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1.
Front Immunol ; 12: 637654, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33732258

RESUMO

A coronavirus SARS-CoV-2, which has caused the pandemic viral pneumonia disease COVID-19, significantly threatens global public health, highlighting the need to develop effective and safe vaccines against its infection. In this study, we developed a novel DNA vaccine candidate against SARS-CoV-2 by expressing a chimeric protein of its receptor-binding domain (RBD) fused to a 33-bp sequence (11 aa) from the hepatitis B virus (HBV) preS1 region with a W4P mutation (W4P-RBD) at the N-terminal region and evaluated its immunogenicity. In vitro transfection experiments in multiple cell lines demonstrated that W4P-RBD vs. wild-type RBD protein (W-RBD) led to enhanced production of IL-6 and TNFα at the transcription and translation levels, suggesting the adjuvant potential of N-terminal HBV preS1 sequences for DNA vaccines against SARS-CoV-2. W4P-RBD also led to enhanced production of IgG and IgA, which can neutralize and block SARS-CoV-2 infection in both blood sera and bronchoalveolar lavage (BAL) fluid from the lung in vaccinated mice. Additionally, W4P-RBD led to an enhanced T-cell-mediated cellular immune response under S1 protein stimulation. In summary, W4P-RBD led to robust humoral and cell-mediated immune responses against SARS-CoV-2 in vaccinated mice, highlighting its feasibility as a novel DNA vaccine to protect against SARS-CoV-2 infection.


Assuntos
/imunologia , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Mutação , Domínios Proteicos/imunologia , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Humanos , Imunogenicidade da Vacina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vacinação/métodos , Células Vero
2.
Nat Commun ; 12(1): 1952, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782393

RESUMO

The non-protein amino acid γ-aminobutyric acid (GABA) has been proposed to be an ancient messenger for cellular communication conserved across biological kingdoms. GABA has well-defined signalling roles in animals; however, whilst GABA accumulates in plants under stress it has not been determined if, how, where and when GABA acts as an endogenous plant signalling molecule. Here, we establish endogenous GABA as a bona fide plant signal, acting via a mechanism not found in animals. Using Arabidopsis thaliana, we show guard cell GABA production is necessary and sufficient to reduce stomatal opening and transpirational water loss, which improves water use efficiency and drought tolerance, via negative regulation of a stomatal guard cell tonoplast-localised anion transporter. We find GABA modulation of stomata occurs in multiple plants, including dicot and monocot crops. This study highlights a role for GABA metabolism in fine tuning physiology and opens alternative avenues for improving plant stress resilience.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Canais de Cloreto/genética , Glutamato Descarboxilase/genética , Estômatos de Plantas/metabolismo , Transpiração Vegetal/genética , Água/metabolismo , Ácido gama-Aminobutírico/metabolismo , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Adaptação Fisiológica/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Canais de Cloreto/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Glutamato Descarboxilase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hordeum/genética , Hordeum/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/genética , Transpiração Vegetal/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Soja/genética , Soja/metabolismo , Estresse Fisiológico , Tabaco/genética , Tabaco/metabolismo , Vicia faba/genética , Vicia faba/metabolismo
4.
Lancet Neurol ; 20(4): 284-293, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33743238

