RESUMEN
The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.
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COVID-19/virología , Contención de Riesgos Biológicos/métodos , SARS-CoV-2 , Células A549 , Animales , Chlorocebus aethiops , Cricetinae , Prueba de Complementación Genética/métodos , Genoma Viral , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , ARN Viral , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Células Vero , Virulencia , Replicación ViralRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuing to evolve around the world, generating new variants that are of concern on the basis of their potential for altered transmissibility, pathogenicity, and coverage by vaccines and therapeutic agents1-5. Here we show that serum samples taken from twenty human volunteers, two or four weeks after their second dose of the BNT162b2 vaccine, neutralize engineered SARS-CoV-2 with a USA-WA1/2020 genetic background (a virus strain isolated in January 2020) and spike glycoproteins from the recently identified B.1.617.1, B.1.617.2, B.1.618 (all of which were first identified in India) or B.1.525 (first identified in Nigeria) lineages. Geometric mean plaque reduction neutralization titres against the variant viruses-particularly the B.1.617.1 variant-seemed to be lower than the titre against the USA-WA1/2020 virus, but all sera tested neutralized the variant viruses at titres of at least 1:40. The susceptibility of the variant strains to neutralization elicited by the BNT162b2 vaccine supports mass immunization as a central strategy to end the coronavirus disease 2019 (COVID-19) pandemic globally.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , COVID-19/virología , Pruebas de Neutralización , SARS-CoV-2/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacuna BNT162 , COVID-19/prevención & control , Vacunas contra la COVID-19/genética , Chlorocebus aethiops , Humanos , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Sintéticas/genética , Células Vero , Vacunas de ARNmRESUMEN
Resistance represents a major challenge for antibody-based therapy for COVID-191-4. Here we engineered an immunoglobulin M (IgM) neutralizing antibody (IgM-14) to overcome the resistance encountered by immunoglobulin G (IgG)-based therapeutics. IgM-14 is over 230-fold more potent than its parental IgG-14 in neutralizing SARS-CoV-2. IgM-14 potently neutralizes the resistant virus raised by its corresponding IgG-14, three variants of concern-B.1.1.7 (Alpha, which first emerged in the UK), P.1 (Gamma, which first emerged in Brazil) and B.1.351 (Beta, which first emerged in South Africa)-and 21 other receptor-binding domain mutants, many of which are resistant to the IgG antibodies that have been authorized for emergency use. Although engineering IgG into IgM enhances antibody potency in general, selection of an optimal epitope is critical for identifying the most effective IgM that can overcome resistance. In mice, a single intranasal dose of IgM-14 at 0.044 mg per kg body weight confers prophylactic efficacy and a single dose at 0.4 mg per kg confers therapeutic efficacy against SARS-CoV-2. IgM-14, but not IgG-14, also confers potent therapeutic protection against the P.1 and B.1.351 variants. IgM-14 exhibits desirable pharmacokinetics and safety profiles when administered intranasally in rodents. Our results show that intranasal administration of an engineered IgM can improve efficacy, reduce resistance and simplify the prophylactic and therapeutic treatment of COVID-19.
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COVID-19/prevención & control , COVID-19/virología , Inmunoglobulina M/administración & dosificación , Inmunoglobulina M/inmunología , SARS-CoV-2/clasificación , SARS-CoV-2/inmunología , Administración Intranasal , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/efectos adversos , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , COVID-19/inmunología , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Inmunoglobulina A/genética , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/efectos adversos , Inmunoglobulina M/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ingeniería de Proteínas , Receptores Virales/antagonistas & inhibidores , Receptores Virales/metabolismo , SARS-CoV-2/genética , Tratamiento Farmacológico de COVID-19RESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.
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COVID-19/transmisión , COVID-19/virología , Aptitud Genética , Mutación , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Cricetinae , Modelos Animales de Enfermedad , Humanos , Pulmón/virología , Masculino , Mesocricetus/virología , Modelos Biológicos , Mucosa Nasal/virología , Pruebas de Neutralización , Estabilidad Proteica , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Técnicas de Cultivo de Tejidos , Tráquea/virología , Carga Viral , Virión/química , Virión/patogenicidad , Virión/fisiología , Replicación Viral/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.
