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Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D-PGD2/PTGDR pathway is a useful target for therapeutic interventions.
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COVID-19 , SARS-CoV-2 , Animales , Eicosanoides , Leucocitos Mononucleares , Ratones , Compuestos Orgánicos , Oxazoles , Piperazinas , Poliésteres , Prostaglandinas , Glicoproteína de la Espiga del Coronavirus , SulfonamidasRESUMEN
Fusarium head blight (FHB), caused by Fusarium graminearum species complexes (FGSG), is an epidemic disease in wheat and poses a serious threat to wheat production and security worldwide. Profilins are a class of actin-binding proteins that participate in actin depolymerization. However, the roles of profilins in plant fungal pathogens remain largely unexplored. Here, we identified FgPfn, a homolog to profilins in F. graminearum, and the deletion of FgPfn resulted in severe defects in mycelial growth, conidia production, and pathogenicity, accompanied by marked disruptions in toxisomes formation and deoxynivalenol (DON) transport, while sexual development was aborted. Additionally, FgPfn interacted with Fgα1 and Fgß2, the significant components of microtubules. The organization of microtubules in the ΔFgPfn was strongly inhibited under the treatment of 0.4 µg/mL carbendazim, a well-known group of tubulin interferers, resulting in increased sensitivity to carbendazim. Moreover, FgPfn interacted with both myosin-5 (FgMyo5) and actin (FgAct), the targets of the fungicide phenamacril, and these interactions were reduced after phenamacril treatment. The deletion of FgPfn disrupted the normal organization of FgMyo5 and FgAct cytoskeleton, weakened the interaction between FgMyo5 and FgAct, and resulting in increased sensitivity to phenamacril. The core region of the interaction between FgPfn and FgAct was investigated, revealing that the integrity of both proteins was necessary for their interaction. Furthermore, mutations in R72, R77, R86, G91, I101, A112, G113, and D124 caused the non-interaction between FgPfn and FgAct. The R86K, I101E, and D124E mutants in FgPfn resulted in severe defects in actin organization, development, and pathogenicity. Taken together, this study revealed the role of FgPfn-dependent cytoskeleton in development, DON production and transport, fungicides sensitivity in F. graminearum.
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Actinas , Proteínas Fúngicas , Fungicidas Industriales , Fusarium , Microtúbulos , Enfermedades de las Plantas , Triticum , Microtúbulos/metabolismo , Fusarium/metabolismo , Fusarium/patogenicidad , Fusarium/genética , Fusarium/efectos de los fármacos , Fusarium/crecimiento & desarrollo , Actinas/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Triticum/microbiología , Fungicidas Industriales/farmacología , Esporas Fúngicas/metabolismo , Esporas Fúngicas/crecimiento & desarrollo , ReproducciónRESUMEN
Spermatozoa in animal species are usually highly elongated cells with a long motile tail attached to a head that contains the haploid genome in a compact and often elongated nucleus. In Drosophila melanogaster, the nucleus is compacted two hundred-fold in volume during spermiogenesis and re-modeled into a needle that is thirty-fold longer than its diameter. Nuclear elongation is preceded by a striking relocalization of nuclear pore complexes (NPCs). While NPCs are initially located throughout the nuclear envelope (NE) around the spherical nucleus of early round spermatids, they are later confined to one hemisphere. In the cytoplasm adjacent to this NPC-containing NE, the so-called dense complex with a strong bundle of microtubules is assembled. While this conspicuous proximity argued for functional significance of NPC-NE and microtubule bundle, experimental confirmation of their contributions to nuclear elongation has not yet been reported. Our functional characterization of the spermatid specific Mst27D protein now resolves this deficit. We demonstrate that Mst27D establishes physical linkage between NPC-NE and dense complex. The C-terminal region of Mst27D binds to the nuclear pore protein Nup358. The N-terminal CH domain of Mst27D, which is similar to that of EB1 family proteins, binds to microtubules. At high expression levels, Mst27D promotes bundling of microtubules in cultured cells. Microscopic analyses indicated co-localization of Mst27D with Nup358 and with the microtubule bundles of the dense complex. Time-lapse imaging revealed that nuclear elongation is accompanied by a progressive bundling of microtubules into a single elongated bundle. In Mst27D null mutants, this bundling process does not occur and nuclear elongation is abnormal. Thus, we propose that Mst27D permits normal nuclear elongation by promoting the attachment of the NPC-NE to the microtubules of the dense complex, as well as the progressive bundling of these microtubules.
