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1.
Food Chem ; 366: 130595, 2022 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-34298393

RESUMEN

Herein, a surface-enhanced Raman scattering (SERS)-integrated LFS platform was developed for rapid and simultaneous screening of multiple genetically modified organism (GMO) components (promoter, codon, and terminator) in soybean. Research demonstrated that, on the same test line (T line) of single LFS, three different GMP components can be well distinguished with the help of three SERS nano tags. Good linear correlations between SERS signal and concentration of each GMO component were also obtained for quantitative analysis. Of greater importance, whether these multiple analytes coexisted or not, varied in the same concentration trend or not, these multiple GMP components can be rapidly (15 min) and accurately screened with satisfied sensitivity and specificity by decoding the signals on the same T line. We envision that this decoding platform can further improve the potential of LFS and SERS for practical applications and provide a promising alternative for multiple screening of GMO identification in food.


Asunto(s)
Espectrometría Raman , Organismos Modificados Genéticamente , Sensibilidad y Especificidad
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(5): 1369-1374, 2021 Oct.
Artículo en Chino | MEDLINE | ID: mdl-34627412

RESUMEN

OBJECTIVE: To establish a leukemia mouse model induced by transplantation of hematopoietic cells from mixed lineage leukemia (MLL)-AF9 transgenic mice so as to provide the basis for the mechanism research and drug screening of acute myeloid leukemia (AML). METHODS: MLL-AF9 knock-in mice were bred and identified. When the mice developed leukemia, white blood cell (WBC) count in peripheral blood, flow cytometry and morphology method were analyzed to identify the disease. When the WBC count in peripheral blood was more than 100×109/L, bone marrow cells and spleen cells were collected and cryopresevated. After resuscitation, the cells were injected into 4.5 Gy irradiated wild C57BL/6J mice through the tail vein to develop MLL-AF9 leukemia mouse model. Finally, the therapeutic effect was evaluated by positive drug on the model. RESULTS: The natural onset times of leukemia on MLL-AF9 knock-in mice were 22-28 weeks. The spleens of the transgenic mice enlarged and the bone marrow showed the immature forms of myeloid leukemia cells. Both the bone marrow and spleen cells highly expressed myeloid markers, CD11b and Gr-1. At least 0.5×106 bone marrow cells and 2.5×106 spleen cells could induce leukemia in all recipient mice, and the median survival times of mice were 20 days and 36 days, respectively. Experimental treatment was carried out on the leukemia mouse model transplanted with MLL-AF9 spleen cells, and it was found that the traditional chemotherapy drug cytarabine could delay the onset of leukemia and prolong the survival time of the mouse model. CONCLUSION: The leukemia model of hematopoietic cell transplantation based on MLL-AF9 transgenic mice is successfully established, which can be used for the study of the pathogenesis and evaluation of therapeutic effect of AML.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Fusión Oncogénica
3.
Transl Psychiatry ; 11(1): 507, 2021 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-34611141

RESUMEN

Population-based studies reveal that apolipoprotein E (APOE) ε4 gene allele is closely associated with late-life depression (LLD). However, its exact role and underlying mechanism remain obscure. The current study found that aged apoE4-targeted replacement (TR) mice displayed obvious depression-like behavior when compared with age-matched apoE3-TR mice. Furthermore, apoE4 increased stress-induced depression-like behaviors, accompanied by declines in the hippocampal 5-HT (1A) radioligand [18F] MPPF uptake evidenced by positron emission tomography (PET). In [18F]-fluorodeoxyglucose PET ([18F]-FDG PET) analyses, the FDG uptake in the prefrontal cortex, temporal cortex and hippocampus of apoE4-TR mice significantly declined when compared with that of apoE3-TR mice after acute stress. Further biochemical analysis revealed that ATP levels in the prefrontal cortex of apoE4-TR mice decreased during aging or stress process and ATP supplementation effectively rescued the depression-like behaviors of elderly apoE4-TR mice. In primary cultured astrocytes from the cortex of apoE-TR mice, apoE4, when compared with apoE3, obviously decreased the mitochondrial membrane potential, mitochondrial respiration, and glycolysis in a culture time-dependent manner. Our findings highlight that apoE4 is a potential risk factor of depression in elderly population by impairing the glucose metabolism, reducing ATP level, and damaging mitochondrial functions in astrocytes, which indicates that in clinical settings ATP supplementation may be effective for elderly depression patients with apoE4 carrier.