RESUMO

BACKGROUND: Spinal muscular atrophy type 1 is a motor neuron disorder resulting in death or the need for permanent ventilation by age 2 years. We aimed to evaluate the safety and efficacy of onasemnogene abeparvovec (previously known as AVXS-101), a gene therapy delivering the survival motor neuron gene (SMN), in symptomatic patients (identified through clinical examination) with infantile-onset spinal muscular atrophy. METHODS: STR1VE was an open-label, single-arm, single-dose, phase 3 trial done at 12 hospitals and universities in the USA. Eligible patients had to be younger than 6 months and have spinal muscular atrophy with biallelic SMN1 mutations (deletion or point mutations) and one or two copies of SMN2. Patients received a one-time intravenous infusion of onasemnogene abeparvovec (1·1 × 1014 vector genomes per kg) for 30-60 min. During the outpatient follow-up, patients were assessed once per week, beginning at day 7 post-infusion for 4 weeks and then once per month until the end of the study (age 18 months or early termination). Coprimary efficacy outcomes were independent sitting for 30 s or longer (Bayley-III item 26) at the 18 month of age study visit and survival (absence of death or permanent ventilation) at age 14 months. Safety was assessed through evaluation of adverse events, concomitant medication usage, physical examinations, vital sign assessments, cardiac assessments, and laboratory evaluation. Primary efficacy endpoints for the intention-to-treat population were compared with untreated infants aged 6 months or younger (n=23) with spinal muscular atrophy type 1 (biallelic deletion of SMN1 and two copies of SMN2) from the Pediatric Neuromuscular Clinical Research (PNCR) dataset. This trial is registered with ClinicalTrials.gov, NCT03306277 (completed). FINDINGS: From Oct 24, 2017, to Nov 12, 2019, 22 patients with spinal muscular atrophy type 1 were eligible and received onasemnogene abeparvovec. 13 (59%, 97·5% CI 36-100) of 22 patients achieved functional independent sitting for 30 s or longer at the 18 month of age study visit (vs 0 of 23 patients in the untreated PNCR cohort; p<0·0001). 20 patients (91%, 79-100]) survived free from permanent ventilation at age 14 months (vs 6 [26%], 8-44; p<0·0001 in the untreated PNCR cohort). All patients who received onasemnogene abeparvovec had at least one adverse event (most common was pyrexia). The most frequently reported serious adverse events were bronchiolitis, pneumonia, respiratory distress, and respiratory syncytial virus bronchiolitis. Three serious adverse events were related or possibly related to the treatment (two patients had elevated hepatic aminotransferases, and one had hydrocephalus). INTERPRETATION: Results from this multicentre trial build on findings from the phase 1 START study by showing safety and efficacy of commercial grade onasemnogene abeparvovec. Onasemnogene abeparvovec showed statistical superiority and clinically meaningful responses when compared with observations from the PNCR natural history cohort. The favourable benefit-risk profile shown in this study supports the use of onasemnogene abeparvovec for treatment of symptomatic patients with genetic or clinical characteristics predictive of infantile-onset spinal muscular atrophy type 1. FUNDING: Novartis Gene Therapies.


Assuntos
Produtos Biológicos/uso terapêutico , Terapia Genética/métodos , Proteínas Recombinantes de Fusão/uso terapêutico , Atrofias Musculares Espinais da Infância/tratamento farmacológico , Atrofias Musculares Espinais da Infância/genética , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Resultado do Tratamento
5.
Biomolecules ; 11(2)2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671255

RESUMO

SARS-CoV-2, or COVID-19, has a devastating effect on our society, both in terms of quality of life and death rates; hence, there is an urgent need for developing safe and effective therapeutics against SARS-CoV-2. The most promising strategy to fight against this deadly virus is to develop an effective vaccine. Internalization of SARS-CoV-2 into the human host cell mainly occurs through the binding of the coronavirus spike protein (a trimeric surface glycoprotein) to the human angiotensin-converting enzyme 2 (ACE2) receptor. The spike-ACE2 protein-protein interaction is mediated through the receptor-binding domain (RBD) of the spike protein. Mutations in the spike RBD can significantly alter interactions with the ACE2 host receptor. Due to its important role in virus transmission, the spike RBD is considered to be one of the key molecular targets for vaccine development. In this study, a spike RBD-based subunit vaccine was designed by utilizing a ferritin protein nanocage as a scaffold. Several fusion protein constructs were designed in silico by connecting the spike RBD via a synthetic linker (different sizes) to different ferritin subunits (H-ferritin and L-ferritin). The stability and the dynamics of the engineered nanocage constructs were tested by extensive molecular dynamics simulation (MDS). Based on our MDS analysis, a five amino acid-based short linker (S-Linker) was the most effective for displaying the spike RBD over the surface of ferritin. The behavior of the spike RBD binding regions from the designed chimeric nanocages with the ACE2 receptor was highlighted. These data propose an effective multivalent synthetic nanocage, which might form the basis for new vaccine therapeutics designed against viruses such as SARS-CoV-2.