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Proteínas de Drosophila , Poro Nuclear , Masculino , Animales , Poro Nuclear/genética , Poro Nuclear/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Microtúbulos/metabolismo , Espermatogénesis/genética , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMEN
SARS-CoV-2, the causative agent of COVID-19 encodes at least 16 nonstructural proteins of variably understood function. Nsp3, the largest nonstructural protein contains several domains, including a SARS-unique domain (SUD), which occurs only in Sarbecovirus. The SUD has a role in preferentially enhancing viral translation. During isolation of mouse-adapted SARS-CoV-2, we isolated an attenuated virus that contained a single mutation in a linker region of nsp3 (nsp3-S676T). The S676T mutation decreased virus replication in cultured cells and primary human cells and in mice. Nsp3-S676T alleviated the SUD translational enhancing ability by decreasing the interaction between two translation factors, Paip1 and PABP1. We also identified a compensatory mutation in the nucleocapsid (N) protein (N-S194L) that restored the virulent phenotype, without directly binding to SUD. Together, these results reveal an aspect of nsp3-N interactions, which impact both SARS-CoV-2 replication and, consequently, pathogenesis.
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COVID-19 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Humanos , Animales , Ratones , SARS-CoV-2 , Virulencia , MutaciónRESUMEN
Hepatitis E virus (HEV) infection has been shown to activate NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in macrophages, a key mechanism of causing pathological inflammation, but the mechanisms regulating this response remain poorly understood. Here, we report that the mature tRNAome dynamically responds to HEV infection in macrophages. This directs IL-1ß expression, the hallmark of NLRP3 inflammasome activation, at mRNA and protein levels. Conversely, pharmacological inhibition of inflammasome activation abrogates HEV-provoked tRNAome remodeling, revealing a reciprocal interaction between the mature tRNAome and the NLRP3 inflammasome response. Remodeling the tRNAome results in improved decoding of codons directing leucine- and proline synthesis, which are the major amino acid constituents of IL-1ß protein, whereas genetic or functional interference with tRNAome-mediated leucine decoding impairs inflammasome activation. Finally, we demonstrated that the mature tRNAome also actively responds to lipopolysaccharide (a key component of gram-negative bacteria)-triggered inflammasome activation, but the response dynamics and mode of actions are distinct from that induced by HEV infection. Our findings thus reveal the mature tRNAome as a previously unrecognized but essential mediator of host response to pathogens and represent a unique target for developing anti-inflammatory therapeutics.
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Virus de la Hepatitis E , Hepatitis E , Humanos , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Leucina , MacrófagosRESUMEN
Identification of a conserved G-quadruplex in E165R of ASFVAfrican swine fever virus (ASFV) is a double-stranded DNA arbovirus with high transmissibility and mortality rates. It has caused immense economic losses to the global pig industry. Currently, no effective vaccines or medications are to combat ASFV infection. G-quadruplex (G4) structures have attracted increasing interest because of their regulatory role in vital biological processes. In this study, we identified a conserved G-rich sequence within the E165R gene of ASFV. Subsequently, using various methods, we verified that this sequence could fold into a parallel G4. In addition, the G4-stabilizers pyridostatin and 5,10,15,20-tetrakis-(N-methyl-4-pyridyl) porphin (TMPyP4) can bind and stabilize this G4 structure, thereby inhibiting E165R gene expression, and the inhibitory effect is associated with G4 formation. Moreover, the G4 ligand pyridostatin substantially impeded ASFV proliferation in Vero cells by reducing gene copy number and viral protein expression. These compelling findings suggest that G4 structures may represent a promising and novel antiviral target against ASFV.