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COVID-19/virología , Furina/metabolismo , Mutación , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/inmunología , COVID-19/patología , COVID-19/fisiopatología , Línea Celular , Chlorocebus aethiops , Cricetinae , Femenino , Humanos , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/fisiopatología , Enfermedades Pulmonares/virología , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteolisis , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Replicación Viral/genéticaRESUMEN
TGF-ß signaling is a key player in tumor progression and immune evasion, and is associated with poor response to cancer immunotherapies. Here, we identified ubiquitin-specific peptidase 8 (USP8) as a metastasis enhancer and a highly active deubiquitinase in aggressive breast tumors. USP8 acts both as a cancer stemness-promoting factor and an activator of the TGF-ß/SMAD signaling pathway. USP8 directly deubiquitinates and stabilizes the type II TGF-ß receptor TßRII, leading to its increased expression in the plasma membrane and in tumor-derived extracellular vesicles (TEVs). Increased USP8 activity was observed in patients resistant to neoadjuvant chemotherapies. USP8 promotes TGF-ß/SMAD-induced epithelial-mesenchymal transition (EMT), invasion, and metastasis in tumor cells. USP8 expression also enables TßRII+ circulating extracellular vesicles (crEVs) to induce T cell exhaustion and chemoimmunotherapy resistance. Pharmacological inhibition of USP8 antagonizes TGF-ß/SMAD signaling, and reduces TßRII stability and the number of TßRII+ crEVs to prevent CD8+ T cell exhaustion and to reactivate anti-tumor immunity. Our findings not only reveal a novel mechanism whereby USP8 regulates the cancer microenvironment but also demonstrate the therapeutic advantages of engineering USP8 inhibitors to simultaneously suppress metastasis and improve the efficacy of cancer immunotherapy.
Asunto(s)
Vesículas Extracelulares , Neoplasias , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina Tiolesterasa , Linfocitos T CD8-positivos/metabolismo , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Vesículas Extracelulares/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral , Ubiquitina Tiolesterasa/metabolismoRESUMEN
BACKGROUND: The emergence of immune-escape variants of severe acute respiratory syndrome coronavirus 2 warrants the use of sequence-adapted vaccines to provide protection against coronavirus disease 2019. METHODS: In an ongoing phase 3 trial, adults older than 55 years who had previously received three 30-µg doses of the BNT162b2 vaccine were randomly assigned to receive 30 µg or 60 µg of BNT162b2, 30 µg or 60 µg of monovalent B.1.1.529 (omicron) BA.1-adapted BNT162b2 (monovalent BA.1), or 30 µg (15 µg of BNT162b2 + 15 µg of monovalent BA.1) or 60 µg (30 µg of BNT162b2 + 30 µg of monovalent BA.1) of BA.1-adapted BNT162b2 (bivalent BA.1). Primary objectives were to determine superiority (with respect to 50% neutralizing titer [NT50] against BA.1) and noninferiority (with respect to seroresponse) of the BA.1-adapted vaccines to BNT162b2 (30 µg). A secondary objective was to determine noninferiority of bivalent BA.1 to BNT162b2 (30 µg) with respect to neutralizing activity against the ancestral strain. Exploratory analyses assessed immune responses against omicron BA.4, BA.5, and BA.2.75 subvariants. RESULTS: A total of 1846 participants underwent randomization. At 1 month after vaccination, bivalent BA.1 (30 µg and 60 µg) and monovalent BA.1 (60 µg) showed neutralizing activity against BA.1 superior to that of BNT162b2 (30 µg), with NT50 geometric mean ratios (GMRs) of 1.56 (95% confidence interval [CI], 1.17 to 2.08), 1.97 (95% CI, 1.45 to 2.68), and 3.15 (95% CI, 2.38 to 4.16), respectively. Bivalent BA.1 (both doses) and monovalent BA.1 (60 µg) were also noninferior to BNT162b2 (30 µg) with respect to seroresponse against BA.1; between-group differences ranged from 10.9 to 29.1 percentage points. Bivalent BA.1 (either dose) was noninferior to BNT162b2 (30 µg) with respect to neutralizing activity against the ancestral strain, with NT50 GMRs of 0.99 (95% CI, 0.82 to 1.20) and 1.30 (95% CI, 1.07 to 1.58), respectively. BA.4-BA.5 and BA.2.75 neutralizing titers were numerically higher with 30-µg bivalent BA.1 than with 30-µg BNT162b2. The safety profile of either dose of monovalent or bivalent BA.1 was similar to that of BNT162b2 (30 µg). Adverse events were more common in the 30-µg monovalent-BA.1 (8.5%) and 60-µg bivalent-BA.1 (10.4%) groups than in the other groups (3.6 to 6.6%). CONCLUSIONS: The candidate monovalent or bivalent omicron BA.1-adapted vaccines had a safety profile similar to that of BNT162b2 (30 µg), induced substantial neutralizing responses against ancestral and omicron BA.1 strains, and, to a lesser extent, neutralized BA.4, BA.5, and BA.2.75 strains. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04955626.).