Asunto(s)
Apolipoproteína E4 , Depresión , Adenosina Trifosfato , Anciano , Animales , Apolipoproteína E4/genética , Depresión/genética , Genotipo , Humanos , Ratones , Ratones Transgénicos
4.
Nat Commun ; 12(1): 5736, 2021 09 30.
Artículo en Inglés | MEDLINE | ID: mdl-34593792

RESUMEN

Despite the emerging importance of reactive electrophilic drugs, deconvolution of their principal targets remains difficult. The lack of genetic tractability/interventions and reliance on secondary validation using other non-specific compounds frequently complicate the earmarking of individual binders as functionally- or phenotypically-sufficient pathway regulators. Using a redox-targeting approach to interrogate how on-target binding of pleiotropic electrophiles translates to a phenotypic output in vivo, we here systematically track the molecular components attributable to innate immune cell toxicity of the electrophilic-drug dimethyl fumarate (Tecfidera®). In a process largely independent of canonical Keap1/Nrf2-signaling, Keap1-specific modification triggers mitochondrial-targeted neutrophil/macrophage apoptosis. On-target Keap1-ligand-engagement is accompanied by dissociation of Wdr1 from Keap1 and subsequent coordination with cofilin, intercepting Bax. This phagocytic-specific cell-killing program is recapitulated by whole-animal administration of dimethyl fumarate, where individual depletions of the players identified above robustly suppress apoptosis.


Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Dimetilfumarato/farmacología , Inmunosupresores/farmacología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Animales Modificados Genéticamente , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Embrión de Mamíferos , Embrión no Mamífero , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Inmunidad Innata/efectos de los fármacos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Pez Cebra
5.
Nat Commun ; 12(1): 5740, 2021 09 30.
Artículo en Inglés | MEDLINE | ID: mdl-34593806

RESUMEN

NG2 glia, also known as oligodendrocyte precursor cells (OPCs), play an important role in proliferation and give rise to myelinating oligodendrocytes during early brain development. In contrast to other glial cell types, the most intriguing aspect of NG2 glia is their ability to directly sense synaptic inputs from neurons. However, whether this synaptic interaction is bidirectional or unidirectional, or its physiological relevance has not yet been clarified. Here, we report that NG2 glia form synaptic complexes with hippocampal interneurons and that selective photostimulation of NG2 glia (expressing channelrhodopsin-2) functionally drives GABA release and enhances inhibitory synaptic transmission onto proximal interneurons in a microcircuit. The mechanism involves GAD67 biosynthesis and VAMP-2 containing vesicular exocytosis. Further, behavioral assays demonstrate that NG2 glia photoactivation triggers anxiety-like behavior in vivo and contributes to chronic social defeat stress.


Asunto(s)
Ansiedad/psicología , Hipocampo/patología , Células Precursoras de Oligodendrocitos/metabolismo , Estrés Psicológico/complicaciones , Ácido gamma-Aminobutírico/metabolismo , Animales , Ansiedad/etiología , Ansiedad/patología , Diferenciación Celular , Modelos Animales de Enfermedad , Exocitosis , Glutamato Descarboxilasa/biosíntesis , Hipocampo/citología , Humanos , Interneuronas/patología , Masculino , Ratones , Ratones Transgénicos , Técnicas de Placa-Clamp , Derrota Social , Estrés Psicológico/patología , Estrés Psicológico/psicología , Sinapsis/patología , Transmisión Sináptica/fisiología , Proteína 2 de Membrana Asociada a Vesículas/metabolismo
6.
Nat Commun ; 12(1): 5763, 2021 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-34599158