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Virus de la Fiebre Porcina Africana , Antivirales , G-Cuádruplex , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/metabolismo , Animales , Chlorocebus aethiops , Células Vero , Antivirales/farmacología , Antivirales/química , Porcinos , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/metabolismo , Porfirinas/química , Porfirinas/farmacología , Ácidos Picolínicos/química , Ácidos Picolínicos/farmacología , Ácidos Picolínicos/metabolismo , Replicación Viral/efectos de los fármacos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Virales/química , AminoquinolinasRESUMEN
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has resulted in substantial morbidity and mortality. The basis of severe disease in humans is difficult to determine without the use of experimental animal models. Mice are resistant to infection with ancestral strains of SARS-CoV-2, although many variants that arose later in the pandemic were able to directly infect mice. In almost all cases, viruses that naturally infected mice or were engineered to enable mouse infection required mouse passage to become virulent. In most cases, changes in structural and nonstructural changes occurred during mouse adaptation. However, the mechanism of increased virulence in mice is not understood. Here, using a recently described strain of mouse-adapted SARS-CoV-2 (rSARS2-MA30N501Y), we engineered a series of recombinant viruses that expressed a subset of the mutations present in rSARS2-MA30N501Y. Mutations were detected in the spike protein and in three nonstructural proteins (nsp4, nsp8, and nsp9). We found that infection of mice with recombinant SARS-CoV-2 expressing only the S protein mutations caused very mild infection. Addition of the mutations in nsp4 and nsp8 was required for complete virulence. Of note, all these recombinant viruses replicated equivalently in cultured cells. The innate immune response was delayed after infection with virulent compared to attenuated viruses. Further, using a lineage tracking system, we found that attenuated virus was highly inhibited in the ability to infect the parenchyma, but not the airway after infection. Together, these results indicate that mutations in both the S protein and nonstructural proteins are required for maximal virulence during mouse adaptation.IMPORTANCEUnderstanding the pathogenesis of coronavirus disease 2019 (COVID-19) requires the study of experimental animals after infection with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). For this purpose, several mouse-adapted SARS-CoV-2 strains have been developed. Here, using a strain of mouse-adapted virus that causes a range of diseases ranging from mild to severe, we show that mutations in both a structural protein [spike (S) protein] and nonstructural proteins are required for maximal virulence. Thus, changes in the S protein, the most widely studied viral protein, while required for mouse adaptation, are not sufficient to result in a virulent virus.
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COVID-19 , Modelos Animales de Enfermedad , Mutación , SARS-CoV-2 , Proteínas no Estructurales Virales , Animales , Ratones , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , COVID-19/virología , SARS-CoV-2/patogenicidad , SARS-CoV-2/genética , Virulencia , Humanos , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Chlorocebus aethiops , Replicación Viral , FemeninoRESUMEN
Betacoronaviruses encode a conserved accessory gene within the +1 open reading frame (ORF) of nucleocapsid called the internal N gene. This gene is referred to as "I" for mouse hepatitis virus (MHV), ORF9b for severe acute respiratory CoV (SARS-CoV) and SARS-CoV-2, and ORF8b for Middle East respiratory syndrome CoV (MERS-CoV). Previous studies have shown ORF8b and ORF9b have immunoevasive properties, while the only known information for MHV I is its localization within the virion of the hepatotropic/neurotropic A59 strain of MHV. Whether MHV I is an innate immune antagonist or has other functions has not been evaluated. In this report, we show that the I protein of the neurotropic JHM strain of MHV (JHMV) lacks a N terminal domain present in other MHV strains, has immunoevasive properties, and is a component of the virion. Genetic deletion of JHMV I (rJHMVIΔ57-137) resulted in a highly attenuated virus both in vitro and in vivo that displayed a post RNA replication/transcription defect that ultimately resulted in fewer infectious virions packaged compared with wild-type virus. This phenotype was only seen for rJHMVIΔ57-137, suggesting the structural changes predicted for A59 I altered its function, as genetic deletion of A59 I did not change viral replication or pathogenicity. Together, these data show that JHMV I both acts as a mild innate immune antagonist and aids in viral assembly and infectious virus production, and suggest that the internal N proteins from different betacoronaviruses have both common and virus strain-specific properties.IMPORTANCECoV accessory genes are largely studied in overexpression assays and have been identified as innate immune antagonists. However, functions identified after overexpression are often not confirmed in the infected animal host. Furthermore, some accessory proteins are components of the CoV virion, but their role in viral replication and release remains unclear. Here, we utilized reverse genetics to abrogate expression of a conserved CoV accessory gene, the internal N ("I") gene, of the neurotropic JHMV strain of MHV and found that loss of the I gene resulted in a post replication defect that reduced virion assembly and ultimately infectious virus production, while also increasing some inflammatory molecule expression. Thus, the JHMV I protein has roles in virion assembly that were previously underappreciated and in immunoevasion.
