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BACKGROUND: Sepsis from infection is a global health priority and clinical trials have failed to deliver effective therapeutic interventions. To address complicating heterogeneity in sepsis pathobiology, and improve outcomes, promising precision medicine approaches are helping identify disease endotypes, however, they require a more complete definition of sepsis subgroups. METHODS: Here, we use RNA sequencing from peripheral blood to interrogate the host response to sepsis from participants in a global observational study carried out in West Africa, Southeast Asia, and North America (N = 494). RESULTS: We identify four sepsis subtypes differentiated by 28-day mortality. A low mortality immunocompetent group is specified by features that describe the adaptive immune system. In contrast, the three high mortality groups show elevated clinical severity consistent with multiple organ dysfunction. The immunosuppressed group members show signs of a dysfunctional immune response, the acute-inflammation group is set apart by molecular features of the innate immune response, while the immunometabolic group is characterized by metabolic pathways such as heme biosynthesis. CONCLUSIONS: Our analysis reveals details of molecular endotypes in sepsis that support immunotherapeutic interventions and identifies biomarkers that predict outcomes in these groups.
Sepsis is a life-threatening multi-organ failure caused by the body's immune response to infection. Clinical symptoms of sepsis vary from one person to another likely due to differences in host factors, infecting pathogen, and comorbidities. This difference in clinical symptoms may contribute to the lack of effective interventions for sepsis. Therefore, approaches tailored to targeting groups of patients who present similarly are of great interest. This study analysed a large group of sepsis patients with diverse symptoms using laboratory markers and mathematical analysis. We report four patient groups that differ by risk of death and immune response profile. Targeting these defined groups with tailored interventions presents an exciting opportunity to improve the health outcomes of patients with sepsis.
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Our limited understanding of the pathophysiological mechanisms that operate during sepsis is an obstacle to rational treatment and clinical trial design. There is a critical lack of data from low- and middle-income countries where the sepsis burden is increased which inhibits generalized strategies for therapeutic intervention. Here we perform RNA sequencing of whole blood to investigate longitudinal host response to sepsis in a Ghanaian cohort. Data dimensional reduction reveals dynamic gene expression patterns that describe cell type-specific molecular phenotypes including a dysregulated myeloid compartment shared between sepsis and COVID-19. The gene expression signatures reported here define a landscape of host response to sepsis that supports interventions via targeting immunophenotypes to improve outcomes.
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COVID-19 , Fenotipo , Sepsis , Transcriptoma , Humanos , Sepsis/genética , Sepsis/sangre , Sepsis/inmunología , COVID-19/inmunología , COVID-19/genética , COVID-19/sangre , COVID-19/virología , Ghana/epidemiología , Masculino , Estudios de Cohortes , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Femenino , Adulto , Persona de Mediana Edad , Perfilación de la Expresión Génica , Análisis de Secuencia de ARNRESUMEN
Rapid and accurate measurements of immune protein markers are essential for diagnosis and treatment in all clinical settings. The recent pandemic has revealed a stark need for developing new tools and assays that could be rapidly used in diverse settings and provide useful information to clinicians. Here, we describe the development and test application of a novel one-step CRP/IP-10 duplex assay for the LightDeck platform capable of delivering reproducible and accurate measurements in under eight minutes. We used the optimized assay to measure CRP and IP-10 levels in human blood and serum samples from healthy, SARS-CoV-2 (COVID-19) positive, and influenza-like illness (ILI) presenting patients. Our results agreed with previously published analyte levels and enabled us to make statistically significant comparisons relevant to multiple clinical parameters. Our duplex assay is a simple and powerful tool for aiding prognostic decision-making in diverse settings.
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COVID-19 , Sistemas de Atención de Punto , Humanos , Biomarcadores , Quimiocina CXCL10/sangre , Quimiocina CXCL10/química , COVID-19/diagnóstico , SARS-CoV-2 , Proteína C-Reactiva/químicaRESUMEN
DNA transposon systems are widely used in mammalian cells for genetic modification experiments, but their regulation remains poorly understood. We used biochemical and cell-based assays together with AlphaFold modeling and rational protein redesign to evaluate aspects of piggyBac transposition including the previously unexplained role of the transposase N-terminus and the need for asymmetric transposon ends for cellular activity. We found that phosphorylation at predicted casein kinase II sites in the transposase N-terminus inhibits transposition, most likely by preventing transposase-DNA interactions. Deletion of the region containing these sites releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. When the N-terminus is deleted, the transposase gains the ability to carry out transposition using symmetric transposon left ends. This novel activity is also conferred by appending a second C-terminal domain. When combined, these modifications together result in a transposase that is highly active when symmetric transposon ends are used. Our results demonstrate that transposase N-terminal phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposition in mammalian cells. These novel insights into the mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.
