RESUMO
The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.
Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , COVID-19/virologia , Interações Hospedeiro-Patógeno/imunologia , Ativação Linfocitária/imunologia , SARS-CoV-2/imunologia , Biomarcadores , Linfócitos T CD8-Positivos/metabolismo , COVID-19/diagnóstico , COVID-19/genética , Citocinas/metabolismo , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Estudos Longitudinais , Ativação Linfocitária/genética , Fosforilação Oxidativa , Fenótipo , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de Doença , TranscriptomaRESUMO
Mechanical deformations of DNA such as bending are ubiquitous and have been implicated in diverse cellular functions1. However, the lack of high-throughput tools to measure the mechanical properties of DNA has limited our understanding of how DNA mechanics influence chromatin transactions across the genome. Here we develop 'loop-seq'-a high-throughput assay to measure the propensity for DNA looping-and determine the intrinsic cyclizabilities of 270,806 50-base-pair DNA fragments that span Saccharomyces cerevisiae chromosome V, other genomic regions, and random sequences. We found sequence-encoded regions of unusually low bendability within nucleosome-depleted regions upstream of transcription start sites (TSSs). Low bendability of linker DNA inhibits nucleosome sliding into the linker by the chromatin remodeller INO80, which explains how INO80 can define nucleosome-depleted regions in the absence of other factors2. Chromosome-wide, nucleosomes were characterized by high DNA bendability near dyads and low bendability near linkers. This contrast increases for deeper gene-body nucleosomes but disappears after random substitution of synonymous codons, which suggests that the evolution of codon choice has been influenced by DNA mechanics around gene-body nucleosomes. Furthermore, we show that local DNA mechanics affect transcription through TSS-proximal nucleosomes. Overall, this genome-scale map of DNA mechanics indicates a 'mechanical code' with broad functional implications.
Assuntos
Fenômenos Biomecânicos , DNA Fúngico/química , DNA Fúngico/genética , Genoma Fúngico , Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina , Códon/genética , DNA Fúngico/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Maleabilidade , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.
Assuntos
Envelhecimento , Sistema Nervoso Entérico/citologia , Feto/citologia , Saúde , Intestinos/citologia , Intestinos/crescimento & desenvolvimento , Linfonodos/citologia , Linfonodos/crescimento & desenvolvimento , Adulto , Animais , Criança , Doença de Crohn/patologia , Conjuntos de Dados como Assunto , Sistema Nervoso Entérico/anatomia & histologia , Sistema Nervoso Entérico/embriologia , Sistema Nervoso Entérico/crescimento & desenvolvimento , Células Epiteliais/citologia , Feminino , Feto/anatomia & histologia , Feto/embriologia , Humanos , Intestinos/embriologia , Intestinos/inervação , Linfonodos/embriologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Organogênese , Receptores de IgG/metabolismo , Transdução de Sinais , Análise Espaço-Temporal , Fatores de TempoRESUMO
Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is a central protein in necroptosis, but posttranslational processes that regulate RIP3 activity and stability remain poorly understood. Here, we identify pellino E3 ubiquitin protein ligase 1 (PELI1) as an E3 ligase that targets RIP3 for proteasome-dependent degradation. Phosphorylation of RIP3 on T182 leads to interaction with the forkhead-associated (FHA) domain of PELI1 and PELI1-mediated K48-linked polyubiquitylation of RIP3 on K363. This same phosphorylation event is also important for RIP3 kinase activity; thus, PELI1 preferentially targets kinase-active RIP3 for degradation. PELI1-mediated RIP3 degradation effectively prevents cell death triggered by RIP3 hyperactivation. Importantly, upregulated RIP3 expression in keratinocytes from toxic epidermal necrolysis (TEN) patients is correlated with low expression of PELI1, suggesting that loss of PELI1 may play a role in the pathogenesis of TEN. We propose that PELI1 may function to control inadvertent activation of RIP3, thus preventing aberrant cell death and maintaining cellular homeostasis.