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Senecavirus A (SVA), a picornavirus, causes vesicular diseases and epidemic transient neonatal losses in swine, resulting in a multifaceted economic impact on the swine industry. SVA counteracts host antiviral response through multiple strategies facilitatng viral infection and transmission. However, the mechanism of how SVA modulates interferon (IFN) response remains elusive. Here, we demonstrate that SVA 3C protease (3Cpro) blocks the transduction of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway to antagonize type I IFN response. Mechanistically, 3Cpro selectively cleaves and degrades STAT1 and STAT2 while does not target JAK1, JAK2, and IRF9, through its protease activity. Notably, SVA 3Cpro cleaves human and porcine STAT1 on a Leucine (L)-Aspartic acid (D) motif, specifically L693/D694. In the case of STAT2, two cleavage sites were identified: glutamine (Q) 707 was identified in both human and porcine, while the second cleavage pattern differed, with residues 754-757 (Valine-Leucine-Glutamine-Serine motifs) in human STAT2 and Q758 in porcine STAT2. These cleavage patterns by SVA 3Cpro partially differ from previously reported classical motifs recognized by other picornaviral 3Cpro, highlighting the distinct characteristics of SVA 3Cpro. Together, these results reveal a mechanism by which SVA 3Cpro antagonizes IFN-induced antiviral response but also expands our knowledge about the substrate recognition patterns for picornaviral 3Cpro.IMPORTANCESenecavirus A (SVA), the only member in the Senecavirus genus within the Picornaviridae family, causes vesicular diseases in pigs that are clinically indistinguishable from foot-and-mouth disease (FMD), a highly contagious viral disease listed by the World Organization for Animal Health (WOAH). Interferon (IFN)-mediated antiviral response plays a pivotal role in restricting and controlling viral infection. Picornaviruses evolved numerous strategies to antagonize host antiviral response. However, how SVA modulates the JAK-STAT signaling pathway, influencing the type I IFN response, remains elusive. Here, we identify that 3Cpro, a protease of SVA, functions as an antagonist for the IFN response. 3Cpro utilizes its protease activity to cleave STAT1 and STAT2, thereby diminishing the host IFN response to promote SVA infection. Our findings underscore the significance of 3Cpro as a key virulence factor in the antagonism of the type I signaling pathway during SVA infection.
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Cisteína Endopeptidasas , Infecciones por Picornaviridae , Picornaviridae , Factor de Transcripción STAT1 , Factor de Transcripción STAT2 , Transducción de Señal , Proteínas Virales , Animales , Porcinos , Factor de Transcripción STAT2/metabolismo , Humanos , Infecciones por Picornaviridae/virología , Infecciones por Picornaviridae/inmunología , Infecciones por Picornaviridae/metabolismo , Factor de Transcripción STAT1/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas Virales/metabolismo , Proteasas Virales 3C , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Células HEK293 , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/metabolismo , Línea Celular , Quinasas Janus/metabolismo , Quinasas Janus/antagonistas & inhibidoresRESUMEN
The Coronavirus Disease 2019 (COVID-19) pandemic continues to cause extraordinary loss of life and economic damage. Animal models of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection are needed to better understand disease pathogenesis and evaluate preventive measures and therapies. While mice are widely used to model human disease, mouse angiotensin converting enzyme 2 (ACE2) does not bind the ancestral SARS-CoV-2 spike protein to mediate viral entry. To overcome this limitation, we "humanized" mouse Ace2 using CRISPR gene editing to introduce a single amino acid substitution, H353K, predicted to facilitate S protein binding. While H353K knockin Ace2 (mACE2H353K) mice supported SARS-CoV-2 infection and replication, they exhibited minimal disease manifestations. Following 30 serial passages of ancestral SARS-CoV-2 in mACE2H353K mice, we generated and cloned a more virulent virus. A single isolate (SARS2MA-H353K) was prepared for detailed studies. In 7-11-month-old mACE2H353K mice, a 104 PFU inocula resulted in diffuse alveolar disease manifested as edema, hyaline membrane formation, and interstitial cellular infiltration/thickening. Unexpectedly, the mouse-adapted virus also infected standard BALB/c and C57BL/6 mice and caused severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.IMPORTANCEWe developed a new mouse model with a humanized angiotensin converting enzyme 2 (ACE2) locus that preserves native regulatory elements. A single point mutation in mouse ACE2 (H353K) was sufficient to confer in vivo infection with ancestral severe acute respiratory syndrome-coronavirus-2 virus. Through in vivo serial passage, a virulent mouse-adapted strain was obtained. In aged mACE2H353K mice, the mouse-adapted strain caused diffuse alveolar disease. The mouse-adapted virus also infected standard BALB/c and C57BL/6 mice, causing severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Humanos , Ratones , Regiones no Traducidas 5' , Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Nucleótidos , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