Asunto(s)
Vacuna BNT162 , COVID-19 , SARS-CoV-2 , Vacunas Combinadas , Humanos , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacuna BNT162/efectos adversos , Vacuna BNT162/inmunología , Vacuna BNT162/uso terapéutico , COVID-19/genética , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Vacunación , Vacunas Combinadas/uso terapéutico , Persona de Mediana EdadRESUMEN
BACKGROUND: Safe and effective vaccines against coronavirus disease 2019 (Covid-19) are urgently needed in young children. METHODS: We conducted a phase 1 dose-finding study and are conducting an ongoing phase 2-3 safety, immunogenicity, and efficacy trial of the BNT162b2 vaccine in healthy children 6 months to 11 years of age. We present results for children 6 months to less than 2 years of age and those 2 to 4 years of age through the data-cutoff dates (April 29, 2022, for safety and immunogenicity and June 17, 2022, for efficacy). In the phase 2-3 trial, participants were randomly assigned (in a 2:1 ratio) to receive two 3-µg doses of BNT162b2 or placebo. On the basis of preliminary immunogenicity results, a third 3-µg dose (≥8 weeks after dose 2) was administered starting in January 2022, which coincided with the emergence of the B.1.1.529 (omicron) variant. Immune responses at 1 month after doses 2 and 3 in children 6 months to less than 2 years of age and those 2 to 4 years of age were immunologically bridged to responses after dose 2 in persons 16 to 25 years of age who received 30 µg of BNT162b2 in the pivotal trial. RESULTS: During the phase 1 dose-finding study, two doses of BNT162b2 were administered 21 days apart to 16 children 6 months to less than 2 years of age (3-µg dose) and 48 children 2 to 4 years of age (3-µg or 10-µg dose). The 3-µg dose level was selected for the phase 2-3 trial; 1178 children 6 months to less than 2 years of age and 1835 children 2 to 4 years of age received BNT162b2, and 598 and 915, respectively, received placebo. Immunobridging success criteria for the geometric mean ratio and seroresponse at 1 month after dose 3 were met in both age groups. BNT162b2 reactogenicity events were mostly mild to moderate, with no grade 4 events. Low, similar incidences of fever were reported after receipt of BNT162b2 (7% among children 6 months to <2 years of age and 5% among those 2 to 4 years of age) and placebo (6 to 7% among children 6 months to <2 years of age and 4 to 5% among those 2 to 4 years of age). The observed overall vaccine efficacy against symptomatic Covid-19 in children 6 months to 4 years of age was 73.2% (95% confidence interval, 43.8 to 87.6) from 7 days after dose 3 (on the basis of 34 cases). CONCLUSIONS: A three-dose primary series of 3-µg BNT162b2 was safe, immunogenic, and efficacious in children 6 months to 4 years of age. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04816643.).