Assuntos
Queratinócitos/enzimologia , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Síndrome de Stevens-Johnson/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Morte Celular , Fibroblastos/enzimologia , Fibroblastos/patologia , Células HEK293 , Células HT29 , Células HeLa , Humanos , Queratinócitos/patologia , Camundongos , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais , Síndrome de Stevens-Johnson/genética , Síndrome de Stevens-Johnson/patologia , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here, we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes.
RESUMO
Mutations in the gene encoding the CDKL5 kinase are among the most common genetic causes of childhood epilepsy and can also give rise to the severe neurodevelopmental condition CDD (CDKL5 deficiency disorder). Despite its importance for human health, the phosphorylation targets and cellular roles of CDKL5 are poorly understood, especially in the cell nucleus. Here, we report that CDKL5 is recruited to sites of DNA damage in actively transcribed regions of the nucleus. A quantitative phosphoproteomic screen for nuclear CDKL5 substrates reveals a network of transcriptional regulators including Elongin A (ELOA), phosphorylated on a specific CDKL5 consensus motif. Recruitment of CDKL5 and ELOA to damaged DNA, and subsequent phosphorylation of ELOA, requires both active transcription and the synthesis of poly(ADP-ribose) (PAR), to which CDKL5 can bind. Critically, CDKL5 kinase activity is essential for the transcriptional silencing of genes induced by DNA double-strand breaks. Thus, CDKL5 is a DNA damage-sensing, PAR-controlled transcriptional modulator, a finding with implications for understanding the molecular basis of CDKL5-related diseases.
Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , Elonguina/metabolismo , Neurônios/patologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ativação Transcricional , Elonguina/genética , Síndromes Epilépticas/genética , Síndromes Epilépticas/metabolismo , Síndromes Epilépticas/patologia , Humanos , Mutação , Neurônios/metabolismo , Fosfoproteínas/genética , Fosforilação , Poli Adenosina Difosfato Ribose/metabolismo , Proteínas Serina-Treonina Quinases/genética , Espasmos Infantis/genética , Espasmos Infantis/metabolismo , Espasmos Infantis/patologiaRESUMO
Gene therapy has become a clinical reality as market-approved advanced therapy medicinal products for the treatment of distinct monogenetic diseases and B-cell malignancies. This Therapeutic Review aims to explain how progress in genome editing technologies offers the possibility to expand both therapeutic options and the types of diseases that will become treatable. To frame these impressive advances in the context of modern medicine, we incorporate examples from human clinical trials into our discussion on how genome editing will complement currently available strategies in gene therapy, which still mainly rely on gene addition strategies. Furthermore, safety considerations and ethical implications, including the issue of accessibility, are addressed as these crucial parameters will define the impact that gene therapy in general and genome editing in particular will have on how we treat patients in the near future.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Terapia GenéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Biochemical reactions are intrinsically stochastic, leading to variation in the production of mRNAs and proteins within cells. In the scientific literature, this source of variation is typically referred to as 'noise'. The observed variability in molecular phenotypes arises from a combination of processes that amplify and attenuate noise. Our ability to quantify cell-to-cell variability in numerous biological contexts has been revolutionized by recent advances in single-cell technology, from imaging approaches through to 'omics' strategies. However, defining, accurately measuring and disentangling the stochastic and deterministic components of cell-to-cell variability is challenging. In this Review, we discuss the sources, impact and function of molecular phenotypic variability and highlight future directions to understand its role.