ConspectusThe innovation of chiral ligands has been crucial for the asymmetric synthesis of functional molecules, as demonstrated by several types of widely applied "privileged" ligands. In this context, chiral pyridine-derived ligands, by far some of the oldest and most widely utilized ligands in catalysis, have attracted considerable research interest in the past half-century. However, the development of broadly applicable chiral pyridine units (CPUs) has been plagued by several intertwining challenges, thus delaying advancements in many asymmetric reactions.This Account aims to summarize the recent progress in new CPU-containing ligands, focusing on a rationally designed, modular, and tunable CPU developed in our laboratory. A significant problem thwarting conventional designs is the paradox between broad reactivity and stereoselectivity; that is, while enhanced stereoselectivity may be achieved by introducing chiral elements close to the N atom, the concomitant increase in local steric hindrance often limits catalytic activity and scope. Our newly developed CPU features a rigid [6-5-3] fused-ring framework and a tunable spirocyclic ketal side wall. The well-defined three-dimensional structure minimizes local (inner layer) steric hindrance and tunes the peripheral environment (outer layer) by remote substituents, thus securing reactivity and stereoselectivity. Different chelating ligands were readily assembled using this chiral structural module, with applications in mechanistically diverse transition-metal-catalyzed reactions. Thus, a series of chiral 2,2'-bipyridine ligands were successfully employed in the development of a general, efficient, and highly enantioselective nickel-catalyzed intermolecular reductive addition, Ullmann coupling of ortho-chlorinated aryl aldehydes, and carboxylation of benzylic (pseudo)halides with CO2. Notably, these chiral 2,2'-bipyridine ligands exhibited superior catalytic activity in the reactions compared to common N-based ligands. In addition, highly enantioselective iridium-catalyzed C-H borylation was developed using a CPU-containing N,B-bidentate ligand. Furthermore, mechanistically challenging, additive-free, and broad-scope transfer hydrogenative direct asymmetric reductive amination was achieved using a half-sandwich iridium catalyst supported by a chiral N,C-bidentate ligand. The new ligands demonstrated excellent performance in securing high catalytic activity and stereoselectivity, which, when combined with experimental and computational mechanistic investigations, supported the "double-layer control" design concept.Considering the broad applications of pyridine-derived ligands, the research progress described herein should inspire the creation of novel chiral catalysts and drive the development of many catalytic asymmetric reactions.
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Aberrant glycans are a hallmark of cancer states. Notably, emerging evidence has demonstrated that the diagnosis of cancers with tumour-specific glycan patterns holds great potential to address unmet medical needs, especially in improving diagnostic sensitivity and selectivity. However, despite vast glycans having been identified as potent markers, glycan-based diagnostic methods remain largely limited in clinical practice. There are several reasons that prevent them from reaching the market, and the lack of anti-glycan antibodies is one of the most challenging hurdles. With the increasing need for accelerating the translational process, numerous efforts have been made to find antibody alternatives, such as lectins, boronic acids and aptamers. However, issues concerning affinity, selectivity, stability and versatility are yet to be fully addressed. Molecularly imprinted polymers (MIPs), synthetic antibody mimics with tailored cavities for target molecules, hold the potential to revolutionize this dismal progress. MIPs can bind a wide range of glycan markers, even those without specific antibodies. This capacity effectively broadens the clinical applicability of glycan-based diagnostics. Additionally, glycoform-resolved diagnosis can also be achieved through customization of MIPs, allowing for more precise diagnostic applications. In this review, we intent to introduce the current status of glycans as potential biomarkers and critically evaluate the challenges that hinder the development of in vitro diagnostic assays, with a particular focus on glycan-specific recognition entities. Moreover, we highlight the key role of MIPs in this area and provide examples of their successful use. Finally, we conclude the review with the remaining challenges, future outlook, and emerging opportunities.
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Impresión Molecular , Neoplasias , Polímeros Impresos Molecularmente , Impresión Molecular/métodos , Lectinas/metabolismo , Biomarcadores , Anticuerpos , Polisacáridos , Neoplasias/diagnósticoRESUMEN
Formate dehydrogenase (FDH; EC 1.2.1.2.) has been implicated in plant responses to a variety of stresses, including aluminum (Al) stress in acidic soils. However, the role of this enzyme in Al tolerance is not yet fully understood, and how FDH gene expression is regulated is unknown. Here, we report the identification and functional characterization of the tomato (Solanum lycopersicum) SlFDH gene. SlFDH encodes a mitochondria-localized FDH with Km values of 2.087 mm formate and 29.1 µm NAD+ . Al induced the expression of SlFDH in tomato root tips, but other metals did not, as determined by quantitative reverse transcriptase-polymerase chain reaction. CRISPR/Cas9-generated SlFDH knockout lines were more sensitive to Al stress and formate than wild-type plants. Formate failed to induce SlFDH expression in the tomato root apex, but NAD+ accumulated in response to Al stress. Co-expression network analysis and interaction analysis between genomic DNA and transcription factors (TFs) using PlantRegMap identified seven TFs that might regulate SlFDH expression. One of these TFs, SlSTOP1, positively regulated SlFDH expression by directly binding to its promoter, as demonstrated by a dual-luciferase reporter assay and electrophoretic mobility shift assay. The Al-induced expression of SlFDH was completely abolished in Slstop1 mutants, indicating that SlSTOP1 is a core regulator of SlFDH expression under Al stress. Taken together, our findings demonstrate that SlFDH plays a role in Al tolerance and reveal the transcriptional regulatory mechanism of SlFDH expression in response to Al stress in tomato.