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Vacuna BNT162 , COVID-19 , Adolescente , Niño , Preescolar , Humanos , Lactante , Adulto Joven , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Vacuna BNT162/administración & dosificación , Vacuna BNT162/efectos adversos , Vacuna BNT162/inmunología , Vacuna BNT162/uso terapéutico , COVID-19/sangre , COVID-19/inmunología , COVID-19/prevención & control , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/efectos adversos , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/uso terapéutico , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Vacunas/efectos adversos , Vacunas/uso terapéutico , Inmunogenicidad Vacunal , Resultado del Tratamiento , Eficacia de las VacunasRESUMEN
Post-transcriptional regulation by small RNAs and post-translational modifications (PTM) such as lysine acetylation play fundamental roles in physiological circuits, offering rapid responses to environmental signals with low energy consumption. Yet, the interplay between these regulatory systems remains underexplored. Here, we unveil the cross-talk between sRNAs and lysine acetylation in Streptococcus mutans, a primary cariogenic pathogen known for its potent acidogenic virulence. Through systematic overexpression of sRNAs in S. mutans, we identified sRNA SmsR1 as a critical player in modulating acidogenicity, a key cariogenic virulence feature in S. mutans. Furthermore, combined with the analysis of predicted target mRNA and transcriptome results, potential target genes were identified and experimentally verified. A direct interaction between SmsR1 and 5'-UTR region of pdhC gene was determined by in vitro binding assays. Importantly, we found that overexpression of SmsR1 reduced the expression of pdhC mRNA and increased the intracellular concentration of acetyl-CoA, resulting in global changes in protein acetylation levels. This was verified by acetyl-proteomics in S. mutans, along with an increase in acetylation level and decreased activity of LDH. Our study unravels a novel regulatory paradigm where sRNA bridges post-transcriptional regulation with post-translational modification, underscoring bacterial adeptness in fine-tuning responses to environmental stress.
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Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Procesamiento Proteico-Postraduccional , Streptococcus mutans , Animales , Acetilación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Caries Dental/microbiología , Caries Dental/metabolismo , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , ARN Pequeño no Traducido/metabolismo , ARN Pequeño no Traducido/genética , Streptococcus mutans/metabolismo , Streptococcus mutans/genética , Streptococcus mutans/patogenicidad , Virulencia , Femenino , RatasRESUMEN
Large-scale omics studies have generated a wealth of mass spectrometry-based proteomics data, which provide additional insights into disease biology spanning genomic boundaries. However, there is a notable lack of web-based analysis and visualization tools that facilitate the reutilization of these data. Given this challenge, we present iProPhos, a user-friendly web server to deliver interactive and customizable functionalities. iProPhos incorporates a large number of samples, including 1444 tumor samples and 746 normal samples across 12 cancer types, sourced from the Clinical Proteomic Tumor Analysis Consortium. Additionally, users can also upload their own proteomics/phosphoproteomics data for analysis and visualization. In iProPhos, users can perform profiling plotting and differential expression, patient survival, clinical feature-related, and correlation analyses, including protein-protein, mRNA-protein, and kinase-substrate correlations. Furthermore, functional enrichment, protein-protein interaction network, and kinase-substrate enrichment analyses are accessible. iProPhos displays the analytical results in interactive figures and tables with various selectable parameters. It is freely accessible at http://longlab-zju.cn/iProPhos without login requirement. We present two case studies to demonstrate that iProPhos can identify potential drug targets and upstream kinases contributing to site-specific phosphorylation. Ultimately, iProPhos allows end-users to leverage the value of big data in cancer proteomics more effectively and accelerates the discovery of novel therapeutic targets.
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Neoplasias , Proteoma , Humanos , Proteómica/métodos , Programas Informáticos , Neoplasias/genética , InternetRESUMEN
Selective autophagy mediates specific degradation of unwanted cytoplasmic components to maintain cellular homeostasis. The suppressor of gene silencing 3 (SGS3) and RNA-dependent RNA polymerase 6 (RDR6)-formed bodies (SGS3/RDR6 bodies) are essential for siRNA amplification in planta. However, whether autophagy receptors regulate selective turnover of SGS3/RDR6 bodies is unknown. By analyzing the transcriptomic response to virus infection in Arabidopsis, we identified a virus-induced small peptide 1 (VISP1) composed of 71 amino acids, which harbor a ubiquitin-interacting motif that mediates interaction with autophagy-related protein 8. Overexpression of VISP1 induced selective autophagy and compromised antiviral immunity by inhibiting SGS3/RDR6-dependent viral siRNA amplification, whereas visp1 mutants exhibited opposite effects. Biochemistry assays demonstrate that VISP1 interacted with SGS3 and mediated autophagic degradation of SGS3/RDR6 bodies. Further analyses revealed that overexpression of VISP1, mimicking the sgs3 mutant, impaired biogenesis of endogenous trans-acting siRNAs and up-regulated their targets. Collectively, we propose that VISP1 is a small peptide receptor functioning in the crosstalk between selective autophagy and RNA silencing.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/inmunología , Péptidos/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Arabidopsis/metabolismo , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Autofagosomas/fisiología , Autofagia/fisiología , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , Péptidos/metabolismo , Inmunidad de la Planta , Plantas Modificadas Genéticamente , ARN Interferente Pequeño , ARN Polimerasa Dependiente del ARN/genética , Nicotiana/genéticaRESUMEN