Assuntos
/química , Ferritinas/química , Nanoestruturas/química , Glicoproteína da Espícula de Coronavírus/química , /metabolismo , /metabolismo , Ferritinas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas de Subunidades/química , Vacinas de Subunidades/metabolismo
6.
PLoS Pathog ; 17(3): e1009328, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33657135

RESUMO

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and 'greasy' site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.


Assuntos
/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Afinidade de Anticorpos , Sítios de Ligação , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética
7.
J Vis Exp ; (168)2021 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-33682851

RESUMO

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.


Assuntos
Citometria de Fluxo/métodos , Leucemia Mieloide Aguda/patologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Anticorpos Antineoplásicos/metabolismo , Biotinilação , Contagem de Células , Humanos , Ligantes , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem , Células Tumorais Cultivadas
8.
Ophthalmologe ; 118(3): 248-256, 2021 Mar.
Artigo em Alemão | MEDLINE | ID: mdl-33555415

RESUMO

The anti-vascular endothelial growth factor (anti-VEGF) agent brolucizumab has been approved in the USA in October 2019 and in Europe in February 2020 for the treatment of neovascular age-related macular degeneration (nAMD). The approval was based on the randomized, double-blind phase III studies HAWK and HARRIER with a total of 1817 patients. Brolucizumab 6 mg (administered every 12 or 8 weeks depending on the activity of the disease) showed a non-inferior efficacy in terms of best-corrected visual acuity compared to aflibercept 2 mg (administered every 8 weeks). Initial reports on the use of brolucizumab after its approval in the USA indicated a safety signal of rare adverse events termed as retinal vasculitis and/or retinal vascular occlusion that may result in severe loss of vision. Typically, these events occurred in the presence of intraocular inflammation (IOI). A safety review committee (SRC) subsequently carried out an independent analysis of data from the pivotal studies. This article sets out the current state of knowledge and aims to provide users with orientation-from the authors' perspective-in treating brolucizumab-associated IOI. It appears mandatory to provide patients with information about possible symptoms of IOI. Even though the case reports and the SRC review of HAWK/HARRIER may not yet provide sufficient evidence for any final conclusions, it seems crucial to educate patients about signs and symptoms to ensure an early detection and diagnosis in cases of IOI. Once a patient is diagnosed with IOI, retinal vasculitis, and/or retinal vascular occlusive events, physicians should act promptly with an adequate and intensive anti-inflammatory treatment and brolucizumab treatment should be discontinued. It is important to note that these recommendations are primarily based on the authors' expert opinions and should be considered as guidance in managing these events rather than a formal protocol or guidelines.


Assuntos
Inibidores da Angiogênese , Receptores de Fatores de Crescimento do Endotélio Vascular , Inibidores da Angiogênese/uso terapêutico , Anticorpos Monoclonais Humanizados , Humanos , Inflamação/tratamento farmacológico , Injeções Intravítreas , Ensaios Clínicos Controlados Aleatórios como Assunto , Proteínas Recombinantes de Fusão , Acuidade Visual
9.
Nat Commun ; 12(1): 1034, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589617

RESUMO

Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma Humano , RNA Guia/genética , Pareamento de Bases , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Bases de Dados Genéticas , Doença de Fabry/genética , Doença de Fabry/metabolismo , Doença de Fabry/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Hemofilia A/genética , Hemofilia A/metabolismo , Hemofilia A/patologia , Humanos , Modelos Biológicos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Mutação , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
10.
Virology ; 556: 73-78, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33548599

RESUMO

The need to stem the current outbreak of SARS-CoV-2 responsible for COVID-19 is driving the search for inhibitors that will block coronavirus replication and pathogenesis. The coronavirus 3C-like protease (3CLpro) encoded in the replicase polyprotein is an attractive target for antiviral drug development because protease activity is required for generating a functional replication complex. Reagents that can be used to screen for protease inhibitors and for identifying the replicase products of SARS-CoV-2 are urgently needed. Here we describe a luminescence-based biosensor assay for evaluating small molecule inhibitors of SARS-CoV-2 3CLpro/main protease. We also document that a polyclonal rabbit antiserum developed against SARS-CoV 3CLpro cross reacts with the highly conserved 3CLpro of SARS-CoV-2. These reagents will facilitate the pre-clinical evaluation of SARS-CoV-2 protease inhibitors.


