RESUMEN
Approximately 15% of US adults have circulating levels of uric acid above its solubility limit, which is causally linked to the disease gout. In most mammals, uric acid elimination is facilitated by the enzyme uricase. However, human uricase is a pseudogene, having been inactivated early in hominid evolution. Though it has long been known that uric acid is eliminated in the gut, the role of the gut microbiota in hyperuricemia has not been studied. Here, we identify a widely distributed bacterial gene cluster that encodes a pathway for uric acid degradation. Stable isotope tracing demonstrates that gut bacteria metabolize uric acid to xanthine or short chain fatty acids. Ablation of the microbiota in uricase-deficient mice causes severe hyperuricemia, and anaerobe-targeted antibiotics increase the risk of gout in humans. These data reveal a role for the gut microbiota in uric acid excretion and highlight the potential for microbiome-targeted therapeutics in hyperuricemia.
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Gota , Hominidae , Hiperuricemia , Adulto , Animales , Humanos , Ratones , Gota/genética , Gota/metabolismo , Hominidae/genética , Hiperuricemia/genética , Mamíferos/metabolismo , Urato Oxidasa/genética , Ácido Úrico/metabolismo , Evolución MolecularRESUMEN
Many human spinal cord injuries are anatomically incomplete but exhibit complete paralysis. It is unknown why spared axons fail to mediate functional recovery in these cases. To investigate this, we undertook a small-molecule screen in mice with staggered bilateral hemisections in which the lumbar spinal cord is deprived of all direct brain-derived innervation, but dormant relay circuits remain. We discovered that a KCC2 agonist restored stepping ability, which could be mimicked by selective expression of KCC2, or hyperpolarizing DREADDs, in the inhibitory interneurons between and around the staggered spinal lesions. Mechanistically, these treatments transformed this injury-induced dysfunctional spinal circuit to a functional state, facilitating the relay of brain-derived commands toward the lumbar spinal cord. Thus, our results identify spinal inhibitory interneurons as a roadblock limiting the integration of descending inputs into relay circuits after injury and suggest KCC2 agonists as promising treatments for promoting functional recovery after spinal cord injury.
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Traumatismos de la Médula Espinal/tratamiento farmacológico , Simportadores/agonistas , Simportadores/metabolismo , Animales , Axones , Regulación de la Expresión Génica/genética , Interneuronas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Regeneración Nerviosa/fisiología , Plasticidad Neuronal/genética , Neuronas/metabolismo , Recuperación de la Función/genética , Recuperación de la Función/fisiología , Médula Espinal , Simportadores/uso terapéutico , Cotransportadores de K ClRESUMEN
Corticospinal neurons (CSNs) represent the direct cortical outputs to the spinal cord and play important roles in motor control across different species. However, their organizational principle remains unclear. By using a retrograde labeling system, we defined the requirement of CSNs in the execution of a skilled forelimb food-pellet retrieval task in mice. In vivo imaging of CSN activity during performance revealed the sequential activation of topographically ordered functional ensembles with moderate local mixing. Region-specific manipulations indicate that CSNs from caudal or rostral forelimb area control reaching or grasping, respectively, and both are required in the transitional pronation step. These region-specific CSNs terminate in different spinal levels and locations, therefore preferentially connecting with the premotor neurons of muscles engaged in different steps of the task. Together, our findings suggest that spatially defined groups of CSNs encode different movement modules, providing a logic for parallel-ordered corticospinal circuits to orchestrate multistep motor skills.
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Médula Cervical/fisiología , Destreza Motora , Vías Nerviosas , Animales , Calcio/análisis , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Médula Cervical/citología , Miembro Anterior/fisiología , Articulaciones/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
The brain controls nearly all bodily functions via spinal projecting neurons (SPNs) that carry command signals from the brain to the spinal cord. However, a comprehensive molecular characterization of brain-wide SPNs is still lacking. Here we transcriptionally profiled a total of 65,002 SPNs, identified 76 region-specific SPN types, and mapped these types into a companion atlas of the whole mouse brain1. This taxonomy reveals a three-component organization of SPNs: (1) molecularly homogeneous excitatory SPNs from the cortex, red nucleus and cerebellum with somatotopic spinal terminations suitable for point-to-point communication; (2) heterogeneous populations in the reticular formation with broad spinal termination patterns, suitable for relaying commands related to the activities of the entire spinal cord; and (3) modulatory neurons expressing slow-acting neurotransmitters and/or neuropeptides in the hypothalamus, midbrain and reticular formation for 'gain setting' of brain-spinal signals. In addition, this atlas revealed a LIM homeobox transcription factor code that parcellates the reticulospinal neurons into five molecularly distinct and spatially segregated populations. Finally, we found transcriptional signatures of a subset of SPNs with large soma size and correlated these with fast-firing electrophysiological properties. Together, this study establishes a comprehensive taxonomy of brain-wide SPNs and provides insight into the functional organization of SPNs in mediating brain control of bodily functions.