BACKGROUND: In diabetic retinopathy (DR), hypoxia-inducible factor (HIF-1α) induces oxidative stress by upregulating glycolysis. This process leads to neurodegeneration, particularly photoreceptor cell damage, which further contributes to retinal microvascular deterioration. Further, the regulation of Wnt-inhibitory factor 1 (WIF1), a secreted Wnt signaling antagonist, has not been fully characterized in neurodegenerative eye diseases. We aimed to explore the impact of WIF1 on photoreceptor function within the context of DR. METHOD: Twelve-week-old C57BL/KsJ-db/db mice were intravitreally injected with WIF1 overexpression lentivirus. After 4 weeks, optical coherence tomography (OCT), transmission electron microscopy (TEM), H&E staining, and electroretinography (ERG) were used to assess the retinal tissue and function. The potential mechanism of action of WIF1 in photoreceptor cells was explored using single-cell RNA sequencing. Under high-glucose conditions, 661 W cells were used as an in vitro DR model. WIF1-mediated signaling pathway components were assessed using quantitative real-time PCR, immunostaining, and western blotting. RESULT: Typical diabetic manifestations were observed in db/db mice. Notably, the expression of WIF1 was decreased at the mRNA and protein levels. These pathological manifestations and visual function improved after WIF1 overexpression in db/db mice. TEM demonstrated that WIF1 restored damaged mitochondria, the Golgi apparatus, and photoreceptor outer segments. Moreover, ERG indicated the recovery of a-wave potential amplitude. Single-cell RNA sequencing and in vitro experiments suggested that WIF1 overexpression prevented the expression of glycolytic enzymes and lactate production by inhibiting the canonical Wnt signaling pathway, HIF-1α, and Glut1, thereby reducing retinal and cellular reactive oxygen species levels and maintaining 661 W cell viability. CONCLUSIONS: WIF1 exerts an inhibitory effect on the Wnt/ß-catenin-HIF-1α-Glut1 glycolytic pathway, thereby alleviating oxidative stress levels and mitigating pathological structural characteristics in retinal photoreceptor cells. This mechanism helps preserve the function of photoreceptor cells in DR and indicates that WIF1 holds promise as a potential therapeutic candidate for DR and other neurodegenerative ocular disorders.
Asunto(s)
Diabetes Mellitus , Retinopatía Diabética , Animales , Ratones , Transportador de Glucosa de Tipo 1 , Ratones Endogámicos C57BL , Células Fotorreceptoras , RetinaRESUMEN
Dental caries is the most common chronic infectious disease around the world and disproportionately affects the marginalized socioeconomic group. Streptococcus mutans, considered a primary etiological agent of caries, depends on the coordinated physiological response to tolerate the oxidative stress generated by commensal species within dental plaque, which is a critical aspect of its pathogenicity. Here, we identified and characterized a novel tetracycline repressor family regulator, SMU_1361c, which appears to be acquired by the bacteria via horizontal gene transfer. Surprisingly, smu_1361c functions as a negative transcriptional regulator to regulate gene expression outside its operon and is involved in the oxidative stress response of S. mutans. The smu_1361c overexpression strain UA159/pDL278-1361c was more susceptible to oxidative stress and less competitive against hydrogen peroxide generated by commensal species Streptococcus gordonii and Streptococcus sanguinis. Transcriptomics analysis revealed that smu_1361c overexpression resulted in the significant downregulation of 22 genes, mainly belonging to three gene clusters responsible for the oxidative stress response. The conversed DNA binding motif of SMU_1361c was determined by electrophoretic mobility shift and DNase I footprinting assay with purified SMU_1361c protein; therefore, smu_1361c is directly involved in gene transcription related to the oxidative stress response. Crucially, our finding provides a new understanding of how S. mutans deals with the oxidative stress that is required for pathogenesis and will facilitate the development of new and improved therapeutic approaches for dental caries.IMPORTANCEStreptococcus mutans is the major organism associated with the development of dental caries, which globally is the most common chronic disease. To persist and survive in biofilms, S. mutans must compete with commensal species that occupy the same ecological niche. Here, we uncover a novel molecular mechanism of how tetracycline repressor family regulator smu_1361c is involved in the oxidative stress response through transcriptomics analysis, electrophoretic mobility shift assay, and DNase I footprinting assay. Furthermore, we demonstrated that smu_1361c mediates S. mutans sensitivity to oxidative stress and competitiveness with commensal streptococci. Therefore, this study has revealed a previously unknown regulation between smu_1361c and genes outside its operon and demonstrated the importance of smu_1361c in the oxidative stress response and the fitness of S. mutans within the plaque biofilms, which can be exploited as a new therapy to modulate ecological homeostasis and prevent dental caries.