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Elementos Transponibles de ADN , Transposasas , Humanos , Elementos Transponibles de ADN/genética , Fosforilación , Transposasas/metabolismo , Línea CelularRESUMEN
Embryonic stem cells (ESCs) can adopt lineage-specific gene-expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single-cell-based assays were employed to catalog gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further specification toward myogenic and neurogenic lineages. These assays identified cis-regulatory regions and transcription factors presiding over gene-expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene-expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs and ESC-derived cell lineages.
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Células Madre Embrionarias , Transcriptoma , Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
PIWI-interacting RNAs (piRNAs) guard germline genomes against the deleterious action of mobile genetic elements. PiRNAs use extensive base-pairing to recognize their targets and variable 3'ends could change the specificity and efficacy of piRNA silencing. Here, we identify conserved rules that ensure the generation of a single major piRNA 3'end in flies and mice. Our data suggest that the PIWI proteins initially define a short interval on pre-piRNAs that grants access to the ZUC-processor complex. Within this Goldilocks zone, the preference to cut in front of Uridine determines the ultimate processing site. We observe a mouse-specific roadblock that relocates the Goldilocks zone and generates an opportunity for consecutive trimming. Our data reveal a conserved hierarchy between length and sequence preferences that controls the piRNA sequence space. The unanticipated precision of 3'end formation bolsters the emerging understanding that the functional piRNA sequence space is tightly controlled to ensure effective defense.
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The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins with high affinity and specificity while preserving their physiological expression patterns. However, stably modifying endogenous genes in cells that do not allow for clonal selection has been challenging. Here, we present a simple and fast strategy to generate stable, endogenously tagged alleles in a non-transformed cell culture model. At the example of piwi in Drosophila ovarian somatic sheath cells, we show that this strategy enables the generation of an N-terminally tagged protein that emulates the expression level and subcellular localization of the wild type protein and forms functional Piwi-piRNA complexes. We further present a concise workflow to establish endogenously N-terminally and C-terminally tagged proteins, and knockout alleles through rapid selection of cell pools in fly and human models.
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Proteínas de Drosophila , Drosophila , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Edición Génica , Genes Reporteros , Humanos , Ovario/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Defense against genome invaders universally relies on RNA-guided immunity. Prokaryotic CRISPR-Cas and eukaryotic RNA interference pathways recognize targets by complementary base-pairing, which places the sequences of their guide RNAs at the center of self/nonself discrimination. Here, we explore the sequence space of PIWI-interacting RNAs (piRNAs), the genome defense of animals, and establish functional priority among individual sequences. Our results reveal that only the topmost abundant piRNAs are commonly present in every cell, whereas rare sequences generate cell-to-cell diversity in flies and mice. We identify a skewed distribution of sequence abundance as a hallmark of piRNA populations and show that quantitative differences of more than a 1000-fold are established by conserved mechanisms of biogenesis. Finally, our genomics analyses and direct reporter assays reveal that abundance determines function in piRNA-guided genome defense. Taken together, we identify an effective sequence space and untangle two classes of piRNAs that differ in complexity and function. The first class represents the topmost abundant sequences and drives silencing of genomic parasites. The second class sparsely covers an enormous sequence space. These rare piRNAs cannot function in every cell, every individual, or every generation but create diversity with potential for adaptation in the ongoing arms race with genome invaders.
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ARN Guía de Kinetoplastida , Animales , Ratones , ARN Guía de Kinetoplastida/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.