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Encéfalo , Perfilación de la Expresión Génica , Vías Nerviosas , Neuronas , Médula Espinal , Animales , Ratones , Hipotálamo , Neuronas/metabolismo , Neuropéptidos , Médula Espinal/citología , Médula Espinal/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Neurotransmisores , Mesencéfalo/citología , Formación Reticular/citología , Electrofisiología , Cerebelo/citología , Corteza Cerebral/citologíaRESUMEN
γδ T cells are essential for immune defense and modulating physiological processes. While they have the potential to recognize large numbers of antigens through somatic gene rearrangement, the antigens which trigger most γδ T cell response remain unidentified, and the role of antigen recognition in γδ T cell function is contentious. Here, we show that some γδ T cell receptors (TCRs) exhibit polyspecificity, recognizing multiple ligands of diverse molecular nature. These ligands include haptens, metabolites, neurotransmitters, posttranslational modifications, as well as peptides and proteins of microbial and host origin. Polyspecific γδ T cells are enriched among activated cells in naive mice and the responding population in infection. They express diverse TCR sequences, have different functional potentials, and include the innate-like γδ T cells, such as the major IL-17 responders in various pathological/physiological conditions. We demonstrate that encountering their antigenic microbiome metabolite maintains their homeostasis and functional response, indicating that their ability to recognize multiple ligands is essential for their function. Human γδ T cells with similar polyspecificity also respond to various immune challenges. This study demonstrates that polyspecificity is a prevalent feature of γδ T cell antigen recognition, which enables rapid and robust T cell responses to a wide range of challenges, highlighting a unique function of γδ T cells.
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Antígenos de Grupos Sanguíneos , Receptores de Antígenos de Linfocitos T gamma-delta , Humanos , Ratones , Animales , Antígenos , HaptenosRESUMEN
We develop soft and stretchable fatigue-resistant hydrogel optical fibers that enable optogenetic modulation of peripheral nerves in naturally behaving animals during persistent locomotion. The formation of polymeric nanocrystalline domains within the hydrogels yields fibers with low optical losses of 1.07 dB cm-1, Young's modulus of 1.6 MPa, stretchability of 200% and fatigue strength of 1.4 MPa against 30,000 stretch cycles. The hydrogel fibers permitted light delivery to the sciatic nerve, optogenetically activating hindlimb muscles in Thy1::ChR2 mice during 6-week voluntary wheel running assays while experiencing repeated deformation. The fibers additionally enabled optical inhibition of pain hypersensitivity in an inflammatory model in TRPV1::NpHR mice over an 8-week period. Our hydrogel fibers offer a motion-adaptable and robust solution to peripheral nerve optogenetics, facilitating the investigation of somatosensation.
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Fibras Ópticas , Optogenética , Ratones , Animales , Hidrogeles , Actividad Motora , Nervio Ciático/fisiología , LocomociónRESUMEN
Human gut bacteria perform diverse metabolic functions with consequences for host health. The prevalent and disease-linked Actinobacterium Eggerthella lenta performs several unusual chemical transformations, but it does not metabolize sugars and its core growth strategy remains unclear. To obtain a comprehensive view of the metabolic network of E. lenta, we generated several complementary resources: defined culture media, metabolomics profiles of strain isolates, and a curated genome-scale metabolic reconstruction. Stable isotope-resolved metabolomics revealed that E. lenta uses acetate as a key carbon source while catabolizing arginine to generate ATP, traits which could be recapitulated in silico by our updated metabolic model. We compared these in vitro findings with metabolite shifts observed in E. lenta-colonized gnotobiotic mice, identifying shared signatures across environments and highlighting catabolism of the host signaling metabolite agmatine as an alternative energy pathway. Together, our results elucidate a distinctive metabolic niche filled by E. lenta in the gut ecosystem. Our culture media formulations, atlas of metabolomics data, and genome-scale metabolic reconstructions form a freely available collection of resources to support further study of the biology of this prevalent gut bacterium.