RESUMEN

Signals from the central circadian pacemaker, the suprachiasmatic nucleus (SCN), must be decoded to generate daily rhythms in hormone release. Here, we hypothesized that the SCN entrains rhythms in the paraventricular nucleus (PVN) to time the daily release of corticosterone. In vivo recording revealed a critical circuit from SCN vasoactive intestinal peptide (SCNVIP)-producing neurons to PVN corticotropin-releasing hormone (PVNCRH)-producing neurons. PVNCRH neurons peak in clock gene expression around midday and in calcium activity about three hours later. Loss of the clock gene Bmal1 in CRH neurons results in arrhythmic PVNCRH calcium activity and dramatically reduces the amplitude and precision of daily corticosterone release. SCNVIP activation reduces (and inactivation increases) corticosterone release and PVNCRH calcium activity, and daily SCNVIP activation entrains PVN clock gene rhythms by inhibiting PVNCRH neurons. We conclude that daily corticosterone release depends on coordinated clock gene and neuronal activity rhythms in both SCNVIP and PVNCRH neurons.


Asunto(s)
Ritmo Circadiano/fisiología , Glucocorticoides/metabolismo , Neuronas/fisiología , Núcleo Hipotalámico Paraventricular/fisiología , Animales , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Calcio/metabolismo , Ritmo Circadiano/efectos de los fármacos , Ritmo Circadiano/genética , Corticosterona/farmacología , Hormona Liberadora de Corticotropina/metabolismo , Heces/química , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Fotometría , Núcleo Supraquiasmático/fisiología
7.
Nat Commun ; 12(1): 5771, 2021 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-34599190

RESUMEN

Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.


Asunto(s)
Elementos de Facilitación Genéticos , Células Germinativas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animales , Diferenciación Celular , Cromatina/metabolismo , Embrión de Mamíferos/citología , Regulación de la Expresión Génica , Células Germinativas/citología , Estratos Germinativos/citología , Masculino , Metilación , Ratones , Ratones Transgénicos , Células Madre Embrionarias de Ratones/citología , Mutación/genética , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , RNA-Seq , Análisis de la Célula Individual , Sitio de Iniciación de la Transcripción , Transcripción Genética
8.
Nat Commun ; 12(1): 5804, 2021 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-34608155

RESUMEN

During the last decade, cardiac optogenetics has turned into an essential tool for investigating cardiac function in general and for assessing functional interactions between different myocardial cell types in particular. To advance exploitation of the unique research opportunities offered by this method, we develop a panoramic opto-electrical measurement and stimulation (POEMS) system for mouse hearts. The core of the experimental platform is composed of 294 optical fibers and 64 electrodes that form a cup which embraces the entire ventricular surface of mouse hearts and enables straightforward 'drop&go' experimentation. The flexible assignment of fibers and electrodes to recording or stimulation tasks permits a precise tailoring of experiments to the specific requirements of individual optogenetic constructs thereby avoiding spectral congestion. Validation experiments with hearts from transgenic animals expressing the optogenetic voltage reporters ASAP1 and ArcLight-Q239 demonstrate concordance of simultaneously recorded panoramic optical and electrical activation maps. The feasibility of single fiber optical stimulation is proven with hearts expressing the optogenetic voltage actuator ReaChR. Adaptation of the POEMS system to larger hearts and incorporation of additional sensors can be achieved by redesigning the system-core accordingly.


Asunto(s)
Corazón/fisiología , Optogenética/métodos , Animales , Técnicas Electrofisiológicas Cardíacas , Frecuencia Cardíaca , Potenciales de la Membrana , Ratones , Ratones Transgénicos , Miocitos Cardíacos/fisiología , Optogenética/instrumentación , Imagen de Colorante Sensible al Voltaje
9.
Nat Commun ; 12(1): 5819, 2021 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-34611155

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The continued spread of SARS-CoV-2 increases the probability of influenza/SARS-CoV-2 coinfection, which may result in severe disease. In this study, we examine the disease outcome of influenza A virus (IAV) and SARS-CoV-2 coinfection in K18-hACE2 mice. Our data indicate enhance susceptibility of IAV-infected mice to developing severe disease upon coinfection with SARS-CoV-2 two days later. In contrast to nonfatal influenza and lower mortality rates due to SARS-CoV-2 alone, this coinfection results in severe morbidity and nearly complete mortality. Coinfection is associated with elevated influenza viral loads in respiratory organs. Remarkably, prior immunity to influenza, but not to SARS-CoV-2, prevents severe disease and mortality. This protection is antibody-dependent. These data experimentally support the necessity of seasonal influenza vaccination for reducing the risk of severe influenza/COVID-19 comorbidity during the COVID-19 pandemic.