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Solanum lycopersicum , Solanum lycopersicum/genética , NAD/metabolismo , Aluminio/toxicidad , Aluminio/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Formiatos/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Sulfate crystals are often criticized for their low birefringence. The small anisotropic SO4 group is becoming the biggest bottleneck hindering the application of sulfates in optical functional materials. In this study, we report a new method to significantly enhance the birefringence of sulfates. The title compound increases the birefringence recording of sulfates to 0.542@546 nm, which is significantly larger than that of the commercial birefringent crystal of TiO2 (0.306@546.1 nm). At the infrared wavelength, the birefringence of Hg4(Te2O5)(SO4) can be up to 0.400@1064 nm, which is also much larger than the infrared birefringent crystal of YVO4 (0.209@1064 nm). In addition, it also has a wide transparency range, high thermal stability, and excellent environmental stability, making it a potential birefringent material. Hg4(Te2O5)(SO4) features a novel two-dimensional layered structure composed of [Hg4(Te2O5)]2+ layers separated by isolated (SO4)2- tetrahedra. This compound was designed by introducing a highly selective cation in a tellurite sulfate system. The low valence low coordination cations connect with tellurite groups only, making the sulfate isolated in the structure. The steric repulsive action of the isolated SO4 tetrahedra may regulate the linear and lone pair groups arranged in a way that favors large birefringence. This method can be proven by theoretical calculations. PAWED studies showed that the large birefringence originated from the synergistic effect of (Hg2O2)2-, (Te2O5)2-, and (SO4)2- units, with a contribution ratio of 42.17, 37.92, and 19.88%, respectively. Our work breaks the limitation of low birefringence in sulfates and opens up new possibilities for their application as birefringent crystals.
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Cytochrome P450s are a large family of protein-encoding genes in plant genomes, many of which have not yet been comprehensively characterized. Here, a novel P450 gene, CYP82D47, was isolated and functionally characterized from cucumber (Cucumis sativus L.). Quantitative real-time reverse-transcription polymerase chain reaction analysis revealed that CYP82D47 expression was triggered by salicylic acid (SA) and ethephon (ETH). Expression analysis revealed a correlation between CYP82D47 transcript levels and plant defense responses against powdery mildew (PM) and Fusarium oxysporum f. sp. cucumerinum (Foc). Although no significant differences were observed in disease resistance between CYP82D47-RNAi and wild-type cucumber, overexpression (OE) of CYP82D47 enhanced PM and Foc resistance in cucumber. Furthermore, the expression levels of SA-related genes (PR1, PR2, PR4, and PR5) increased in CYP82D47-overexpressing plants 7 days post fungal inoculation. The levels of ETH-related genes (EIN3 and EBF2) were similarly upregulated. The observed enhanced resistance was associated with the upregulation of SA/ETH-signaling-dependent defense genes. These findings indicate the crucial role of CYP82D47 in pathogen defense in cucumber. CYP82D47-overexpressing cucumber plants exhibited heightened susceptibility to both diseases. The study results offer important insights that could aid in the development of disease-resistant cucumber cultivars and elucidate the molecular mechanisms associated with the functions of CYP82D47.