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Actinobacteria , Microbioma Gastrointestinal , Humanos , Ratones , Animales , Biología de Sistemas , Ecosistema , Actinobacteria/metabolismoRESUMEN
BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disorder. However, the pathogenesis of HCM, especially its nongenetic mechanisms, remains largely unclear. Transcription factors are known to be involved in various biological processes including cell growth. We hypothesized that SP1 (specificity protein 1), the first purified TF in mammals, plays a role in the cardiomyocyte growth and cardiac hypertrophy of HCM. METHODS: Cardiac-specific conditional knockout of Sp1 mice were constructed to investigate the role of SP1 in the heart. The echocardiography, histochemical experiment, and transmission electron microscope were performed to analyze the cardiac phenotypes of cardiac-specific conditional knockout of Sp1 mice. RNA sequencing, chromatin immunoprecipitation sequencing, and adeno-associated virus experiments in vivo were performed to explore the downstream molecules of SP1. To examine the therapeutic effect of SP1 on HCM, an SP1 overexpression vector was constructed and injected into the mutant allele of Myh6 R404Q/+ (Myh6 c. 1211C>T) HCM mice. The human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with HCM were used to detect the potential therapeutic effects of SP1 in human HCM. RESULTS: The cardiac-specific conditional knockout of Sp1 mice developed a typical HCM phenotype, displaying overt myocardial hypertrophy, interstitial fibrosis, and disordered myofilament. In addition, Sp1 knockdown dramatically increased the cell area of hiPSC-CMs and caused intracellular myofibrillar disorganization, which was similar to the hypertrophic cardiomyocytes of HCM. Mechanistically, Tuft1 was identified as the key target gene of SP1. The hypertrophic phenotypes induced by Sp1 knockdown in both hiPSC-CMs and mice could be rescued by TUFT1 (tuftelin 1) overexpression. Furthermore, SP1 overexpression suppressed the development of HCM in the mutant allele of Myh6 R404Q/+ mice and also reversed the hypertrophic phenotype of HCM hiPSC-CMs. CONCLUSIONS: Our study demonstrates that SP1 deficiency leads to HCM. SP1 overexpression exhibits significant therapeutic effects on both HCM mice and HCM hiPSC-CMs, suggesting that SP1 could be a potential intervention target for HCM.
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Cardiomiopatía Hipertrófica , Células Madre Pluripotentes Inducidas , Humanos , Ratones , Animales , Células Madre Pluripotentes Inducidas/metabolismo , Cardiomiopatía Hipertrófica/metabolismo , Miofibrillas/metabolismo , Miocitos Cardíacos/metabolismo , Cardiomegalia/metabolismo , Factores de Transcripción/metabolismo , MamíferosRESUMEN
Cannabis glandular trichomes (GTs) are economically and biotechnologically important structures that have a remarkable morphology and capacity to produce, store, and secrete diverse classes of secondary metabolites. However, our understanding of the developmental changes and the underlying molecular processes involved in cannabis GT development is limited. In this study, we developed Cannabis Glandular Trichome Detection Model (CGTDM), a deep learning-based model capable of differentiating and quantifying three types of cannabis GTs with a high degree of efficiency and accuracy. By profiling at eight different time points, we captured dynamic changes in gene expression, phenotypes, and metabolic processes associated with GT development. By integrating weighted gene co-expression network analysis with CGTDM measurements, we established correlations between phenotypic variations in GT traits and the global transcriptome profiles across the developmental gradient. Notably, we identified a module containing methyl jasmonate (MeJA)-responsive genes that significantly correlated with stalked GT density and cannabinoid content during development, suggesting the existence of a MeJA-mediated GT formation pathway. Our findings were further supported by the successful promotion of GT development in cannabis through exogenous MeJA treatment. Importantly, we have identified CsMYC4 as a key transcription factor that positively regulates GT formation via MeJA signaling in cannabis. These findings provide novel tools for GT detection and counting, as well as valuable information for understanding the molecular regulatory mechanism of GT formation, which has the potential to facilitate the molecular breeding, targeted engineering, informed harvest timing, and manipulation of cannabinoid production.