Asunto(s)
COVID-19/inmunología , COVID-19/virología , Coinfección/inmunología , Coinfección/virología , Inmunidad , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , SARS-CoV-2/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Antivirales/inmunología , COVID-19/patología , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/genética , Pulmón/patología , Pulmón/virología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Regulación hacia Arriba/genética , Carga Viral/inmunología
10.
Curr Protoc ; 1(10): e253, 2021 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-34661993

RESUMEN

The amyloid-ß (Aß) peptides of 40 and 42 amino acids that are implicated in Alzheimer's disease may potentially aggregate into toxic oligomers and form neuritic plaques. The enzyme-linked immunosorbent assay (ELISA) is a facile method used for the determination of Aß concentrations in biological matrices, namely plasma, cerebrospinal fluid, and brain. The method is mostly used for the measurement of Aß concentrations in transgenic mice, but it is unknown whether the ELISA method is suitable for measuring low, endogenous levels of Aß in the brains of wild-type mice. The Aß ELISA kit manufacturer recommends use of 5 M guanidine hydrochloride (GuHCl), a protein-denaturing agent, for homogenization of the brain tissue, followed by dilution back down to 0.1 M to avoid quenching by GuHCl. Components of brain matrices and GuHCl that could interfere with the quantitation have not been investigated. In this article, we describe an improved method involving homogenization of mouse brain with 1 M instead of 5 M GuHCl, reducing the dilution factor by 5× to provide a higher sensitivity. The modified ELISA assay is improved for the quantitation of brain Aß peptides in wild-type mice, where Aß peptide levels are much lower than those in transgenic mouse models. © 2021 Wiley Periodicals LLC.


Asunto(s)
Péptidos beta-Amiloides , Placa Amiloide , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Transgénicos
11.
Signal Transduct Target Ther ; 6(1): 345, 2021 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-34552055

RESUMEN

The SARS-CoV-2 infection causes severe immune disruption. However, it is unclear if disrupted immune regulation still exists and pertains in recovered COVID-19 patients. In our study, we have characterized the immune phenotype of B cells from 15 recovered COVID-19 patients, and found that healthy controls and recovered patients had similar B-cell populations before and after BCR stimulation, but the frequencies of PBC in patients were significantly increased when compared to healthy controls before stimulation. However, the percentage of unswitched memory B cells was decreased in recovered patients but not changed in healthy controls upon BCR stimulation. Interestingly, we found that CD19 expression was significantly reduced in almost all the B-cell subsets in recovered patients. Moreover, the BCR signaling and early B-cell response were disrupted upon BCR stimulation. Mechanistically, we found that the reduced CD19 expression was caused by the dysregulation of cell metabolism. In conclusion, we found that SARS-CoV-2 infection causes immunodeficiency in recovered patients by downregulating CD19 expression in B cells via enhancing B-cell metabolism, which may provide a new intervention target to cure COVID-19.


Asunto(s)
Antígenos CD19/inmunología , Linfocitos B/inmunología , COVID-19/inmunología , Regulación hacia Abajo/inmunología , Síndromes de Inmunodeficiencia/inmunología , SARS-CoV-2/inmunología , Animales , COVID-19/complicaciones , Chlorocebus aethiops , Femenino , Humanos , Síndromes de Inmunodeficiencia/etiología , Síndromes de Inmunodeficiencia/virología , Memoria Inmunológica , Masculino , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos B/inmunología , Células Vero
12.
Proc Natl Acad Sci U S A ; 118(41)2021 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-34561300

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.