Assuntos
Técnicas Biossensoriais/métodos , Soros Imunes/imunologia , Luciferases/metabolismo , /metabolismo , Animais , Antivirais/farmacologia , /genética , Reações Cruzadas , Luciferases/genética , Inibidores de Proteases/farmacologia , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus da SARS/imunologia , Vírus da SARS/metabolismo , /genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
12.
J Vis Exp ; (167)2021 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-33522504

RESUMO

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment's purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.


Assuntos
Imunoglobulina G/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tabaco/genética , Agrobacterium tumefaciens/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Sequência de Bases , Cromatografia , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/metabolismo , Humanos , Canamicina/farmacologia , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Tabaco/crescimento & desenvolvimento , Tabaco/microbiologia , Raios Ultravioleta
13.
Nat Commun ; 12(1): 792, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542232

RESUMO

The immune system is a sophisticated network of different cell types performing complex biocomputation at single-cell and consortium levels. The ability to reprogram such an interconnected multicellular system holds enormous promise in treating various diseases, as exemplified by the use of chimeric antigen receptor (CAR) T cells as cancer therapy. However, most CAR designs lack computation features and cannot reprogram multiple immune cell types in a coordinated manner. Here, leveraging our split, universal, and programmable (SUPRA) CAR system, we develop an inhibitory feature, achieving a three-input logic, and demonstrate that this programmable system is functional in diverse adaptive and innate immune cells. We also create an inducible multi-cellular NIMPLY circuit, kill switch, and a synthetic intercellular communication channel. Our work highlights that a simple split CAR design can generate diverse and complex phenotypes and provide a foundation for engineering an immune cell consortium with user-defined functionalities.


Assuntos
Engenharia Celular/métodos , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos Quiméricos/genética , Proteínas Recombinantes de Fusão/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Cultura Primária de Células , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Biologia Sintética/métodos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
ACS Synth Biol ; 10(2): 379-390, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33534552

RESUMO

Generating and characterizing immunoreagents to enable studies of novel emerging viruses is an area where ensembles of synthetic genes, recombinant antibody pipelines, and modular antibody-reporter fusion proteins can respond rapidly. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread through the global population causing widespread morbidity, mortality, and socioeconomic chaos. Using SARS-CoV-2 as our model and starting with a gBlocks encoded nucleocapsid (N) gene, we purified recombinant protein from E. coli, to serve as bait for selecting semisynthetic nanobodies from our Nomad single-pot library. Clones were isolated in days and first fused to Gaussia luciferase to determine EC50 in the tens of nM range, and second fused to the ascorbate peroxidase derivative APEX2 for sensitive detection of SARS-CoV-2 infected cells. To generate inherently fluorescent immunoreagents, we introduce novel periplasmic sdAb fusions made with mNeonGreen and mScarlet-I, which were produced at milligram amounts. The fluorescent fusion proteins enabled concise visualization of SARS-CoV-2 N in the cytoplasm but not in the nucleus 24 h post infection, akin to the distribution of SARS-CoV N, thereby validating these useful imaging tools. SdAb reactivity appeared specific to SARS-CoV-2 with very much weaker binding to SARS-CoV, and no noticeable cross-reactivity to a panel of overexpressed human codon optimized N proteins from other CoV. High periplasmic expression levels and in silico immortalization of the nanobody constructs guarantees a cost-effective and reliable source of SARS-CoV-2 immunoreagents. Our proof-of-principle study should be applicable to known and newly emerging CoV to broaden the tools available for their analysis and help safeguard human health in a more proactive than reactive manner.


