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1.
EMBO J ; 40(19): e107985, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34302370

RESUMO

Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Mutação/imunologia , SARS-CoV-2/imunologia , Anticorpos de Domínio Único/imunologia , Animais , COVID-19/virologia , Camelídeos Americanos/imunologia , Camelídeos Americanos/virologia , Linhagem Celular , Escherichia coli/virologia , Feminino , Humanos , Glicoproteína da Espícula de Coronavírus/imunologia
2.
J Clin Invest ; 117(4): 961-70, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17404619

RESUMO

Aristaless-related homeobox (Arx) was recently demonstrated to be involved in pancreatic alpha cell fate specification while simultaneously repressing the beta and delta cell lineages. To establish whether Arx is not only necessary, but also sufficient to instruct the alpha cell fate in endocrine progenitors, we used a gain-of-function approach to generate mice conditionally misexpressing this factor. Mice with forced Arx expression in the embryonic pancreas or in developing islet cells developed a dramatic hyperglycemia and eventually died. Further analysis demonstrated a drastic loss of beta and delta cells. Concurrently, a remarkable increase in the number of cells displaying alpha cell or, strikingly, pancreatic polypeptide (PP) cell features was observed. Notably, the ectopic expression of Arx induced in embryonic or adult beta cells led to a loss of the beta cell phenotype and a concomitant increase in a number of cells with alpha or PP cell characteristics. Combining quantitative real-time PCR and lineage-tracing experiments, we demonstrate that, in adult mice, the misexpression of Arx, rather than its overexpression, promotes a conversion of beta cells into glucagon- or PP-producing cells in vivo. These results provide important insights into the complex mechanisms underlying proper pancreatic endocrine cell allocation and cell identity acquisition.


Assuntos
Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/embriologia , Polipeptídeo Pancreático/metabolismo , Fatores de Transcrição/genética , Animais , Regulação da Expressão Gênica , Glucagon/metabolismo , Ilhotas Pancreáticas/crescimento & desenvolvimento , Camundongos , Fenótipo , Reação em Cadeia da Polimerase
3.
Development ; 133(9): 1779-87, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16571633

RESUMO

The specification of neuronal cell types in the developing neural tube is orchestrated by signaling centers. However, how patterned territories of the central nervous system (CNS) are organized into structures with appropriate size and shape is still unclear. We report that in the absence of the mouse transcription factor mBtd/Sp8, a posterior shift of the isthmic organizer (IsO) occurs, suggesting a crucial role for Sp8 in this process. In addition, large patches of cells ectopically expressing Fgf8, Otx2 and/or Wnt1 in the rostral hindbrain are detected in Sp8 mutant embryos. In this context, midbrain dopaminergic neurons are found posterior to the IsO. Furthermore, we provide evidence that cell proliferation in the mid- and hindbrain is tightly controlled by Sp8 activity. Our observations are consistent with a role for Sp8 in restricting Fgf8 expression at the IsO.


Assuntos
Padronização Corporal , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesencéfalo/embriologia , Rombencéfalo/embriologia , Fatores de Transcrição/metabolismo , Animais , Padronização Corporal/genética , Proteínas de Ligação a DNA/genética , Mesencéfalo/metabolismo , Camundongos , Camundongos Knockout , Rombencéfalo/metabolismo , Fatores de Transcrição/genética
4.
Development ; 132(13): 2969-80, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15930104

RESUMO

The specification of the different mouse pancreatic endocrine subtypes is determined by the concerted activities of transcription factors. However, the molecular mechanisms regulating endocrine fate allocation remain unclear. In the present study, we uncover the molecular consequences of the simultaneous depletion of Arx and Pax4 activity during pancreas development. Our findings reveal a so far unrecognized essential role of the paired-box-encoding Pax4 gene. Specifically, in the combined absence of Arx and Pax4, an early-onset loss of mature alpha- and beta-cells occurs in the endocrine pancreas, concomitantly with a virtually exclusive generation of somatostatin-producing cells. Furthermore, despite normal development of the PP-cells in the double-mutant embryos, an atypical expression of the pancreatic polypeptide (PP) hormone was observed in somatostatin-labelled cells after birth. Additional characterizations indicate that such an expression of PP was related to the onset of feeding, thereby unravelling an epigenetic control. Finally, our data provide evidence that both Arx and Pax4 act as transcriptional repressors that control the expression level of one another, thereby mediating proper endocrine fate allocation.


Assuntos
Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/genética , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/embriologia , Somatostatina/biossíntese , Fatores de Transcrição/genética , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glucagon/metabolismo , Proteínas de Homeodomínio/metabolismo , Hiperglicemia/genética , Hiperglicemia/mortalidade , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Fatores de Transcrição Box Pareados , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia
5.
Genes Dev ; 17(20): 2591-603, 2003 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-14561778

RESUMO

Genes encoding homeodomain-containing proteins potentially involved in endocrine pancreas development were isolated by combined in silico and nested-PCR approaches. One such transcription factor, Arx, exhibits Ngn3-dependent expression throughout endocrine pancreas development in alpha, beta-precursor, and delta cells. We have used gene targeting in mouse embryonic stem cells to generate Arx loss-of-function mice. Arx-deficient animals are born at the expected Mendelian frequency, but develop early-onset hypoglycemia, dehydration, and weakness, and die 2 d after birth. Immunohistological analysis of pancreas from Arx mutants reveals an early-onset loss of mature endocrine alpha cells with a concomitant increase in beta-and delta-cell numbers, whereas islet morphology remains intact. Our study indicates a requirement of Arx for alpha-cell fate acquisition and a repressive action on beta-and delta-cell destiny, which is exactly the opposite of the action of Pax4 in endocrine commitment. Using multiplex reverse transcriptase PCR (RT-PCR), we demonstrate an accumulation of Pax4 and Arx transcripts in Arx and Pax4 mutant mice, respectively. We propose that the antagonistic functions of Arx and Pax4 for proper islet cell specification are related to the pancreatic levels of the respective transcripts.


Assuntos
Proteínas de Homeodomínio/metabolismo , Pâncreas/embriologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/fisiologia , Genes Letais , Proteínas de Homeodomínio/genética , Hipoglicemia/genética , Hipoglicemia/metabolismo , Camundongos , Mutação , Fatores de Transcrição Box Pareados , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Regulação para Cima/fisiologia
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