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Proteínas Arqueales/química , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/genética , Methanosarcina/enzimología , Transposasas/química , Transposasas/metabolismo , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Asociadas a CRISPR/química , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/química , ADN/metabolismo , Elementos Transponibles de ADN , ADN de Archaea/química , ADN de Archaea/genética , ADN de Archaea/metabolismo , ADN Intergénico , Methanosarcina/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , Multimerización de Proteína , Transposasas/genéticaRESUMEN
Germline genes that are aberrantly expressed in nongermline cancer cells have the potential to be ideal targets for diagnosis and therapy due to their restricted physiological expression, their broad reactivation in various cancer types, and their immunogenic properties. Among such cancer/testis genes, components of the PIWI-interacting small RNA (piRNA) pathway are of particular interest, as they control mobile genetic elements (transposons) in germ cells and thus hold great potential to counteract genome instability in cancer. Here, we systematically investigate the potential reactivation of functional piRNA-silencing mechanisms in the aberrant context. While we observe expression of individual piRNA-pathway genes in cancer, we fail to detect the formation of functional piRNA-silencing complexes. Accordingly, the expression of a PIWI protein alone remains inconsequential to the cancer cell transcriptome. Our data provide a framework for the investigation of complex aberrant gene-expression signatures and establish that reactivation of piRNA silencing, if at all, is not a prevalent phenomenon in cancer cells.
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Silenciador del Gen/fisiología , Neoplasias/genética , ARN Interferente Pequeño/genética , Línea Celular Tumoral , Elementos Transponibles de ADN/genética , Expresión Génica/genética , Inestabilidad Genómica/genética , Células Germinativas/patología , Humanos , Masculino , Testículo/fisiología , Transcriptoma/genéticaRESUMEN
PIWI-interacting RNAs (piRNAs) are at the center of a small RNA-based immune system that defends genomes against the deleterious action of mobile genetic elements (transposons). PiRNAs are highly variable in sequence with extensive targeting potential. Their diversity is restricted by their preference to start with a Uridine (U) at the 5' most position (1U-bias), a bias that remains poorly understood. Here we uncover that the 1U-bias of Piwi-piRNAs is established by consecutive discrimination against all nucleotides but U, first during piRNA biogenesis and then upon interaction with Piwi's specificity loop. Sequence preferences during piRNA processing also restrict U across the piRNA body with the potential to directly impact target recognition. Overall, the uncovered signatures could modulate specificity and efficacy of piRNA-mediated transposon restriction, and provide a substrate for purifying selection in the ongoing arms race between genomes and their mobile parasites.
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Proteínas Argonautas/genética , Proteínas de Drosophila/genética , ARN Interferente Pequeño/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Argonautas/metabolismo , Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Mutación , Ovario/metabolismo , Dominios Proteicos , ARN Interferente Pequeño/genética , Uracilo/metabolismo , Uridina/genética , Uridina/metabolismoRESUMEN
In mammals, gene silencing by the RNA-induced silencing complex (RISC) is a well-understood cytoplasmic posttranscriptional gene regulatory mechanism. Here, we show that embryonic stem cells (ESCs) contain high levels of nuclear AGO proteins and that in ESCs nuclear AGO protein activity allows for the onset of differentiation. In the nucleus, AGO proteins interact with core RISC components, including the TNRC6 proteins and the CCR4-NOT deadenylase complex. In contrast to cytoplasmic miRNA-mediated gene silencing that mainly operates on cis-acting elements in mRNA 3' untranslated (UTR) sequences, in the nucleus AGO binding in the coding sequence and potentially introns also contributed to post-transcriptional gene silencing. Thus, nuclear localization of AGO proteins in specific cell types leads to a previously unappreciated expansion of the miRNA-regulated transcriptome.
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Proteínas Argonautas/fisiología , Silenciador del Gen/fisiología , MicroARNs/fisiología , Animales , Proteínas Argonautas/genética , Diferenciación Celular/genética , Línea Celular , Núcleo Celular , Citoplasma , Células Madre Embrionarias/metabolismo , Humanos , Mamíferos , Ratones , MicroARNs/genética , Interferencia de ARN , Estabilidad del ARN , ARN Mensajero , ARN Interferente Pequeño , Proteínas de Unión al ARN , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Factores de TranscripciónRESUMEN
Piwi-interacting piRNAs are a major and essential class of small RNAs in the animal germ cells with a prominent role in transposon control. Efficient piRNA biogenesis and function require a cohort of proteins conserved throughout the animal kingdom. Here we studied Maelstrom (MAEL), which is essential for piRNA biogenesis and germ cell differentiation in flies and mice. MAEL contains a high mobility group (HMG)-box domain and a Maelstrom-specific domain with a presumptive RNase H-fold. We employed a combination of sequence analyses, structural and biochemical approaches to evaluate and compare nucleic acid binding of mouse MAEL HMG-box to that of canonical HMG-box domain proteins (SRY and HMGB1a). MAEL HMG-box failed to bind double-stranded (ds)DNA but bound to structured RNA. We also identified important roles of a novel cluster of arginine residues in MAEL HMG-box in these interactions. Cumulatively, our results suggest that the MAEL HMG-box domain may contribute to MAEL function in selective processing of retrotransposon RNA into piRNAs. In this regard, a cellular role of MAEL HMG-box domain is reminiscent of that of HMGB1 as a sentinel of immunogenic nucleic acids in the innate immune response.
