RESUMEN
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global health emergency that is in urgent need of intervention1-3. The entry of SARS-CoV-2 into its target cells depends on binding between the receptor-binding domain (RBD) of the viral spike protein and its cellular receptor, angiotensin-converting enzyme 2 (ACE2)2,4-6. Here we report the isolation and characterization of 206 RBD-specific monoclonal antibodies derived from single B cells from 8 individuals infected with SARS-CoV-2. We identified antibodies that potently neutralize SARS-CoV-2; this activity correlates with competition with ACE2 for binding to RBD. Unexpectedly, the anti-SARS-CoV-2 antibodies and the infected plasma did not cross-react with the RBDs of SARS-CoV or Middle East respiratory syndrome-related coronavirus (MERS-CoV), although there was substantial plasma cross-reactivity to their trimeric spike proteins. Analysis of the crystal structure of RBD-bound antibody revealed that steric hindrance inhibits viral engagement with ACE2, thereby blocking viral entry. These findings suggest that anti-RBD antibodies are largely viral-species-specific inhibitors. The antibodies identified here may be candidates for development of clinical interventions against SARS-CoV-2.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Neumonía Viral/inmunología , Neumonía Viral/virología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Anciano , Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Linfocitos B/citología , Linfocitos B/inmunología , Betacoronavirus/química , COVID-19 , Niño , Células Clonales/citología , Células Clonales/inmunología , Reacciones Cruzadas , Cristalización , Cristalografía por Rayos X , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Pruebas de Neutralización , Pandemias , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Plasma/inmunología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
As a model plant for bryophytes, Marchantia polymorpha offers insights into the role of RNA silencing in aiding early land plants navigate the challenges posed by high-temperature environments. Genomic analysis revealed unique ARGONAUTE1 ortholog gene (MpAGO1) in M. polymorpha that is regulated by two species-specific microRNAs (miRNAs), miR11707.1 and miR11707.2. Comparative studies of small RNA profiles from M. polymorpha cellular and MpAGO1 immunoprecipitation (MpAGO1-IP) profiles at various temperatures, along with analyses of Arabidopsis AGO1 (AtAGO1), revealed that MpAGO1 has a low-selectivity for a diverse range of small RNA species than AtAGO1. Protein structural comparisons revealed no discernible differences in the MID domains of MpAGO1 and AtAGO1, suggesting the complexity of miRNA species specificity and necessitating further exploration. Small RNA profiling and size exclusion chromatography have pinpointed a subset of M. polymorpha miRNAs, notably miR11707, that remain in free form within the cell at 22°C but are loaded into MpAGO1 at 28°C to engage in RNA silencing. Investigations into the mir11707 gene editing (mir11707ge) mutants provided evidence of the regulation of miR11707 in MpAGO1. Notably, while MpAGO1 mRNA expression decreases at 28°C, the stability of the MpAGO1 protein and its associated miRNAs is essential for enhancing the RISC activity, revealing the importance of RNA silencing in enabling M. polymorpha to survive thermal stress. This study advances our understanding of RNA silencing in bryophytes and provides groundbreaking insights into the evolutionary resilience of land plants to climatic adversities.