RESUMO
The devastating impact of COVID-19 on global health shows the need to increase our pandemic preparedness. Recombinant therapeutic antibodies were successfully used to treat and protect at-risk patients from COVID-19. However, the currently circulating Omicron subvariants of SARS-CoV-2 are largely resistant to therapeutic antibodies, and novel approaches to generate broadly neutralizing antibodies are urgently needed. Here, we describe a tetravalent bispecific antibody, A7A9 TVB, which actively neutralized many SARS-CoV-2 variants of concern, including early Omicron subvariants. Interestingly, A7A9 TVB neutralized more variants at lower concentration as compared to the combination of its parental monoclonal antibodies, A7K and A9L. A7A9 also reduced the viral load of authentic Omicron BA.1 virus in infected pseudostratified primary human nasal epithelial cells. Overall, A7A9 displayed the characteristics of a potent broadly neutralizing antibody, which may be suitable for prophylactic and therapeutic applications in the clinics, thus highlighting the usefulness of an effective antibody-designing approach.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Anticorpos Monoclonais/uso terapêutico , Pais , Anticorpos Antivirais/uso terapêutico , Anticorpos Neutralizantes/uso terapêuticoRESUMO
Testosterone may reduce pain in cisgender women and transgender men. Rodents can provide a useful model for investigating physiological effects of hormone therapy. To this end, continuous-release testosterone or blank (placebo) capsules were implanted s.c. into young adult female rats, and three weeks later rats were either ovariectomized or sham-ovariectomized. Testosterone treatment that mimicked previously reported endogenous levels in males eliminated estrous cycling and decreased uterine weight. Testosterone also significantly increased body weight and suppressed the increases in daily wheel running observed in placebo controls over time. Subsequent ovariectomy or sham-ovariectomy decreased wheel running in all groups, but testosterone-treated rats recovered significantly more quickly than did placebo-treated rats. Neither testosterone nor ovariectomy significantly altered hindpaw mechanical threshold. Two weeks after sham/ovariectomy surgery, injection of Complete Freund Adjuvant (CFA) into one hindpaw reduced wheel running and mechanical threshold in all groups; running significantly decreased from the first to second day after CFA in testosterone- but not in placebo-treated rats. Morphine 1.0 but not 3.2 mg/kg increased CFA-suppressed wheel running similarly in all groups, whereas both doses of morphine increased CFA-suppressed mechanical threshold. These data suggest that weeks-long testosterone treatment with or without ovariectomy may provide a useful physiological model of testosterone therapy as used in human gender transition. Although testosterone administered at levels similar to those in gonadally intact males tended to hasten female rats' recovery from surgery, it did not decrease maximal pain-related behaviors after surgery or hindpaw inflammatory insult, nor did it alter opioid antinociception.
Assuntos
Atividade Motora , Testosterona , Animais , Feminino , Ratos , Morfina/farmacologia , Ovariectomia , Dor/tratamento farmacológico , Testosterona/farmacologiaRESUMO
Usher syndrome 1B (USH1B) is a devastating genetic disorder with congenital deafness, loss of balance, and blindness caused by mutations in the myosin-VIIa (MYO7A) gene, for which there is currently no cure. We developed a gene therapy approach addressing the vestibulo-cochlear deficits of USH1B using a third-generation, high-capacity lentiviral vector system capable of delivering the large 6,645-bp MYO7A cDNA. Lentivirally delivered MYO7A and co-encoded dTomato were successfully expressed in the cochlear cell line HEI-OC1. In normal-hearing mice, both cochlea and the vestibular organ were efficiently transduced, and ectopic MYO7A overexpression did not show any adverse effects. In Shaker-1 mice, an USH1B disease model based on Myo7a mutation, cochlear and vestibular hair cells, the main inner ear cell types affected in USH1B, were successfully transduced. In homozygous mutant mice, delivery of MYO7A at postnatal day 16 resulted in a trend for partial recovery of auditory function and in strongly reduced balance deficits. Heterozygous mutant mice were found to develop severe hearing loss at 6 months of age without balance deficits, and lentiviral MYO7A gene therapy completely rescued hearing to wild-type hearing thresholds. In summary, this study demonstrates improved hearing and balance function through lentiviral gene therapy in the inner ear.