Asunto(s)
COVID-19 , Regulación Viral de la Expresión Génica , Genes Reporteros , SARS-CoV-2 , Proteínas Virales , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/genética , COVID-19/metabolismo , Chlorocebus aethiops , Proteínas de la Nucleocápside de Coronavirus/biosíntesis , Proteínas de la Nucleocápside de Coronavirus/genética , Femenino , Humanos , Ratones , Ratones Transgénicos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Teschovirus/genética , Células Vero , Proteínas Virales/biosíntesis , Proteínas Virales/genética
13.
Signal Transduct Target Ther ; 6(1): 347, 2021 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-34564690

RESUMEN

SARS-CoV-2 mutations contribute to increased viral transmissibility and immune escape, compromising the effectiveness of existing vaccines and neutralizing antibodies. An in-depth investigation on COVID-19 pathogenesis is urgently needed to develop a strategy against SARS-CoV-2 variants. Here, we identified CD147 as a universal receptor for SARS-CoV-2 and its variants. Meanwhile, Meplazeumab, a humanized anti-CD147 antibody, could block cellular entry of SARS-CoV-2 and its variants-alpha, beta, gamma, and delta, with inhibition rates of 68.7, 75.7, 52.1, 52.1, and 62.3% at 60 µg/ml, respectively. Furthermore, humanized CD147 transgenic mice were susceptible to SARS-CoV-2 and its two variants, alpha and beta. When infected, these mice developed exudative alveolar pneumonia, featured by immune responses involving alveoli-infiltrated macrophages, neutrophils, and lymphocytes and activation of IL-17 signaling pathway. Mechanistically, we proposed that severe COVID-19-related cytokine storm is induced by a "spike protein-CD147-CyPA signaling axis": Infection of SARS-CoV-2 through CD147 initiated the JAK-STAT pathway, which further induced expression of cyclophilin A (CyPA); CyPA reciprocally bound to CD147 and triggered MAPK pathway. Consequently, the MAPK pathway regulated the expression of cytokines and chemokines, which promoted the development of cytokine storm. Importantly, Meplazumab could effectively inhibit viral entry and inflammation caused by SARS-CoV-2 and its variants. Therefore, our findings provided a new perspective for severe COVID-19-related pathogenesis. Furthermore, the validated universal receptor for SARS-CoV-2 and its variants can be targeted for COVID-19 treatment.


Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Monoclonales Humanizados/farmacología , Basigina/antagonistas & inhibidores , Basigina/metabolismo , COVID-19/tratamiento farmacológico , COVID-19/metabolismo , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Animales , Basigina/genética , COVID-19/genética , Chlorocebus aethiops , Síndrome de Liberación de Citoquinas/genética , Síndrome de Liberación de Citoquinas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Transgénicos , SARS-CoV-2/genética , Células Vero
14.
Emerg Microbes Infect ; 10(1): 1931-1946, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34538222

RESUMEN

Identification of relevant epitopes is crucial for the development of subunit peptide vaccines inducing neutralizing and cellular immunity against SARS-CoV-2. Our aim was the characterization of epitopes in the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to generate a peptide vaccine. Epitope mapping using a panel of 10 amino acid overlapped 15-mer peptides covering region 401-515 from RBD did not identify linear epitopes when tested with sera from infected individuals or from RBD-immunized mice. However, immunization of mice with these 15-mer peptides identified four peptides located at region 446-480 that induced antibodies recognizing the peptides and RBD/S1 proteins. Immunization with peptide 446-480 from S protein formulated with Freund's adjuvant or with CpG oligodeoxinucleotide/Alum induced polyepitopic antibody responses in BALB/c and C56BL/6J mice, recognizing RBD (titres of 3 × 104-3 × 105, depending on the adjuvant) and displaying neutralizing capacity (80-95% inhibition capacity; p < 0.05) against SARS-CoV-2. Murine CD4 and CD8T-cell epitopes were identified in region 446-480 and vaccination experiments using HLA transgenic mice suggested the presence of multiple human T-cell epitopes. Antibodies induced by peptide 446-480 showed broad recognition of S proteins and S-derived peptides belonging to SARS-CoV-2 variants of concern. Importantly, vaccination with peptide 446-480 or with a cyclic version of peptide 446-488 containing a disulphide bridge between cysteines 480 and 488, protected humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2 (62.5 and 75% of protection; p < 0.01 and p < 0.001, respectively). This region could be the basis for a peptide vaccine or other vaccine platforms against Covid-19.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , Inmunidad Celular , Inmunidad Humoral , SARS-CoV-2/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Neutralizantes/sangre , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , Vacunas contra la COVID-19/normas , Reacciones Cruzadas/inmunología , Mapeo Epitopo , Epítopos de Linfocito B , Epítopos de Linfocito T/inmunología , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/inmunología
15.
Nat Commun ; 12(1): 5341, 2021 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-34504070