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The miR390-derived TAS3 trans-acting short-interfering RNAs (tasiRNAs) module represents a conserved RNA silencing pathway in the plant kingdom; however, its characterization in the bryophyte Marchantia polymorpha is limited. This study elucidated that MpDCL4 processes MpTAS3 double-stranded RNA (dsRNA) to generate tasiRNAs, primarily from the 5'- and 3'-ends of dsRNA. Notably, we discovered a novel tasiRNA, tasi78A, which can negatively regulate a cytochrome P450 gene, MpCYP78A101. Additionally, tasi78A was abundant in MpAGO1, and transient expression assays underscored the role of tasi78A in repressing MpCYP78A101. A microRNA, miR11700, also regulates MpCYP78A101 expression. This coordinate regulation suggests a role in modulating auxin signaling at apical notches of gemma, influencing the growth and sexual organ development of M. polymorpha and emphasizing the significance of RNA silencing in MpCYP78A101 regulation. However, phylogenetic analysis identified another paralog of the CYP78 family, Mp1g14150, which may have a redundant role with MpCYP78A101, explaining the absence of noticeable morphological changes in loss-of-function plants. Taken together, our findings provide new insights into the combined regulatory roles of miR390/MpTAS3/miR11700 in controlling MpCYP78A101 and expand our knowledge about the biogenesis and regulation of tasiRNAs in M. polymorpha.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Regulación de la Expresión Génica de las Plantas , Marchantia , MicroARNs , ARN Interferente Pequeño , Marchantia/genética , MicroARNs/genética , MicroARNs/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Filogenia , ARN de Planta/genética , ARN de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Currently, the clinical application of protein/peptide therapeutics is mainly limited to the modulation of diseases in extracellular spaces. Intracellular targets are hardly accessed, owing largely to the endosomal entrapment of internalized proteins/peptides. Here, we report a strategy to design and construct peptides that enable endosome-to-cytosol delivery based on an extension of the "histidine switch" principle. By substituting the Arg/Lys residues in cationic cell-penetrating peptides (CPPs) with histidine, we obtained peptides with pH-dependent membrane-perturbation activity. These peptides do not randomly penetrate cells like CPPs, but imitate the endosomal escape of CPPs following cellular uptake. Working with one such 16-residue peptide (hsLMWP) with high endosomal escape capacity, we engineered modular fusion proteins and achieved antibody-targeted delivery of diverse protein cargoes-including the pro-apoptotic protein BID (BH3-interacting domain death agonist) and Cre recombinase-into the cytosol of multiple cancer cell types. After extensive in vitro testing, an in vivo analysis with xenograft mice ultimately demonstrated that a trastuzumab-hsLMWP-BID fusion conferred strong anti-tumor efficacy without apparent side effects. Notably, our fusion protein features a modular design, allowing flexible applications for any antibody/cargo combination of choice. Therefore, the potential applications extend throughout life science and biomedicine, including gene editing, cancer treatment, and immunotherapy.
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Péptidos de Penetración Celular , Histidina , Humanos , Ratones , Animales , Histidina/metabolismo , Espacio Extracelular/metabolismo , Endosomas/metabolismo , Citosol/metabolismo , Péptidos de Penetración Celular/química , Anticuerpos/metabolismoRESUMEN
The biodiversity of microalgal species is enormous, and their versatile metabolism produces a wide diversity of compounds that can be used in food, healthcare, and other applications. Microalgae are also a potential source of bio-stimulants that enhance nutrition efficiency, abiotic stress tolerance, and/or crop quality traits. In this study, the extracellular metabolites of Auxenochlorella protothecoides (EAp) were prepared using three different culture strategies, and their effects on plant growth were examined. Furthermore, the composition of EAp was analyzed by GC-MS. The elongation of lateral roots and the cold-tolerance of Arabidopsis thaliana and Nicotiana benthamiana were promoted by EAp. Moreover, EAp from high-cell-density fermentation stimulated the growth of the leafy vegetables Brassica rapa and Lactuca sativa at dilutions as high as 500- and 1000-fold. Three major groups of compounds were identified by GC-MS, including organic acids or organic acid esters, phenols, and saccharides. Some of these compounds have known plant-stimulating effects, while the rest requires further investigation in the future. Our study demonstrates that EAp is a potential bio-stimulant, while also providing an environmentally friendly and economical microalgae fermentation process.