Assuntos
Miosinas , Síndromes de Usher , Camundongos , Animais , Miosinas/genética , Miosinas/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Miosina VIIa/genética , Síndromes de Usher/genética , Síndromes de Usher/terapia , Modelos Animais de Doenças , Mutação , Terapia GenéticaRESUMO
STOML3 is a membrane bound scaffolding protein that has been shown to facilitate the opening of mechanically sensitive ion channels and contribute to noxious mechanical sensation, allodynia and hyperalgesia. In this study, we aimed to determine the role of STOML3 in noxious mechanical sensitivity of bone afferent neurons and carrageenan-induced acute inflammation in the bone. An in vivo, electrophysiological bone-nerve preparation was used to make recordings of the activity and sensitivity of bone afferent neurons that innervate the tibial marrow cavity in anaesthetised rats, in response to noxious mechanical stimuli delivered to the marrow cavity, before and after injection of either the STOML3 oligomerisation inhibitor OB-1 or vehicle, in either naïve animals or animals with carrageenan-induced inflammation of the marrow cavity. A dynamic weight-bearing apparatus was used to measure weight bearing in response to inflammatory pain before and after injection of OB-1 or saline into the tibial marrow cavity in the presence of carrageenan-induced inflammation. Electrophysiological recordings revealed that Aδ, but not C bone afferent neurons have a reduced discharge frequency in response to mechanical stimulation, and that carrageenan-induced sensitisation of Aδ, but not C bone afferent neurons was attenuated by inhibition of STOML3 oligomerisation with OB-1. Animals treated with OB-1 spent a significantly greater amount of time on the limb injected with carrageenan than animals treated with saline. Our findings demonstrate that inhibition of STOML3 oligomerisation reduces inflammatory bone pain by reducing the sensitivity of Aδ bone afferent neurons to mechanical stimulation. Targeting STOML3 may be an effective approach to reduce pain from noxious pressure and/or painful inflammatory pathology in bone.
Assuntos
Dor Aguda , Dor Musculoesquelética , Ratos , Animais , Carragenina/toxicidade , Carragenina/metabolismo , Ratos Sprague-Dawley , Neurônios Aferentes/metabolismo , Hiperalgesia/metabolismo , Dor Musculoesquelética/metabolismo , Dor Aguda/metabolismo , Modelos Animais , Inflamação/metabolismoRESUMO
OBJECTIVE: There have been significant developments in understanding artemin/GFRα3 signaling in recent years, and there is now accumulating evidence that artemin has important roles to play in pain signaling, including that derived from joint and bone, and that associated with osteorthritis (OA). METHODS: A total of 163 Sprague-Dawley rats were used in this study. We used an animal model of mono-iodoacetate (MIA)-induced OA, in combination with electrophysiology, behavioral testing, Western blot analysis, and retrograde tracing and immunohistochemistry, to identify roles for artemin/GFRα3 signaling in the pathogenesis of OA pain. RESULTS: We have found that: 1) GFRα3 is expressed in a substantial proportion of knee joint afferent neurons; 2) exogenous artemin sensitizes knee joint afferent neurons in naïve rats; 3) artemin is expressed in articular tissues of the joint, but not surrounding bone, early in MIA-induced OA; 4) artemin expression increases in bone later in MIA-induced OA when pathology involves subchondral bone; and 5) sequestration of artemin reverses MIA-induced sensitization of both knee joint and bone afferent neurons late in disease when there is inflammation of knee joint tissues and damage to the subchondral bone. CONCLUSIONS: Our findings show that artemin/GFRα3 signaling has a role to play in the pathogenesis of OA pain, through effects on both knee joint and bone afferent neurons, and suggest that targeted manipulation of artemin/GFRα3 signaling may provide therapeutic benefit for the management of OA pain. DATA AVAILABILITY: Data are available on request of the corresponding author.