Asunto(s)
Caries Dental , Streptococcus mutans , Humanos , Streptococcus mutans/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Estrés Oxidativo , Tetraciclinas , Desoxirribonucleasa I/metabolismoRESUMEN
Dental caries, as a biofilm-related disease, is closely linked to dysbiosis in microbial ecology within dental biofilms. Beyond its impact on oral health, bacteria within the oral cavity pose systemic health risks by potentially entering the bloodstream, thereby increasing susceptibility to bacterial endocarditis, among other related diseases. Streptococcus mutans, a principal cariogenic bacterium, possesses virulence factors crucial to the pathogenesis of dental caries. Its ability to adhere to tooth surfaces, produce glucans for biofilm formation, and metabolize sugars into lactic acid contributes to enamel demineralization and the initiation of carious lesions. Its aciduricity and ability to produce bacteriocins enable a competitive advantage, allowing it to thrive in acidic environments and dominate in changing oral microenvironments. In contrast, commensal streptococci, such as Streptococcus sanguinis, Streptococcus gordonii, and Streptococcus salivarius, act as primary colonizers and compete with S. mutans for adherence sites and nutrients during biofilm formation. This competition involves the production of alkali, peroxides, and antibacterial substances, thereby inhibiting S. mutans growth and maintaining microbial balance. This dynamic interaction influences the balance of oral microbiota, with disruptions leading to shifts in microbial composition that are marked by rapid increases in S. mutans abundance, contributing to the onset of dental caries. Thus, understanding the dynamic interactions between commensal and pathogenic bacteria in oral microecology is important for developing effective strategies to promote oral health and prevent dental caries. This review highlights the roles and competitive interactions of commensal bacteria and S. mutans in oral microecology, emphasizing the importance of maintaining oral microbial balance for health, and discusses the pathological implications of perturbations in this balance.
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Cu(II)-catalyzed peracetic acid (PAA) processes have shown significant potential to remove contaminants in water treatment. Nevertheless, the role of coexistent H2O2 in the transformation from Cu(II) to Cu(I) remained contentious. Herein, with the Cu(II)/PAA process as an example, the respective roles of PAA and H2O2 on the Cu(II)/Cu(I) cycling were comprehensively investigated over the pH range of 7.0-10.5. Contrary to previous studies, it was surprisingly found that the coexistent deprotonated H2O2 (HO2-), instead of PAA, was crucial for accelerating the transformation from Cu(II) to Cu(I) (kHO2-/Cu(II) = (0.17-1) × 106 M-1 s-1, kPAA/Cu(II) < 2.33 ± 0.3 M-1 s-1). Subsequently, the formed Cu(I) preferentially reacted with PAA (kPAA/Cu(I) = (5.84 ± 0.17) × 102 M-1 s-1), rather than H2O2 (kH2O2/Cu(I) = (5.00 ± 0.2) × 101 M-1 s-1), generating reactive species to oxidize organic contaminants. With naproxen as the target pollutant, the proposed synergistic role of H2O2 and PAA was found to be highly dependent on the solution pH with weakly alkaline conditions being more conducive to naproxen degradation. Overall, this study systematically investigated the overlooked but crucial role of coexistent H2O2 in the Cu(II)/PAA process, which might provide valuable insights for better understanding the underlying mechanism in Cu-catalyzed PAA processes.