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Cucumis sativus , Fusarium , Compuestos Organofosforados , Cucumis sativus/genética , Regulación hacia Arriba , Resistencia a la Enfermedad/genética , Ácido Salicílico/farmacologíaRESUMEN
Background CT-defined visceral pleural invasion (VPI) is an important indicator of prognosis for non-small cell lung cancer (NSCLC). However, there is a lack of studies focused on small subpleural NSCLCs (≤30 mm). Purpose To identify CT features predictive of VPI in patients with subpleural NSCLCs 30 mm or smaller. Materials and Methods This study is a retrospective review of patients enrolled in the Initiative for Early Lung Cancer Research on Treatment (IELCART) at Mount Sinai Hospital between July 2014 and February 2023. Subpleural nodules 30 mm or smaller were classified into two groups: a pleural-attached group and a pleural-tag group. Preoperative CT features suggestive of VPI were evaluated for each group separately. Multivariable logistic regression analysis adjusted for sex, age, nodule size, and smoking status was used to determine predictive factors for VPI. Model performance was analyzed with the area under the receiver operating characteristic curve (AUC), and models were compared using Akaike information criterion (AIC). Results Of 379 patients with NSCLC with subpleural nodules, 37 had subsolid nodules and 342 had solid nodules. Eighty-eight patients (22%) had documented VPI, all in solid nodules. Of the 342 solid nodules (46% in male patients, 54% in female patients; median age, 71 years; IQR: 66, 76), 226 were pleural-attached nodules and 116 were pleural-tag nodules. VPI was more frequent for pleural-attached nodules than for pleural-tag nodules (31% [69 of 226] vs 16% [19 of 116], P = .005). For pleural-attached nodules, jellyfish sign (odds ratio [OR], 21.60; P < .001), pleural thickening (OR, 6.57; P < .001), and contact surface area (OR, 1.05; P = .01) independently predicted VPI. The jellyfish sign led to a better VPI prediction (AUC, 0.84; 95% CI: 0.78, 0.90). For pleural-tag nodules, multiple tags to different pleura surfaces enabled independent prediction of VPI (OR, 9.30; P = .001). Conclusions For patients with solid NSCLC (≤30 mm), CT predictors of VPI were the jellyfish sign, pleural thickening, contact surface area (pleural-attached nodules), and multiple tags to different pleura surfaces (pleural-tag nodules). © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Nishino in this issue.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Femenino , Masculino , Anciano , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico por imagen , Pleura/diagnóstico por imagen , Neoplasias Pulmonares/diagnóstico por imagen , Hospitales , Tomografía Computarizada por Rayos XRESUMEN
Background Pulmonary noncalcified nodules (NCNs) attached to the fissural or costal pleura with smooth margins and triangular or lentiform, oval, or semicircular (LOS) shapes at low-dose CT are recommended for annual follow-up instead of immediate workup. Purpose To determine whether management of mediastinal or diaphragmatic pleura-attached NCNs (M/DP-NCNs) with the same features as fissural or costal pleura-attached NCNs at low-dose CT can follow the same recommendations. Materials and Methods This retrospective study reviewed chest CT examinations in participants from two databases. Group A included 1451 participants who had lung cancer that was first present as a solid nodule with an average diameter of 3.0-30.0 mm. Group B included 345 consecutive participants from a lung cancer screening program who had at least one solid nodule with a diameter of 3.0-30.0 mm at baseline CT and underwent at least three follow-up CT examinations. Radiologists reviewed CT images to identify solid M/DP-NCNs, defined as nodules 0 mm in distance from the mediastinal or diaphragmatic pleura, and recorded average diameter, margin, and shape. General descriptive statistics were used. Results Among the 1451 participants with lung cancer in group A, 163 participants (median age, 68 years [IQR, 61.5-75.0 years]; 92 male participants) had 164 malignant M/DP-NCNs 3.0-30.0 mm in average diameter. None of the 164 malignant M/DP-NCNs had smooth margins and triangular or LOS shapes (upper limit of 95% CI of proportion, 0.02). Among the 345 consecutive screening participants in group B, 146 participants (median age, 65 years [IQR, 59-71 years]; 81 female participants) had 240 M/DP-NCNs with average diameter 3.0-30.0 mm. None of the M/DP-NCNs with smooth margins and triangular or LOS shapes were malignant after a median follow-up of 57.8 months (IQR, 46.3-68.1 months). Conclusion For solid M/DP-NCNs with smooth margins and triangular or LOS shapes at low-dose CT, the risk of lung cancer is extremely low, which supports the recommendation of Lung Imaging Reporting and Data System version 2022 for annual follow-up instead of immediate workup. © RSNA, 2024 See also the editorial by Goodman and Baruah in this issue.
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Neoplasias Pulmonares , Nódulos Pulmonares Múltiples , Femenino , Masculino , Humanos , Anciano , Detección Precoz del Cáncer , Neoplasias Pulmonares/diagnóstico por imagen , Pleura , Estudios Retrospectivos , Tomografía Computarizada por Rayos XRESUMEN
Carbon dots (CDs) have received considerable attention in many application areas owing to their unique optical properties and potential applications; however, the fluorescent mechanism is an obstacle to their applications. Herein, three-color emissive CDs are prepared from single o-phenylenediamine (oPD) by regulating the ratio of ethanol and dimethylformamide (DMF). Fluorescent mechanism of these CDs is proposed as molecular state fluorescence. Reaction intermediates are identified using liquid chromatrography-mass spectroscopy (LC-MS) and 1H nuclear magnetic resonance (NMR) spectra. 1H-Benzo[d]imidazole (BI), 2,3-diaminophenazine (DAP), and 5,14-dihydroquinoxalino[2,3-b] phenazine (DHQP) are proposed to be the fluorophores of blue, green, and red emissive CDs by comparing their optical properties. As per the LC-MS and 1H-NMR analysis, DHQP with red emission tends to form from DAP and oPD in pure ethanol. By adding DMF, BI formation is enhanced and DHQP formation is suppressed. The prepared CDs exhibit green emission with DAP. When the DMF amount is >50%, BI formation is considerably promoted, resulting in DAP formation being suppressed. BI with blue emission then turns into the fluorophore of CDs. This result provides us an improved understanding of the fluorescent mechanism of oPD-based CDs, which guides us in designing the structure and optical properties of CDs.