Assuntos
/epidemiologia , /genética , Sondas Moleculares/genética , Pandemias , /imunologia , Anticorpos Antivirais/genética , Especificidade de Anticorpos/genética , Doenças Transmissíveis Emergentes/virologia , Escherichia coli/genética , Imunofluorescência , Genes Sintéticos , Genes Virais , Células HEK293 , Humanos , Sondas Moleculares/imunologia , Pandemias/prevenção & controle , Biblioteca de Peptídeos , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Domínio Único/genética , Biologia Sintética
15.
Medicine (Baltimore) ; 100(7): e24790, 2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33607835

RESUMO

RATIONALE: Half-dose or reduced-fluence photodynamic therapy (PDT) with verteporfin has been well acknowledged to be the most effective and permanent treatment with very low rates of complications. However, we report a case of chronic central serous chorioretinopathy (CSC) who developed choroidal neovascularization (CNV) secondary to half-dose PDT within only 3 weeks. Such an occurrence following this short a course of treatment has not been reported previously. PATIENT CONCERNS: A 46-year-old Chinese man who had been diagnosed as acute more than 1 year ago revisited our department recently and complained of blurred vision again in his left eye. DIAGNOSES: Fluorescein fundus angiography (FFA) and indocyanine green angiography (ICGA) revealed patchy hyperfluorescent dots and optical coherence tomography (OCT) indicated irregular flat pigment epithelium detachment (PED) in the central macula. The patient was diagnosed with chronic CSC. INTERVENTIONS: The patient was treated by half-dose PDT with verteporfin. Three weeks later, the patient complained of sudden blurred vision and fundus examination showed macular hemorrhages with a best-corrected visual acuity (BCVA) of 20/250. OCT angiography (OCTA) showed a distinct area of flower-like CNV located within the deep retinal slab. Secondary CNV had developed after a quite short course of half-dose PDT treatment. Subsequently, the patient was administered by 2 intravitreal injections of aflibercept (2 mg). OUTCOMES: Two months after the second intravitreal injection, macular hemorrhages and secondary CNV were completely resolved, and the BCVA improved to 20/25. LESSONS: Patients of chronic CSC with irregular PED who undergo PDT should be warned of secondary CNV within a short course after treatment. If happened, it should be treated by intravitreal injections of anti-vascular endothelial growth factor agents as soon as possible.


Assuntos
Coriorretinopatia Serosa Central/tratamento farmacológico , Neovascularização de Coroide/induzido quimicamente , Fármacos Fotossensibilizantes/efeitos adversos , Verteporfina/efeitos adversos , Inibidores da Angiogênese/administração & dosagem , Neovascularização de Coroide/tratamento farmacológico , Angiofluoresceinografia , Humanos , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Fármacos Fotossensibilizantes/administração & dosagem , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Tomografia de Coerência Óptica , Verteporfina/administração & dosagem
16.
Biophys J ; 120(6): 1105-1119, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33631204

RESUMO

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , /metabolismo , Sequência de Aminoácidos , Permeabilidade da Membrana Celular , Lipídeos de Membrana/química , Simulação de Dinâmica Molecular , Pressão , Probabilidade , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Água/química
17.
Medicine (Baltimore) ; 100(7): e24379, 2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33607771

RESUMO

RATIONALE: To summarize and analyze a case of rapid progression of high-risk proliferative diabetic retinopathy after the insulin intensive therapy (IT). PATIENT CONCERNS: A 58-year-old type 2 diabetes female patient suffered a rapid and dramatic decline of vision acuity in the left eye in 2 months after the insulin IT. However, the best corrected visual acuity (BCVA) of her right eye, which was in much severer condition and received panretinal photocoagulation (PRP) before, improved after the IT. INTERVENTIONS: The patient received intravitreal injection of conbercept (IVC) to her left eye, and 5 days later, underwent pars plana vitrectomy (PPV), combined with PRP and silicon oil injection. OUTCOMES: The postoperative BCVA of the left eye was 20/200 and improved to 20/160 one month later. During the subsequent 2 months of follow-up, her BCVA remained 20/160 in both eyes. Her blood glucose level also remained stable. LESSONS: Insulin IT for untreated proliferative diabetic retinopathy (PDR) patients can cause severe and irreversible consequences, so for such patients, the conservative treatment for glycemic control may be much safer. But if insulin IT is inevitable, the patient should undergo PRP promptly before the IT, and close eye monitoring during the IT is also essential.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Retinopatia Diabética/induzido quimicamente , Hipoglicemiantes/efeitos adversos , Insulina/efeitos adversos , Retinopatia Diabética/diagnóstico por imagem , Retinopatia Diabética/patologia , Retinopatia Diabética/terapia , Feminino , Humanos , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Injeções Intravítreas , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/administração & dosagem , Vitrectomia
18.
Methods Mol Biol ; 2261: 93-103, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33420987