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Acute lung injury (ALI) is associated with the leukocyte infiltration and inflammation. Previous studies have shown that miR-146a is a valid regulator of the macrophage polarization in vitro inflammatory model. However, it is unclear whether miR-146a plays a protective role in ALI via modulating macrophage inflammation. To explore the potential therapeutic effect mechanism of miR-146a on ALI. We analyzed the expression of miR-146a in acute injured lung tissues and differentiated macrophage. Lipopolysaccharide (LPS) and interleukin-4 (IL-4) were employed in provoking the macrophage to polarization. We used miR-146a mimics to improve the overexpression of miR-146a and investigated the effect of increased miR-146a on LPS-induced ALI mice via the target of macrophage polarization. We showed that the expression of miR-146a markedly decreased in injured lung tissue and type M1 macrophage, while increased miR-146a expression exhibited in type M2 macrophage. Moreover, overexpression of miR-146a in LPS-induced macrophage reversed inflammatory M1 phenotype to anti-inflammatory M2 phenotype and mitigated inflammatory level via inhibiting Notch 1 signaling pathway. Hence, inflammation, infiltration, integrity of capillary barrier, and histology in ALI model were corrected after miR-146a overexpression treatment. These results suggested that miR-146a promotes type M2 macrophage polarization via restraining Notch 1 signaling pathway. Overexpression of miR-146a prevents inflammation damage and ameliorates lung damage after LPS induction. Therefore, miR-146a may serve as a promising target for the therapy of ALI in the future.
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Lesión Pulmonar Aguda , MicroARNs , Receptor Notch1 , Transducción de Señal , Animales , Ratones , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/metabolismo , MicroARNs/metabolismo , Receptor Notch1/metabolismoRESUMEN
BACKGROUND: Coronavirus disease 2019 (COVID-19) has had a global social and economic impact. An easy assessment procedure to handily identify the mortality risk of inpatients is urgently needed in clinical practice. Therefore, the aim of this study was to develop a simple nomogram model to categorize patients who might have a poor short-term outcome. METHODS: A retrospective cohort study of 189 COVID-19 patients was performed at Shanghai Ren Ji Hospital from December 12, 2022 to February 28, 2023. Chest radiography and biomarkers, including KL-6 were assessed. Risk factors of 28-day mortality were selected by a Cox regression model. A nomogram was developed based on selected variables by SMOTE strategy. The predictive performance of the derived nomogram was evaluated by calibration curve. RESULTS: In total, 173 patients were enrolled in this study. The 28-day mortality event occurred in 41 inpatients (23.7%). Serum KL-6 and radiological severity grade (RSG) were selected as the final risk factors. A nomogram model was developed based on KL-6 and RSG. The calibration curve suggested that the nomogram model might have potential clinical value. The AUCs for serum KL-6, RSG, and the combined score in the development group and validation group were 0.885 (95% CI: 0.804-0.952), 0.818 (95% CI: 0.711-0.899), 0.868 (95% CI: 0.776-0.942) and 0.932 (95% CI: 0.862-0.997), respectively. CONCLUSIONS: Our results suggested that the nomogram based on KL-6 and RSG might be a potential method for evaluating 28-day mortality in COVID-19 patients. A high combined score might indicate a poor outcome in COVID-19 patients with pneumonia.