RESUMEN

Polycomb repressive complexes-1 and -2 (PRC1 and 2) silence developmental genes in a spatiotemporal manner during embryogenesis. How Polycomb group (PcG) proteins orchestrate down-regulation of target genes upon differentiation, however, remains elusive. Here, by differentiating embryonic stem cells into embryoid bodies, we reveal a crucial role for the PCGF1-containing variant PRC1 complex (PCGF1-PRC1) to mediate differentiation-associated down-regulation of a group of genes. Upon differentiation cues, transcription is down-regulated at these genes, in association with PCGF1-PRC1-mediated deposition of histone H2AK119 mono-ubiquitination (H2AK119ub1) and PRC2 recruitment. In the absence of PCGF1-PRC1, both H2AK119ub1 deposition and PRC2 recruitment are disrupted, leading to aberrant expression of target genes. PCGF1-PRC1 is, therefore, required for initiation and consolidation of PcG-mediated gene repression during differentiation.


Asunto(s)
Cuerpos Embrioides/metabolismo , Regulación del Desarrollo de la Expresión Génica , Histonas/genética , Células Madre Embrionarias de Ratones/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 2/genética , Animales , Diferenciación Celular , Embrión de Mamíferos , Cuerpos Embrioides/citología , Histonas/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Transgénicos , Células Madre Embrionarias de Ratones/citología , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Cultivo Primario de Células , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Ubiquitinación
16.
J Vis Exp ; (174)2021 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-34487118

RESUMEN

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.


Asunto(s)
Optogenética , Pez Cebra , Animales , Animales Modificados Genéticamente , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Pez Cebra/genética
17.
Phytomedicine ; 92: 153695, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34500300

RESUMEN

BACKGROUND: Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a significant pathogenic factor in Down syndrome (DS), wherein DYRK1A is overexpressed by 1.5-fold because of trisomy of human chromosome 21. Thus, DYRK1A inhibition is considered a therapeutic strategy to modify the disease. PURPOSE: This study aims to identify a novel DYRK1A inhibitor and validate its therapeutic potential in DS-related pathological conditions. STUDY DESIGN: In order to identify a novel DYRK1A inhibitor, we carried out two-step screening: a structure-based virtual screening of > 300,000 chemical library (first step) and cell-based nuclear factor of activated T-cells (NFAT)-response element (RE) promoter assay (second step). Primary hits were evaluated for their DYRK1A inhibitory activity using in vitro kinase assay and Tau phosphorylation in mammalian cells. Confirmed hit was further evaluated in pathological conditions including DYRK1A-overexpressing fibroblasts, flies, and mice. RESULTS: We identified aristolactam BIII, a natural product derived from herbal plants, as a novel DYRK1A inhibitor. It potently inhibited the kinase activity of DYRK1A in vitro (IC50 = 9.67 nM) and effectively suppressed DYRK1A-mediated hyperphosphorylation of Tau in mammalian cells. Aristolactam BIII rescued the proliferative defects of DYRK1A transgenic (TG) mouse-derived fibroblasts and neurological and phenotypic defects of DS-like Drosophila models. Oral administration of aristolactam BIII acutely suppressed Tau hyperphosphorylation in the brain of DYRK1A TG mice. In the open field test, aristolactam BIII significantly ameliorated the exploratory behavioral deficit of DYRK1A TG mice. CONCLUSION: Our work revealed that aristolactam BIII as a novel DYRK1A inhibitor rescues DS phenotypes in cells and in vivo and suggested its therapeutic potential for the treatment of DYRK1A-related diseases.