RESUMO

Imaged capillary isoelectric focusing (icIEF) is a gold standard method for characterizing the charge heterogeneity of protein therapeutics. A broad range of protein therapeutics such as monoclonal antibodies, antibody-drug conjugates (ADCs), and fusion proteins are routinely analyzed by icIEF due to its high resolution and high reproducibility. Platform methods, which can be applied without modification to the analysis of different protein therapeutics, save valuable time and resources in method development and quality control. Here, we provide platform methods for icIEF analysis of three classes of protein therapeutics, a biosimilar to the monoclonal antibody trastuzumab, recombinant human erythropoietin (rhEPO), and a fusion protein. The details of sample preparation and separation conditions for each molecule are described in this chapter.


Assuntos
Produtos Biológicos/análise , Eletroforese Capilar , Eritropoetina/análise , Focalização Isoelétrica , Proteínas Recombinantes de Fusão/análise , Trastuzumab/análise , Métodos Analíticos de Preparação de Amostras , Ensaios de Triagem em Larga Escala , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Fluxo de Trabalho
19.
Methods Mol Biol ; 2261: 357-379, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33421001

RESUMO

Biotinylation identification (BioID) is a method designed to provide new cellular location and functional knowledge of the protein of interest through the identification of those proteins surrounding and in direct contact. A biotin ligase is fused onto the protein of interest and expressed in cells where it can biotinylate even short-lived transient protein complexes. In addition, due to the proximity labeling nature of the experiment, cellular localization and functional enrichment information can also be obtained. Since labeling occurs only after the addition of biotin, temporal relationships and localization changes (e.g., cytoplasmic to nuclear) can also be identified. Labeled proteins are easily purified, and contaminants minimized, using the strong interaction between biotin and streptavidin. Mass spectrometry analysis of the purified proteins allows for the identification of potential interactors for further validation and characterization.


Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Mapas de Interação de Proteínas , Proteômica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Métodos Analíticos de Preparação de Amostras , Animais , Biotinilação , Carbono-Nitrogênio Ligases/genética , Proteínas de Escherichia coli/genética , Células HEK293 , Humanos , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/genética , Fatores de Tempo
20.
Methods Mol Biol ; 2261: 381-394, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33421002

RESUMO

Protein-protein interactions (PPI) are involved in a myriad of cellular processes, and their deregulation can lead to many diseases. One such process is protein ubiquitination that requires an orchestrated action of three key enzymes to add ubiquitin moieties to substrate proteins. Importantly, this process is reversible through deubiquitinating enzymes. Both ubiquitination and deubiquitination require many PPIs that once classified can be utilized to identify small molecule inhibitors counteracting these reactions. Here, we study the protein-protein interaction between the two deubiquitinating enzymes OTUB1 and OTUD6B and report for the first time that both proteins directly interact with each other. We describe the GFP-Trap immunoprecipitation as a cell-based method to analyze the OTUD6B-OTUB1 interaction in the cellular context and the AlphaScreen (amplified luminescent proximity homogeneous assay) assay as a tool to detect direct interactions and to search for PPI inhibitors.


Assuntos
Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Ensaios de Triagem em Larga Escala , Imunoprecipitação , Mapas de Interação de Proteínas , Proteômica , Cisteína Endopeptidases/genética , Endopeptidases/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitinação
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