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COVID-19 , Humanos , Estudios Retrospectivos , SARS-CoV-2 , China/epidemiología , RadiografíaRESUMEN
Resistance to epidermal growth factor receptor (EGFR) inhibitors, from the first-generation erlotinib to the third generation osimertinib, is a clinical challenge in the treatment of patients with EGFR-mutant lung adenocarcinoma. Our previous work found that a novel allosteric inhibitor of phosphoglycerate mutase 1 (PGAM1), HKB99, restrains erlotinib resistance in lung adenocarcinoma cells. However, the role of HKB99 in osimertinib resistance and its underlying molecular mechanism remains to be elucidated. Herein, we found that IL-6/JAK2/STAT3 signaling pathway is aberrantly activated in both erlotinib and osimertinib resistant cells. Importantly, HKB99 significantly blocks the interaction of PGAM1 with JAK2 and STAT3 via the allosteric sites of PGAM1, which leads to inactivation of JAK2/STAT3 and thereby disrupts IL-6/JAK2/STAT3 signaling pathway. Consequently, HKB99 remarkably restores EGFR inhibitor sensitivity and exerts synergistic tumoricidal effect. Additionally, HKB99 alone or in combination with osimertinib down-regulated the level of p-STAT3 in xenograft tumor models. Collectively, this study identifies PGAM1 as a key regulator in IL-6/JAK2/STAT3 axis in the development of resistance to EGFR inhibitors, which could serve as a therapeutic target in lung adenocarcinoma with acquired resistance to EGFR inhibitors.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Humanos , Clorhidrato de Erlotinib/farmacología , Clorhidrato de Erlotinib/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Interleucina-6/genética , Interleucina-6/farmacología , Interleucina-6/uso terapéutico , Fosfoglicerato Mutasa/metabolismo , Fosfoglicerato Mutasa/farmacología , Resistencia a Antineoplásicos , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Receptores ErbB , Transducción de Señal , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Mutación , Línea Celular Tumoral , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Janus Quinasa 2/farmacologíaRESUMEN
BACKGROUND: Projection Domain Decomposition (PDD) is a dual energy reconstruction method which implements the decomposition process before image reconstruction. The advantage of PDD is that it can alleviate beam hardening artifacts and metal artifacts effectively as energy spectra estimation is considered in PDD. However, noise amplification occurs during the decomposition process, which significantly impacts the accuracy of effective atomic number and electron density. Therefore, effective noise reduction techniques are required in PDD. OBJECTIVE: This study aims to develop a new algorithm capable of minimizing noise while simultaneously preserving edges and fine details. METHODS: In this study, a denoising algorithm based on low rank and similarity-based regularization (LRSBR) is presented. This algorithm incorporates the low-rank characteristic of tensors into similarity-based regularization (SBR) framework. This method effectively addresses the issue of instability in edge pixels within the SBR algorithm and enhances the structural consistency of dual-energy images. RESULTS: A series of simulation and practical experiments were conducted on a dual-layer dual-energy CT system. Experiments demonstrate that the proposed method outperforms exiting noise removal methods in Peak Signal-to-noise Ratio (PSNR), Root Mean Square Error (RMSE), and Structural Similarity (SSIM). Meanwhile, there has been a notable enhancement in the visual quality of CT images. CONCLUSIONS: The proposed algorithm has a significantly improved noise reduction compared to other competing approach in dual-energy CT. Meanwhile, the LRSBR method exhibits outstanding performance in preserving edges and fine structures, making it practical for PDD applications.
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Algoritmos , Procesamiento de Imagen Asistido por Computador , Fantasmas de Imagen , Relación Señal-Ruido , Tomografía Computarizada por Rayos X , Procesamiento de Imagen Asistido por Computador/métodos , Tomografía Computarizada por Rayos X/métodos , Artefactos , Simulación por ComputadorRESUMEN
BACKGROUND: It is unclear whether gut microbiota (GM) affects the risk of optic neuritis (ON) through the "gut-brain" axis and the "gut-retina" axis. To examine the causal relationship between GM and ON, we conducted Mendelian randomization (MR) study. METHODS: Up to 18,340 samples of 24 population-based cohorts were included in genome-wide association study (GWAS) of 196 GM taxa. ON outcomes were selected from the FinnGen GWAS (951 ON cases and 307,092 controls). In addition, the GWAS based on UK Biobank (UKB) (105 ON cases and 456,243 controls) was used for further exploration. Inverse variance weighted (IVW) was carried out to estimate their effects on ON risk and the MR assumptions were evaluated in sensitivity analyses. RESULTS: Among the 196 GM taxa, the IVW results confirmed that Family -Peptococcaceae (P = 2.17 × 10-3), Genus- Hungatella (P = 4.57 × 10-3) and genus-Eubacterium_rectale_group (P = 0.02) were correlated with the risk of ON based on Finngen GWAS. Based on data from UKB, Genus- Eubacterium_hallii_group (P = 1.50 × 10-3) and Genus- Ruminococcaceae_UCG_002 (P = 0.02) were correlated with the risk of ON. At the phylum, class and order levels, no GM taxa were causally related to ON (P > 0.05). Heterogeneity (P > 0.05) and pleiotropy (P > 0.05) analysis confirmed the robustness of the MR results. CONCLUSION: Our MR findings support the causal effect of specific GM taxa on ON. GM may affect the risk of ON through the "gut-brain" axis and the "gut-retina" axis. However, further research is needed to confirm the relevant mechanism of the relationship between GM and ON.