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Acetatos , Cannabis , Ciclopentanos , Aprendizaje Profundo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Oxilipinas , Tricomas , Oxilipinas/farmacología , Oxilipinas/metabolismo , Ciclopentanos/farmacología , Ciclopentanos/metabolismo , Cannabis/genética , Cannabis/crecimiento & desarrollo , Cannabis/metabolismo , Acetatos/farmacología , Tricomas/genética , Tricomas/metabolismo , Tricomas/crecimiento & desarrollo , Perfilación de la Expresión Génica/métodos , Transcriptoma , Reguladores del Crecimiento de las Plantas/metabolismoRESUMEN
The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Live imaging of the AIS is challenging due to the limited number of suitable labeling methods. To overcome this limitation, we established a novel approach for live labeling of the AIS using unnatural amino acids (UAAs) and click chemistry. The small size of UAAs and the possibility of introducing them virtually anywhere into target proteins make this method particularly suitable for labeling of complex and spatially restricted proteins. Using this approach, we labeled two large AIS components, the 186â kDa isoform of neurofascin (NF186; encoded by Nfasc) and the 260â kDa voltage-gated Na+ channel (NaV1.6, encoded by Scn8a) in primary neurons and performed conventional and super-resolution microscopy. We also studied the localization of epilepsy-causing NaV1.6 variants with a loss-of-function effect. Finally, to improve the efficiency of UAA incorporation, we developed adeno-associated viral (AAV) vectors for click labeling in neurons, an achievement that could be transferred to more complex systems such as organotypic slice cultures, organoids, and animal models.
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Segmento Inicial del Axón , Química Clic , Animales , Potenciales de Acción/fisiología , Aminoácidos/metabolismo , Segmento Inicial del Axón/metabolismo , Neuronas , Ratones , RatasRESUMEN
Transmissible gastroenteritis virus (TGEV)-induced enteritis is characterized by watery diarrhea, vomiting, and dehydration, and has high mortality in newborn piglets, resulting in significant economic losses in the pig industry worldwide. Conventional cell lines have been used for many years to investigate inflammation induced by TGEV, but these cell lines may not mimic the actual intestinal environment, making it difficult to obtain accurate results. In this study, apical-out porcine intestinal organoids were employed to study TEGV-induced inflammation. We found that apical-out organoids were susceptible to TGEV infection, and the expression of representative inflammatory cytokines was significantly upregulated upon TGEV infection. In addition, retinoic acid-inducible gene I (RIG-I) and the nuclear factor-kappa B (NF-κB) pathway were responsible for the expression of inflammatory cytokines induced by TGEV infection. We also discovered that the transcription factor hypoxia-inducible factor-1α (HIF-1α) positively regulated TGEV-induced inflammation by activating glycolysis in apical-out organoids, and pig experiments identified the same molecular mechanism as the ex vivo results. Collectively, we unveiled that the inflammatory responses induced by TGEV were modulated via the RIG-I/NF-κB/HIF-1α/glycolysis axis ex vivo and in vivo. This study provides novel insights into TGEV-induced enteritis and verifies intestinal organoids as a reliable model for investigating virus-induced inflammation. IMPORTANCE: Intestinal organoids are a newly developed culture system for investigating immune responses to virus infection. This culture model better represents the physiological environment compared with well-established cell lines. In this study, we discovered that inflammatory responses induced by TGEV infection were regulated by the RIG-I/NF-κB/HIF-1α/glycolysis axis in apical-out porcine organoids and in pigs. Our findings contribute to understanding the mechanism of intestinal inflammation upon viral infection and highlight apical-out organoids as a physiological model to mimic virus-induced inflammation.
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Gastroenteritis Porcina Transmisible , Glucólisis , Inflamación , Organoides , Virus de la Gastroenteritis Transmisible , Animales , Citocinas/metabolismo , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Gastroenteritis Porcina Transmisible/virología , Gastroenteritis Porcina Transmisible/metabolismo , Gastroenteritis Porcina Transmisible/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inflamación/metabolismo , Inflamación/virología , Intestinos/virología , Intestinos/patología , FN-kappa B/metabolismo , Organoides/virología , Organoides/metabolismo , Organoides/patología , Transducción de Señal , Porcinos , Virus de la Gastroenteritis Transmisible/fisiologíaRESUMEN
Fetal development and parturition are precisely regulated processes that involve continuous crosstalk between the mother and the fetus. Our previous discovery that wild-type mice carrying steroid receptor coactivator (Src)-1 and Src-2 double-deficient fetuses exhibit impaired lung development and delayed labor, which indicates that the signals for parturition emanate from the fetus. In this study, we perform RNA sequencing and targeted metabolomics analyses of the lungs from fetal Src-1/-2 double-knockout mice and find that expression of arginase 1 (Arg1) is significantly decreased, accompanied by increased levels of the Arg1 substrate L-arginine. Knockdown of Arg1 in the lungs of fetal mice induces apoptosis of epithelial cells and dramatically delays initiation of labor. Moreover, treatment of human myometrial smooth muscle cells with L-arginine significantly inhibits spontaneous contractions by attenuating activation of NF-κB and downregulating expression of contraction-associated protein genes. Transcription factors GR and C/EBPß increase transcription of Arg1 in an Src-1/Src-2-dependent manner. These findings provide new evidence that fetus-derived factors may play dual roles in coordinating fetal lung development and the initiation of labor.