Assuntos
Nociceptores , Dor , Ratos , Animais , Nociceptores/metabolismo , Ratos Sprague-Dawley , Dor/etiologia , Dor/metabolismo , Neurônios Aferentes , Inflamação/metabolismo , Modelos Animais de DoençasRESUMO
Identifying the factors that shape protein expression variability in complex multi-cellular organisms has primarily focused on promoter architecture and regulation of single-cell expression in cis. However, this targeted approach has to date been unable to identify major regulators of cell-to-cell gene expression variability in humans. To address this, we have combined single-cell protein expression measurements in the human immune system using flow cytometry with a quantitative genetics analysis. For the majority of proteins whose variability in expression has a heritable component, we find that genetic variants act in trans, with notably fewer variants acting in cis. Furthermore, we highlight using Mendelian Randomization that these variability-Quantitative Trait Loci might be driven by the cis regulation of upstream genes. This indicates that natural selection may balance the impact of gene regulation in cis with downstream impacts on expression variability in trans.
Assuntos
Regulação da Expressão Gênica/genética , Expressão Gênica/genética , Alelos , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica/métodos , Testes Genéticos/métodos , Estudo de Associação Genômica Ampla/métodos , Humanos , Sistema Imunitário/metabolismo , Imunidade/genética , Masculino , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genética , Seleção Genética/genéticaRESUMO
Mutations in the gene encoding the protein kinase CDKL5 cause a debilitating neurodevelopmental disease termed CDKL5 disorder. The impact of these mutations on CDKL5 function is poorly understood because the substrates and cellular processes controlled by CDKL5 are unclear. Here, we describe a quantitative phosphoproteomic screening which identified MAP1S, CEP131 and DLG5-regulators of microtubule and centrosome function-as cellular substrates of CDKL5. Antibodies against MAP1S phospho-Ser900 and CEP131 phospho-Ser35 confirmed CDKL5-dependent phosphorylation of these targets in human cells. The phospho-acceptor serine residues in MAP1S, CEP131 and DLG5 lie in the motif RPXSA, although CDKL5 can tolerate residues other than Ala immediately C-terminal to the phospho-acceptor serine. We provide insight into the control of CDKL5 activity and show that pathogenic mutations in CDKL5 cause a major reduction in CDKL5 activity in vitro and in cells. These data reveal the first cellular substrates of CDKL5, which may represent important biomarkers in the diagnosis and treatment of CDKL5 disorder, and illuminate the functions of this poorly characterized kinase.
Assuntos
Síndromes Epilépticas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espasmos Infantis/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Centrossomo/metabolismo , Proteínas do Citoesqueleto , Síndromes Epilépticas/genética , Síndromes Epilépticas/patologia , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas dos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteômica , Espasmos Infantis/genética , Espasmos Infantis/patologia , Proteínas Supressoras de Tumor/genéticaRESUMO
MicroRNAs are key regulators of many biological processes, including cell differentiation. Here we show that during human monocyte-macrophage differentiation, expression of the microRNAs miR-223, miR-15a and miR-16 decreased considerably, which led to higher expression of the serine-threonine kinase IKKalpha in macrophages. In macrophages, higher IKKalpha expression in conjunction with stabilization of the kinase NIK induced larger amounts of p52. Because of low expression of the transcription factor RelB in untreated macrophages, high p52 expression repressed basal transcription of both canonical and noncanonical NF-kappaB target genes. However, proinflammatory stimuli in macrophages resulted in greater induction of noncanonical NF-kappaB target genes. Thus, a decrease in certain microRNAs probably prevents macrophage hyperactivation yet primes the macrophage for certain responses to proinflammatory stimuli.
