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1.
Curr Opin Biotechnol ; 81: 102937, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37187103

RESUMO

Two of the big challenges in modern bioprocesses are process economics and in-depth process understanding. Getting access to online process data helps to understand process dynamics and monitor critical process parameters (CPPs). This is an important part of the quality-by- design concept that was introduced to the pharmaceutical industry in the last decade. Raman spectroscopy has proven to be a versatile tool to allow noninvasive measurements and access to a broad spectrum of analytes. This information can then be used for enhanced process control strategies. This review article will focus on the latest applications of Raman spectroscopy in established protein production bioprocesses as well as show its potential for virus, cell therapy, and mRNA processes.


Assuntos
Produtos Biológicos , Análise Espectral Raman , Análise Espectral Raman/métodos , Produtos Biológicos/análise
2.
Glia ; 68(5): 1046-1064, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31841614

RESUMO

Mutations in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS). Accumulating evidence implicates astrocytes as important non-cell autonomous contributors to ALS pathogenesis, although the potential deleterious effects of astrocytes on the function of motor neurons remains to be determined in a completely humanized model of C9orf72-mediated ALS. Here, we use a human iPSC-based model to study the cell autonomous and non-autonomous consequences of mutant C9orf72 expression by astrocytes. We show that mutant astrocytes both recapitulate key aspects of C9orf72-related ALS pathology and, upon co-culture, cause motor neurons to undergo a progressive loss of action potential output due to decreases in the magnitude of voltage-activated Na+ and K+ currents. Importantly, CRISPR/Cas-9 mediated excision of the C9orf72 repeat expansion reverses these phenotypes, confirming that the C9orf72 mutation is responsible for both cell-autonomous astrocyte pathology and non-cell autonomous motor neuron pathophysiology.


Assuntos
Astrócitos/metabolismo , Proteína C9orf72/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Potenciais de Ação/fisiologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Astrócitos/patologia , Proteína C9orf72/metabolismo , Técnicas de Cocultura , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios Motores/patologia , Mutação
3.
Curr Biol ; 27(7): 1068-1073, 2017 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-28318976

RESUMO

Nodes of Ranvier in the axons of myelinated neurons are exemplars of the specialized cell surface domains typical of polarized cells. They are rich in voltage-gated sodium channels (Nav) and thus underpin rapid nerve impulse conduction in the vertebrate nervous system [1]. Although nodal proteins cluster in response to myelination, how myelin-forming glia influence nodal assembly is poorly understood. An axoglial adhesion complex comprising glial Neurofascin155 and axonal Caspr/Contactin flanks mature nodes [2]. We have shown that assembly of this adhesion complex at the extremities of migrating oligodendroglial processes promotes process convergence along the axon during central nervous system (CNS) node assembly [3]. Here we show that anchorage of this axoglial complex to the axon cytoskeleton is essential for efficient CNS node formation. When anchorage is disrupted, both the adaptor Protein 4.1B and the cytoskeleton protein ßII spectrin are mislocalized in the axon, and assembly of the node of Ranvier is significantly delayed. Nodal proteins and migrating oligodendroglial processes are no longer juxtaposed, and single detached nodal complexes replace the symmetrical heminodes found in both the CNS and peripheral nervous system (PNS) during development. We propose that axoglial adhesion complexes contribute to the formation of an interface between cytoskeletal elements enriched in Protein 4.1B and ßII spectrin and those enriched in nodal ankyrinG and ßIV spectrin. This clusters nascent nodal complexes at heminodes and promotes their timely coalescence to form the mature node of Ranvier. These data demonstrate a role for the axon cytoskeleton in the assembly of a critical neuronal domain, the node of Ranvier.


Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Nós Neurofibrosos/metabolismo , Animais , Axônios/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citoesqueleto/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo
4.
Glia ; 60(2): 203-17, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22052506

RESUMO

Myelin sheath thickness is precisely adjusted to axon caliber, and in the peripheral nervous system, neuregulin 1 (NRG1) type III is a key regulator of this process. It has been proposed that the protease BACE1 activates NRG1 dependent myelination. Here, we characterize the predicted product of BACE1-mediated NRG1 type III processing in transgenic mice. Neuronal overexpression of a NRG1 type III-variant, designed to mimic prior cleavage in the juxtamembrane stalk region, induces hypermyelination in vivo and is sufficient to restore myelination of NRG1 type III-deficient neurons. This observation implies that the NRG1 cytoplasmic domain is dispensable and that processed NRG1 type III is sufficient for all steps of myelination. Surprisingly, transgenic neuronal overexpression of full-length NRG1 type III promotes hypermyelination also in BACE1 null mutant mice. Moreover, NRG1 processing is impaired but not abolished in BACE1 null mutants. Thus, BACE1 is not essential for the activation of NRG1 type III to promote myelination. Taken together, these findings suggest that multiple neuronal proteases collectively regulate NRG1 processing.


Assuntos
Secretases da Proteína Precursora do Amiloide/deficiência , Secretases da Proteína Precursora do Amiloide/fisiologia , Ácido Aspártico Endopeptidases/deficiência , Ácido Aspártico Endopeptidases/fisiologia , Bainha de Mielina/metabolismo , Neuregulina-1/metabolismo , Neuregulina-1/fisiologia , Processamento de Proteína Pós-Traducional/genética , Transdução de Sinais/genética , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Técnicas de Cocultura , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/fisiologia , Bainha de Mielina/genética , Neuregulina-1/genética , Peptídeo Hidrolases/fisiologia , Cultura Primária de Células , Estrutura Terciária de Proteína/genética
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