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Proteínas de Unión al ADN/metabolismo , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Dominios HMG-Box , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis , Conformación de Ácido Nucleico , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/química , Retroelementos/genética , Alineación de Secuencia , Factores de Transcripción/genéticaRESUMEN
Pachytene piRNAs are a class of Piwi-interacting small RNAs abundant in spermatids of the adult mouse testis. They are processed from piRNA primary transcripts by a poorly understood mechanism and, unlike fetal transposon-derived piRNAs, lack complementary targets in the spermatid transcriptome. We report that immunopurified complexes of a conserved piRNA pathway protein Maelstrom (MAEL) are enriched in MIWI (Piwi partner of pachytene piRNAs), Tudor-domain proteins and processing intermediates of pachytene piRNA primary transcripts. We provide evidence of functional significance of these complexes in Mael129 knockout mice that exhibit spermiogenic arrest with acrosome and flagellum malformation. Mael129-null mutant testes possess low levels of piRNAs derived from MAEL-associated piRNA precursors and exhibit reduced translation of numerous spermiogenic mRNAs including those encoding acrosome and flagellum proteins. These translation defects in haploid round spermatids are likely indirect, as neither MAEL nor piRNA precursors associate with polyribosomes, and they may arise from an imbalance between pachytene piRNAs and MIWI.
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Proteínas de Unión al ADN/deficiencia , Mutación , Fase Paquiteno , Biosíntesis de Proteínas , ARN Interferente Pequeño/metabolismo , Espermatogénesis , Testículo/fisiología , Factores de Transcripción/deficiencia , Animales , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Noqueados , Espermátides/fisiología , Factores de Transcripción/metabolismoRESUMEN
Chromatin remodelers are ATP-dependent machines responsible for directionally shifting nucleosomes along DNA. We are interested in defining which elements of the chromodomain helicase DNA-binding protein 1 (Chd1) remodeler are necessary and sufficient for sliding nucleosomes. This work focuses on the polypeptide segment that joins the ATPase motor to the C-terminal DNA-binding domain. We identify amino acid positions outside the ATPase motor that, when altered, dramatically reduce nucleosome sliding ability and yet have only â¼3-fold reduction in ATPase stimulation by nucleosomes. These residues therefore appear to play a role in functionally coupling ATP hydrolysis to nucleosome sliding, and suggest that the ATPase motor requires cooperation with external elements to slide DNA past the histone core.
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Adenosina Trifosfato/química , Proteínas de Unión al ADN/química , Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Cromatina/química , Dicroismo Circular , Regulación Fúngica de la Expresión Génica , Hidrólisis , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Integrity of the germline genome is essential for the production of viable gametes and successful reproduction. In mammals, the generation of gametes involves extensive epigenetic changes (DNA methylation and histone modification) in conjunction with changes in chromosome structure to ensure flawless progression through meiotic recombination and packaging of the genome into mature gametes. Although epigenetic reprogramming is essential for mammalian reproduction, reprogramming also provides a permissive window for exploitation by transposable elements (TEs), autonomously replicating endogenous elements. Expression and propagation of TEs during the reprogramming period can result in insertional mutagenesis that compromises genome integrity leading to reproductive problems and sporadic inherited diseases in offspring. Recent work has identified the germ cell associated PIWI Interacting RNA (piRNA) pathway in conjunction with the DNA methylation and histone modification machinery in silencing TEs. In this review we will highlight these recent advances in piRNA mediated regulation of TEs in the mouse germline, as well as mention the repercussions of failure to properly regulate TEs.