Assuntos
Diferenciação Celular/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Macrófagos/imunologia , MicroRNAs/imunologia , NF-kappa B/imunologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Macrófagos/citologia , NF-kappa B/genética , Transdução de Sinais , Células U937 , Regulação para CimaRESUMO
OBJECTIVES: We proposed a new approach to train deep learning model for aneurysm rupture prediction which only uses a limited amount of labeled data. METHOD: Using segmented aneurysm mask as input, a backbone model was pretrained using a self-supervised method to learn deep embeddings of aneurysm morphology from 947 unlabeled cases of angiographic images. Subsequently, the backbone model was finetuned using 120 labeled cases with known rupture status. Clinical information was integrated with deep embeddings to further improve prediction performance. The proposed model was compared with radiomics and conventional morphology models in prediction performance. An assistive diagnosis system was also developed based on the model and was tested with five neurosurgeons. RESULT: Our method achieved an area under the receiver operating characteristic curve (AUC) of 0.823, outperforming deep learning model trained from scratch (0.787). By integrating with clinical information, the proposed model's performance was further improved to AUC = 0.853, making the results significantly better than model based on radiomics (AUC = 0.805, p = 0.007) or model based on conventional morphology parameters (AUC = 0.766, p = 0.001). Our model also achieved the highest sensitivity, PPV, NPV, and accuracy among the others. Neurosurgeons' prediction performance was improved from AUC=0.877 to 0.945 (p = 0.037) with the assistive diagnosis system. CONCLUSION: Our proposed method could develop competitive deep learning model for rupture prediction using only a limited amount of data. The assistive diagnosis system could be useful for neurosurgeons to predict rupture. KEY POINTS: ⢠A self-supervised learning method was proposed to mitigate the data-hungry issue of deep learning, enabling training deep neural network with a limited amount of data. ⢠Using the proposed method, deep embeddings were extracted to represent intracranial aneurysm morphology. Prediction model based on deep embeddings was significantly better than conventional morphology model and radiomics model. ⢠An assistive diagnosis system was developed using deep embeddings for case-based reasoning, which was shown to significantly improve neurosurgeons' performance to predict rupture.
Assuntos
Aneurisma Roto , Aneurisma Intracraniano , Aneurisma Roto/diagnóstico por imagem , Humanos , Aneurisma Intracraniano/diagnóstico por imagem , Redes Neurais de Computação , Curva ROCRESUMO
Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro. Here we demonstrate that genotoxic vectors induce a unique gene expression signature linked to stemness and oncogenesis in transduced murine hematopoietic stem and progenitor cells. Based on this finding, we developed the surrogate assay for genotoxicity assessment (SAGA). SAGA classifies integrating retroviral vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential, SAGA achieved an accuracy of 90.9%. SAGA is more robust and sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemic severe adverse events in clinical trials. Our work provides a fast and robust tool for preclinical risk assessment of gene therapy vectors, potentially paving the way for safer gene therapy trials.
Assuntos
Terapia Genética , Vetores Genéticos , Animais , Dano ao DNA , Expressão Gênica , Terapia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Vetores Genéticos/genética , Células-Tronco Hematopoéticas , Humanos , Aprendizado de Máquina , Camundongos , Mutagênese InsercionalRESUMO
In vitro generation of hematopoietic cells and especially hematopoietic stem cells (HSCs) from human pluripotent stem cells (PSCs) are subject to intensive research in recent decades, as these cells hold great potential for regenerative medicine and autologous cell replacement therapies. Despite many attempts, in vitro, de novo generation of bona fide HSCs remains challenging, and we are still far away from their clinical use, due to insufficient functionality and quantity of the produced HSCs. The challenges of generating PSC-derived HSCs are already apparent in early stages of hemato-endothelial specification with the limitation of recapitulating complex, dynamic processes of embryonic hematopoietic ontogeny in vitro. Further, these current shortcomings imply the incompleteness of our understanding of human ontogenetic processes from embryonic mesoderm over an intermediate, specialized hemogenic endothelium (HE) to their immediate progeny, the HSCs. In this review, we examine the recent investigations of hemato-endothelial ontogeny and recently reported progress for the conversion of PSCs and other promising somatic cell types towards HSCs with the focus on the crucial and inevitable role of the HE to achieve the long-standing goal-to generate therapeutically applicable PSC-derived HSCs in vitro.