS: Our integrative analysis of toxicogenomics data provides a comprehensive overview of biochemical perturbations affected by a large panel of chemicals. Furthermore, we show that the early toxicological response occurring in animals is recapitulated in human and rat primary hepatocyte cultures at the molecular level, indicating that these models reproduce key pathways in response to chemical stress. These findings expand our understanding and interpretation of toxicogenomics data from human hepatocytes exposed to environmental toxicants.
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Poluentes Ambientais/toxicidade , Hepatócitos/efeitos dos fármacos , Animais , Células Cultivadas , Análise por Conglomerados , Relação Dose-Resposta a Droga , Expressão Gênica , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ratos , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Especificidade da Espécie , Transcrição GênicaRESUMO
Traumatic brain injury (TBI), including mild TBI (mTBI), is importantly associated with vigilance and sleep complaints. Because sleep is required for learning, plasticity and recovery, we here evaluated the bidirectional relationship between mTBI and sleep with two specific objectives: (1) Test that mTBI rapidly impairs sleep-wake architecture and the dynamics of the electrophysiological marker of sleep homeostasis (i.e., non-rapid eye movement sleep delta (1-4Hz) activity); (2) evaluate the impact of sleep loss following mTBI on the expression of plasticity markers that have been linked to sleep homeostasis and on genome-wide gene expression. A closed-head injury model was used to perform a 48h electrocorticographic (ECoG) recording in mice submitted to mTBI or Sham surgery. mTBI was found to immediately decrease the capacity to sustain long bouts of wakefulness as well as the amplitude of the time course of ECoG delta activity during wakefulness. Significant changes in ECoG spectral activity during wakefulness, non-rapid eye movement and rapid eye movement sleep were observed mainly on the second recorded day. A second experiment was performed to measure gene expression in the cerebral cortex and hippocampus after a mTBI followed either by two consecutive days of 6h sleep deprivation (SD) or of undisturbed behavior (quantitative PCR and next-generation sequencing). mTBI modified the expression of genes involved in immunity, inflammation and glial function (e.g., chemokines, glial markers) and SD changed that of genes linked to circadian rhythms, synaptic activity/neuronal plasticity, neuroprotection and cell death and survival. SD appeared to affect gene expression in the cerebral cortex more importantly after mTBI than Sham surgery including that of the astrocytic marker Gfap, which was proposed as a marker of clinical outcome after TBI. Interestingly, SD impacted the hippocampal expression of the plasticity elements Arc and EfnA3 only after mTBI. Overall, our findings reveal alterations in spectral signature across all vigilance states in the first days after mTBI, and show that sleep loss post-mTBI reprograms the transcriptome in a brain area-specific manner and in a way that could be deleterious to brain recovery.
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Lesões Encefálicas/fisiopatologia , Encéfalo/fisiopatologia , Ritmo Circadiano/fisiologia , Sono/fisiologia , Transcriptoma , Animais , Encéfalo/metabolismo , Lesões Encefálicas/metabolismo , Eletrocorticografia , Masculino , Camundongos , Privação do Sono/metabolismo , Privação do Sono/fisiopatologiaRESUMO
BACKGROUND: Microarrays have revolutionized breast cancer (BC) research by enabling studies of gene expression on a transcriptome-wide scale. Recently, RNA-Sequencing (RNA-Seq) has emerged as an alternative for precise readouts of the transcriptome. To date, no study has compared the ability of the two technologies to quantify clinically relevant individual genes and microarray-derived gene expression signatures (GES) in a set of BC samples encompassing the known molecular BC's subtypes. To accomplish this, the RNA from 57 BCs representing the four main molecular subtypes (triple negative, HER2 positive, luminal A, luminal B), was profiled with Affymetrix HG-U133 Plus 2.0 chips and sequenced using the Illumina HiSeq 2000 platform. The correlations of three clinically relevant BC genes, six molecular subtype classifiers, and a selection of 21 GES were evaluated. RESULTS: 16,097 genes common to the two platforms were retained for downstream analysis. Gene-wise comparison of microarray and RNA-Seq data revealed that 52% had a Spearman's correlation coefficient greater than 0.7 with highly correlated genes displaying significantly higher expression levels. We found excellent correlation between microarray and RNA-Seq for the estrogen receptor (ER; rs = 0.973; 95% CI: 0.971-0.975), progesterone receptor (PgR; rs = 0.95; 0.947-0.954), and human epidermal growth factor receptor 2 (HER2; rs = 0.918; 0.912-0.923), while a few discordances between ER and PgR quantified by immunohistochemistry and RNA-Seq/microarray were observed. All the subtype classifiers evaluated agreed well (Cohen's kappa coefficients >0.8) and all the proliferation-based GES showed excellent Spearman correlations between microarray and RNA-Seq (all rs >0.965). Immune-, stroma- and pathway-based GES showed a lower correlation relative to prognostic signatures (all rs >0.6). CONCLUSIONS: To our knowledge, this is the first study to report a systematic comparison of RNA-Seq to microarray for the evaluation of single genes and GES clinically relevant to BC. According to our results, the vast majority of single gene biomarkers and well-established GES can be reliably evaluated using the RNA-Seq technology.
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Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Transcriptoma/genética , Neoplasias de Mama Triplo Negativas/genética , Sequência de Bases , Receptor alfa de Estrogênio/biossíntese , Feminino , Humanos , Proteínas de Neoplasias/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Receptor ErbB-2/biossíntese , Receptores de Progesterona/biossíntese , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The mechanisms of damage at the polyethylene post in 3 contemporary tibial insert designs were evaluated and compared with a historical standard (Insall-Burstein II; Zimmer, Warsaw, Ind). One hundred five gamma sterilized posterior-stabilized tibial inserts were revised after an average of 4.7 years (0.05-13.6 years). Retrievals were classified according to their designs: Insall-Burstein II (n = 28); PFC (Johnson & Johnson, Raynham, Mass; n = 30); NexGen (Zimmer; n = 32); and Scorpio (Stryker Orthopaedics, Mahwah, NJ; n = 15). Reasons for revision and patient details were available. Surface damage scoring and photogrammetry were performed on all the retrieved tibial inserts. Oxidation analysis was carried out for traceable historical, gamma air-sterilized and conventional, gamma inert-sterilized tibial inserts (n = 61) with the use of infrared spectroscopy. The posts for all 3 contemporary designs exhibited damage similar to the historical controls. Articular, post, and backside damage scores significantly increased with implantation time. Post damage was insensitive to design and patient factors but was exacerbated by oxidation. An association between damage at the post and articular surface was also confirmed. Logistic models suggested an interaction between post damage, backside surface damage, and implant loosening.