Asunto(s)
Síndrome de Down , Animales , Encéfalo , Síndrome de Down/tratamiento farmacológico , Ratones , Ratones Transgénicos , Fenotipo , Fosforilación
18.
Mol Cell ; 81(18): 3848-3865.e19, 2021 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-34547241

RESUMEN

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP+. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.


Asunto(s)
Senescencia Celular/fisiología , NAD/metabolismo , Envejecimiento/metabolismo , Envejecimiento/fisiología , Animales , Línea Celular Tumoral , Senescencia Celular/genética , Citosol , Glucosa/metabolismo , Humanos , Hidrógeno/química , Hidrógeno/metabolismo , Malato Deshidrogenasa/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , NAD/fisiología , Oxidación-Reducción , Piruvato Carboxilasa/metabolismo , Ácido Pirúvico/metabolismo
19.
Nat Commun ; 12(1): 5446, 2021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-34521844

RESUMEN

EOMES and T-BET are related T-box transcription factors that control natural killer (NK) cell development. Here we demonstrate that EOMES and T-BET regulate largely distinct gene sets during this process. EOMES is dominantly expressed in immature NK cells and drives early lineage specification by inducing hallmark receptors and functions. By contrast, T-BET is dominant in mature NK cells, where it induces responsiveness to IL-12 and represses the cell cycle, likely through transcriptional repressors. Regardless, many genes with distinct functions are co-regulated by the two transcription factors. By generating two gene-modified mice facilitating chromatin immunoprecipitation of endogenous EOMES and T-BET, we show a strong overlap in their DNA binding targets, as well as extensive epigenetic changes during NK cell differentiation. Our data thus suggest that EOMES and T-BET may distinctly govern, via differential expression and co-factors recruitment, NK cell maturation by inserting partially overlapping epigenetic regulations.


Asunto(s)
Ciclo Celular/genética , Linaje de la Célula/genética , Células Asesinas Naturales/inmunología , Proteínas de Dominio T Box/genética , Animales , Secuencia de Bases , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Antígeno CD11b/genética , Antígeno CD11b/inmunología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/inmunología , Diferenciación Celular , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/inmunología , Epigénesis Genética/inmunología , Interleucina-12/farmacología , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas , Unión Proteica , Bazo/citología , Bazo/inmunología , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/inmunología , Transcripción Genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología
20.
Nat Commun ; 12(1): 5501, 2021 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-34535655

RESUMEN

Fibrotic scar tissue limits central nervous system regeneration in adult mammals. The extent of fibrotic tissue generation and distribution of stromal cells across different lesions in the brain and spinal cord has not been systematically investigated in mice and humans. Furthermore, it is unknown whether scar-forming stromal cells have the same origin throughout the central nervous system and in different types of lesions. In the current study, we compared fibrotic scarring in human pathological tissue and corresponding mouse models of penetrating and non-penetrating spinal cord injury, traumatic brain injury, ischemic stroke, multiple sclerosis and glioblastoma. We show that the extent and distribution of stromal cells are specific to the type of lesion and, in most cases, similar between mice and humans. Employing in vivo lineage tracing, we report that in all mouse models that develop fibrotic tissue, the primary source of scar-forming fibroblasts is a discrete subset of perivascular cells, termed type A pericytes. Perivascular cells with a type A pericyte marker profile also exist in the human brain and spinal cord. We uncover type A pericyte-derived fibrosis as a conserved mechanism that may be explored as a therapeutic target to improve recovery after central nervous system lesions.


Asunto(s)
Sistema Nervioso Central/patología , Cicatriz/patología , Pericitos/patología , Envejecimiento/fisiología , Animales , Astrocitos/patología , Lesiones Traumáticas del Encéfalo/patología , Isquemia Encefálica/patología , Neoplasias Encefálicas/patología , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Matriz Extracelular/metabolismo , Fibroblastos/patología , Fibrosis , Glioblastoma/patología , Humanos , Accidente Cerebrovascular Isquémico/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Médula Espinal/patología , Médula Espinal/ultraestructura , Traumatismos de la Médula Espinal/patología , Células del Estroma/patología
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