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Chlorophyta , Microalgas , Chlorophyta/metabolismo , Ésteres/metabolismo , Procesos Heterotróficos , Microalgas/metabolismo , Fenoles/metabolismoRESUMEN
The emergence and rapid spread of the B.1.1.7 lineage (VOC-202012/01) SARS-CoV-2 variant has aroused global concern. The N501Y substitution is the only mutation in the interface between the RBD of B.1.1.7 and ACE2, raising concerns that its recognition by neutralizing antibodies may be affected. Here, we assessed the neutralizing activity and binding affinity of a panel of 12 monoclonal antibodies against the wild type and N501Y mutant SARS-CoV-2 pseudovirus and RBD protein, respectively. We found that the neutralization activity and binding affinity of most detected antibodies (10 out of 12) were unaffected, although the N501Y substitution decreased the neutralizing and binding activities of CB6 and increased that of BD-23. These findings could be of value in the development of therapeutic antibodies.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Afinidad de Anticuerpos , Sitios de Unión , Epítopos/inmunología , Células HEK293 , Humanos , Mutación , Pruebas de Neutralización , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Brucellosis is a serious public health problem worldwide and can affect any organ system. Due to brucellosis's variable clinical presentation, ranging from subclinical to fully symptomatic, and limited available information, it poses a diagnostic challenge. In this study, we reported a case series of patients with diverse presentations. In addition, we briefly described the pathophysiology and mechanisms of Brucella in the body. These case presentations will be valuable in increasing the awareness of physicians. A prompt diagnosis is crucial, as detecting some clues of the infection in its early stages can help avoid misdiagnoses.
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Pluripotent stem cells (PSCs), characterized by self-renewal and capacity of differentiating into three germ layers, are the programmable building blocks of life. PSC-derived cells and multicellular systems, particularly organoids, exhibit great potential for regenerative medicine. However, this field is still in its infancy, partly due to limited strategies to robustly and precisely control stem cell behaviors, which are tightly regulated by inner gene regulatory networks in response to stimuli from the extracellular environment. Synthetic receptors and genetic circuits are powerful tools to customize the cellular sense-and-response process, suggesting their underlying roles in precise control of cell fate decision and function reconstruction. Herein, we review the progress and challenges needed to be overcome in the fields of PSC-based cell therapy and multicellular system generation, respectively. Furthermore, we summarize several well-established synthetic biology tools and their applications in PSC engineering. Finally, we highlight the challenges and perspectives of harnessing synthetic biology to PSC engineering for regenerative medicine.
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Although thousands of anti-SARS-CoV-2 monoclonal neutralizing antibodies (nAbs) have been identified and well characterized, some crucial events in the development of these nAbs during viral infection remain unclear. Using deep sequencing, we explore the dynamics of antibody repertoire in a SARS-CoV-2-infected donor, from whom the potent and broad nAb P2C-1F11 (the parent version of Brii-196) was previously isolated. Further analysis shows a rapid clonal expansion of some SARS-CoV-2-specific antibodies in early infection. Longitudinal tracing of P2C-1F11 lineage antibodies reveals that these elite nAbs were rare. Using sequence alignment, structure modeling, and bioactivity analysis based on site-mutated assay, we demonstrate that a key substitution F27I in heavy chain contributes significantly to the maturation of P2C-1F11-like antibodies. Overall, our findings elucidate the developmental process and maturation pathway of P2C-1F11, providing some important information for the design of novel immunogens to elicit more potent nAbs against SARS-CoV-2 infection.
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COVID-19 , SARS-CoV-2 , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , HumanosRESUMEN
Neutralizing antibodies (nAbs) to SARS-CoV-2 hold powerful potentials for clinical interventions against COVID-19 disease. However, their common genetic and biologic features remain elusive. Here we interrogate a total of 165 antibodies from eight COVID-19 patients, and find that potent nAbs from different patients have disproportionally high representation of IGHV3-53/3-66 usage, and therefore termed as public antibodies. Crystal structural comparison of these antibodies reveals they share similar angle of approach to RBD, overlap in buried surface and binding residues on RBD, and have substantial spatial clash with receptor angiotensin-converting enzyme-2 (ACE2) in binding to RBD. Site-directed mutagenesis confirms these common binding features although some minor differences are found. One representative antibody, P5A-3C8, demonstrates extraordinarily protective efficacy in a golden Syrian hamster model against SARS-CoV-2 infection. However, virus escape analysis identifies a single natural mutation in RBD, namely K417N found in B.1.351 variant from South Africa, abolished the neutralizing activity of these public antibodies. The discovery of public antibodies and shared escape mutation highlight the intricate relationship between antibody response and SARS-CoV-2, and provide critical reference for the development of antibody and vaccine strategies to overcome the antigenic variation of SARS-CoV-2.