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Arginasa , Pulmón , Animales , Humanos , Ratones , Arginasa/genética , Arginasa/metabolismo , Arginina/metabolismo , Desarrollo Fetal , Feto/metabolismo , Ratones NoqueadosRESUMEN
There is growing evidence that tumour neoantigens have important roles in generating spontaneous antitumour immune responses and predicting clinical responses to immunotherapies1,2. Despite the presence of numerous neoantigens in patients, complete tumour elimination is rare, owing to failures in mounting a sufficient and lasting antitumour immune response3,4. Here we show that durable neoantigen-specific immunity is regulated by mRNA N6-methyadenosine (m6A) methylation through the m6A-binding protein YTHDF15. In contrast to wild-type mice, Ythdf1-deficient mice show an elevated antigen-specific CD8+ T cell antitumour response. Loss of YTHDF1 in classical dendritic cells enhanced the cross-presentation of tumour antigens and the cross-priming of CD8+ T cells in vivo. Mechanistically, transcripts encoding lysosomal proteases are marked by m6A and recognized by YTHDF1. Binding of YTHDF1 to these transcripts increases the translation of lysosomal cathepsins in dendritic cells, and inhibition of cathepsins markedly enhances cross-presentation of wild-type dendritic cells. Furthermore, the therapeutic efficacy of PD-L1 checkpoint blockade is enhanced in Ythdf1-/- mice, implicating YTHDF1 as a potential therapeutic target in anticancer immunotherapy.
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Adenosina/análogos & derivados , Adenosina/metabolismo , Células Dendríticas/inmunología , Neoplasias/inmunología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Antígeno B7-H1/metabolismo , Sitios de Unión , Linfocitos T CD8-positivos/inmunología , Catepsinas/antagonistas & inhibidores , Catepsinas/biosíntesis , Catepsinas/genética , Reactividad Cruzada/inmunología , Células Dendríticas/enzimología , Femenino , Humanos , Metilación , Ratones , Ratones Endogámicos C57BL , Neoplasias/terapia , Biosíntesis de Proteínas , Proteínas/genética , ARN Mensajero/química , Proteínas de Unión al ARN/genética , Transcriptoma/genéticaRESUMEN
In this Letter, a citation to 'Fig. 1e' has been corrected to 'Fig. 1d' in the sentence starting "By contrast, the anti-tumour response ". This has been corrected online.
RESUMEN
Obstructive nephropathy is one of the leading causes of kidney injury and renal fibrosis in pediatric patients. Although considerable advances have been made in understanding the pathophysiology of obstructive nephropathy, most of them were based on animal experiments and a comprehensive understanding of obstructive nephropathy in pediatric patients at the molecular level remains limited. Here, we performed a comparative proteomics analysis of obstructed kidneys from pediatric patients with ureteropelvic junction obstruction and healthy kidney tissues. Intriguingly, the proteomics revealed extensive metabolic reprogramming in kidneys from individuals with ureteropelvic junction obstruction. Moreover, we uncovered the dysregulation of NAD+ metabolism and NAD+-related metabolic pathways, including mitochondrial dysfunction, the Krebs cycle, and tryptophan metabolism, which led to decreased NAD+ levels in obstructed kidneys. Importantly, the major NADase CD38 was strongly induced in human and experimental obstructive nephropathy. Genetic deletion or pharmacological inhibition of CD38 as well as NAD+ supplementation significantly recovered NAD+ levels in obstructed kidneys and reduced obstruction-induced renal fibrosis, partially through the mechanisms of blunting the recruitment of immune cells and NF-κB signaling. Thus, our work not only provides an enriched resource for future investigations of obstructive nephropathy but also establishes CD38-mediated NAD+ decline as a potential therapeutic target for obstruction-induced renal fibrosis.