RESUMEN
Antigen epitope identification is a critical step in the vaccine development process and is a momentous cornerstone for the development of safe and efficient epitope vaccines. In particular, vaccine design is difficult when the function of the protein encoded by the pathogen is unknown. The genome of Tilapia lake virus (TiLV), an emerging virus from fish, encodes protein functions that have not been elucidated, resulting in a lag and uncertainty in vaccine development. Here, we propose a feasible strategy for emerging viral disease epitope vaccine development using TiLV. We determined the targets of specific antibodies in serum from a TiLV survivor by panning a Ph.D.-12 phage library, and we identified a mimotope, TYTTRMHITLPI, referred to as Pep3, which provided protection against TiLV after prime-boost vaccination; its immune protection rate was 57.6%. Based on amino acid sequence alignment and structure analysis of the target protein from TiLV, we further identified a protective antigenic site (399TYTTRNEDFLPT410) which is located on TiLV segment 1 (S1). The epitope vaccine with keyhole limpet hemocyanin (KLH-S1399-410) corresponding to the mimotope induced the tilapia to produce a durable and effective antibody response after immunization, and the antibody depletion test confirmed that the specific antibody against S1399-410 was necessary to neutralize TiLV. Surprisingly, the challenge studies in tilapia demonstrated that the epitope vaccine elicited a robust protective response against TiLV challenge, and the survival rate reached 81.8%. In conclusion, this study revealed a concept for screening antigen epitopes of emerging viral diseases, providing promising approaches for development and evaluation of protective epitope vaccines against viral diseases. IMPORTANCE Antigen epitope determination is an important cornerstone for developing efficient vaccines. In this study, we attempted to explore a novel approach for epitope discovery of TiLV, which is a new virus in fish. We investigated the immunogenicity and protective efficacy of all antigenic sites (mimotopes) identified in serum of primary TiLV survivors by using a Ph.D.-12 phage library. We also recognized and identified the natural epitope of TiLV by bioinformatics, evaluated the immunogenicity and protective effect of this antigenic site by immunization, and revealed 2 amino acid residues that play important roles in this epitope. Both Pep3 and S1399-410 (a natural epitope identified by Pep3) elicited antibody titers in tilapia, but S1399-410 was more prominent. Antibody depletion studies showed that anti-S1399-410-specific antibodies were essential for neutralizing TiLV. Our study demonstrated a model for combining experimental and computational screens to identify antigen epitopes, which is attractive for epitope-based vaccine development.
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Formación de Anticuerpos , Enfermedades de los Peces , Infecciones por Virus ARN , Tilapia , Vacunas Virales , Técnicas de Visualización de Superficie Celular , Simulación por Computador , Epítopos/inmunología , Vacunas Virales/inmunología , Formación de Anticuerpos/inmunología , Tilapia/virología , Línea Celular , Virus ARN/inmunología , Animales , Anticuerpos Antivirales/sangre , Inmunidad Humoral/inmunología , Infecciones por Virus ARN/prevención & control , Infecciones por Virus ARN/veterinaria , Infecciones por Virus ARN/virología , Enfermedades de los Peces/prevención & control , Enfermedades de los Peces/virologíaRESUMEN
Phase vortices exhibit significant applications and hold promising prospects across various scientific fields. However, while extensive attention has been devoted to the two-dimensional transverse plane of these vortices, their longitudinal properties have received comparatively limited exploration. Our study focuses on the longitudinal evolution of phase vortices, encompassing an investigation of variational topological charges and phase distributions. The investigation employs the rotationally interleaved multi-spiral, characterized by multiple identical spirals arranged in an azimuthally symmetric rotation, to modulate phase distributions by the variable spiral radius versus the azimuthal angle. Initially, we analyze the modulation effect theoretically, delving into propagation properties and vortex formations. Subsequently, through numerical simulations of vortices generated by both single and multi-spiral setups, we examine the longitudinal evolution of topological charges and phase distributions. The analyses reveal a step-wise reductant topological charges and a tortuous increasing spatial variations of phase singularities in transmission direction, with the dependency on both propagation distance and number of multi-spiral. The outcomes hold significant potential applications in optical communications and optical tweezers.