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NAD , Obstrucción Ureteral , Animales , Niño , Humanos , Fibrosis , Riñón/metabolismo , NAD/metabolismo , Proteómica , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/tratamiento farmacológico , Obstrucción Ureteral/metabolismoRESUMEN
A common feature of large-scale extreme events, such as pandemics, wildfires, and major storms is that, despite their differences in etiology and duration, they significantly change routine human movement patterns. Such changes, which can be major or minor in size and duration and which differ across contexts, affect both the consequences of the events and the ability of governments to mount effective responses. Based on naturally tracked, anonymized mobility behavior from over 90 million people in the United States, we document these mobility differences in space and over time in six large-scale crises, including wildfires, major tropical storms, winter freeze and pandemics. We introduce a model that effectively captures the high-dimensional heterogeneity in human mobility changes following large-scale extreme events. Across five different metrics and regardless of spatial resolution, the changes in human mobility behavior exhibit a consistent hyperbolic decline, a pattern we characterize as "spatiotemporal decay." When applied to the case of COVID-19, our model also uncovers significant disparities in mobility changes-individuals from wealthy areas not only reduce their mobility at higher rates at the start of the pandemic but also maintain the change longer. Residents from lower-income regions show a faster and greater hyperbolic decay, which we suggest may help account for different COVID-19 rates. Our model represents a powerful tool to understand and forecast mobility patterns post emergency, and thus to help produce more effective responses.
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COVID-19 , Migración Humana , Modelos Estadísticos , Desastres Naturales , Pandemias , COVID-19/epidemiología , Predicción , Migración Humana/tendencias , Humanos , Renta , Estaciones del Año , Análisis Espacio-Temporal , Estados UnidosRESUMEN
Protein kinase R (PKR) is a critical host restriction factor against invading viral pathogens. However, this molecule is inactivated in the cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), an economically devastating pathogen to the world swine industry. Here, we report that this event is to suppress cellular inflammation and is mediated by the viral replicase protein nsp1ß. We show that nsp1ß is a stress-responsive protein, enters virus-induced stress granules (SGs) during infection, and repurposes SGs into a proviral platform, where it co-opts the SG core component G3BP1 to interact with PKR in a regulated manner. RNA interference silencing of G3BP1 or mutation of specific nsp1ß residues (VS19GG) can abolish the antagonization of PKR activation. The viral mutant carrying the corresponding mutations induces elevated level of PKR phosphorylation and pronounced production of inflammatory cytokines (e.g., tumor necrosis factor-α, interleukin [IL]-6, and IL-8), whereas small-interfering RNA knockdown of PKR or treatment with C16, a PKR inhibitor, blocks this effect. Thus, PRRSV has evolved a unique strategy to evade PKR restriction to suppress host inflammatory responses.
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Factores de Restricción Antivirales , ADN Helicasas , Evasión Inmune , Proteínas de Unión a Poli-ADP-Ribosa , Virus del Síndrome Respiratorio y Reproductivo Porcino , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Proteínas no Estructurales Virales , eIF-2 Quinasa , Animales , Factores de Restricción Antivirales/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Gránulos de Estrés/virología , Porcinos , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , eIF-2 Quinasa/metabolismoRESUMEN
Adenine base editors (ABEs) are genome-editing tools that have been harnessed to introduce precise Aâ¢T to Gâ¢C conversion. The discovery of split genes revealed that all introns contain two highly conserved dinucleotides, canonical "AG" (acceptor) and "GT" (donor) splice sites. ABE can directly edit splice acceptor sites of the adenine (A) base, leading to aberrant gene splicing, which may be further adopted to remodel splicing. However, spliced isoforms triggered with ABE have not been well explored. To address it, we initially generated a cell line harboring C-terminal enhanced GFP (eGFP)-tagged ß-actin (ACTB), in which the eGFP signal can track endogenous ß-actin expression. Expectedly, after the editing of splice acceptor sites, we observed a dramatical decrease in the percentage of eGFP-positive cells and generation of splicing products with the noncanonical splice site. Furthermore, we manipulated Peroxidasin in mouse embryos with ABE, in which a noncanonical acceptor was activated to remodel splicing, successfully generating a mouse disease model of anophthalmia and severely malformed microphthalmia. Collectively, we demonstrate that ABE-mediated splicing remodeling can activate a noncanonical acceptor to manipulate human and mouse genomes, which will facilitate the investigation of basic and translational medicine studies.