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1.
Adv Exp Med Biol ; 1309: 67-96, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33782869

RESUMO

From molecular probes, also known as fluorophores (typically emitting a longer wavelength than the absorbing wavelength), to inorganic nanoparticles, various light-emitting materials have been actively studied and developed for various applications in life science owing to their superior imaging and sensing ability. Especially after the breakthrough development of quantum dots (QDs), studies have pursued the development of the optical properties and biological applications of luminescent inorganic nanoparticles such as upconversion nanoparticles (UCNPs), metal nanoclusters, carbon dots, and so on. In this review, we first provide a brief explanation about the theoretical background and traditional concepts of molecular fluorophores. Then, currently developed luminescent nanoparticles are described as sensing and imaging platforms from general aspects to technical views.


Assuntos
Nanopartículas , Nanoestruturas , Pontos Quânticos , Corantes Fluorescentes , Sondas Moleculares
2.
Int J Nanomedicine ; 16: 1901-1911, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33707945

RESUMO

Purpose: Developing a sensitive SERS-based method to quantitatively detect serum biomarkers (Aß1-42 and P-Tau-181) for the early diagnosis of Alzheimer's disease (AD). Methods: In this study, a novel SERS-based sandwich immunoassay, which consists of tannin-capped silver nanoparticles and magnetic graphene oxide (Fe3O4@GOs), was developed. We firstly applied this method for the detection of protein standards in buffer solution, obtaining the regression equation. Then, its potential value on real serum samples of AD was further explored. Results: The detection linear range of Aß1-42 and P-Tau-181 protein standards were observed to range from 100 pg mL-1 to 10 fg mL-1, 100 pg mL-1 to 1 fg mL-1 respectively. We finally explored clinical application of the proposed method in 63 serum samples. As a result, P-tau-181 differentiated AD from non-AD dementia patients (AUC = 0.770), with a more favored ROC than Aß1-42 (AUC = 0.383). Conclusion: The developed SERS-based immunoassay is successfully applied to the determination of Aß1-42 and P-Tau-181 in human serum specimens, which provides a promising tool for the early diagnosis of AD.


Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/diagnóstico , Biomarcadores/sangue , Imunoensaio/métodos , Sondas Moleculares/química , Prata/química , Análise Espectral Raman/métodos , Peptídeos beta-Amiloides/sangue , Benzoatos/química , Calibragem , Feminino , Grafite/química , Humanos , Limite de Detecção , Masculino , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Compostos de Sulfidrila/química , Difração de Raios X , Proteínas tau/sangue
3.
Molecules ; 26(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652775

RESUMO

Europium (III) luminescent chelates possess intrinsic photophysical properties that are extremely useful in a wide range of applications. The lack of examples of coumarin-based lanthanide complexes is mainly due to poor photo-sensitization attempts. However, with the appeal of using such a versatile scaffold as antenna, especially in the development of responsive molecular probes, it is worth the effort to research new structural motifs. In this work, we present a series of two new tris coumarin-dipicolinate europium (III) complexes, specifically tailored to be either a mono or a dual emitter, tuning their properties with a simple chemical modification. We also encountered a rich chemical speciation in solution, studied in detail by means of paramagnetic NMR and emission spectroscopy.


Assuntos
Complexos de Coordenação/química , Cumarínicos/química , Európio/química , Sondas Moleculares/química , Quelantes/química , Elementos da Série dos Lantanídeos/química , Luminescência , Espectroscopia de Ressonância Magnética
4.
Int J Nanomedicine ; 16: 2311-2322, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33776435

RESUMO

Background: Alzheimer's disease (AD) is a neurodegenerative chronic disorder that causes dementia and problems in thinking, cognitive impairment and behavioral changes. Amyloid-beta (Aß) is a peptide involved in AD progression, and a high level of Aß is highly correlated with severe AD. Identifying and quantifying Aß levels helps in the early treatment of AD and reduces the factors associated with AD. Materials and Methods: This research introduced a dual probe detection system involving aptamers and antibodies to identify Aß. Aptamers and antibodies were attached to the gold (Au) urchin and hybrid on the carbon nanohorn-modified surface. The nanohorn was immobilized on the sensor surface by using an amine linker, and then a Au urchin dual probe was immobilized. Results: This dual probe-modified surface enhanced the current flow during Aß detection compared with the surface with antibody as the probe. This dual probe interacted with higher numbers of Aß peptides and reached the detection limit at 10 fM with R2=0.992. Furthermore, control experiments with nonimmune antibodies, complementary aptamer sequences and control proteins did not display the current responses, indicating the specific detection of Aß. Conclusion: Aß-spiked artificial cerebrospinal fluid showed a similar response to current changes, confirming the selective identification of Aß.


Assuntos
Doença de Alzheimer/diagnóstico , Ouro/química , Sondas Moleculares/química , Nanopartículas/química , Peptídeos beta-Amiloides/metabolismo , Eletrodos , Humanos , Limite de Detecção , Modelos Lineares , Nanopartículas/ultraestrutura , Fragmentos de Peptídeos , Multimerização Proteica , Reprodutibilidade dos Testes , Espectrometria por Raios X , Propriedades de Superfície
5.
Molecules ; 26(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669590

RESUMO

Nitroreductases belong to a member of flavin-containing enzymes that can reduce nitroaromatic compounds to amino derivatives with NADH as an electron donor. NTR activity is known to be elevated in the cancerous environment and is considered an advantageous target in therapeutic prodrugs for the treatment of cancer. Here, we developed a ratiometric fluorescent molecule for observing NTR activity in living cells. This can provide a selective and sensitive response to NTR with a distinct increase in fluorescence ratio (FI530/FI630) as well as color changes. We also found a significant increase in NTR activity in cervical cancer HeLa and lung cancer A549 cells compared to non-cancerous NIH3T3. We proposed that this new ratiometric fluorescent molecule could potentially be used as a NTR-sensitive molecular probe in the field of cancer diagnosis and treatment development related to NTR activity.


Assuntos
Ensaios Enzimáticos/métodos , Corantes Fluorescentes/química , Sondas Moleculares/química , Nitrorredutases/metabolismo , Células A549 , Animais , Morte Celular , Cromatografia Líquida de Alta Pressão , Endocitose , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Sondas Moleculares/síntese química , Células NIH 3T3 , Espectrometria de Fluorescência
6.
Nat Commun ; 12(1): 960, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33574252

RESUMO

Nitric oxide (NO) is an important signaling molecule overexpressed in many diseases, thus the development of NO-activatable probes is of vital significance for monitoring related diseases. However, sensitive photoacoustic (PA) probes for detecting NO-associated complicated diseases (e.g., encephalitis), have yet to be developed. Herein, we report a NO-activated PA probe for in vivo detection of encephalitis by tuning the molecular geometry and energy transformation processes. A strong donor-acceptor structure with increased conjugation can be obtained after NO treatment, along with the active intramolecular motion, significantly boosting "turn-on" near-infrared PA property. The molecular probe exhibits high specificity and sensitivity towards NO over interfering reactive species. The probe is capable of detecting and differentiating encephalitis in different severities with high spatiotemporal resolution. This work will inspire more insights into the development of high-performing activatable PA probes for advanced diagnosis by making full use of intramolecular motion and energy transformation processes.


Assuntos
Técnicas Biossensoriais/métodos , Encefalite/diagnóstico , Encefalite/metabolismo , Óxido Nítrico/isolamento & purificação , Técnicas Fotoacústicas/métodos , Animais , Técnicas Biossensoriais/instrumentação , Modelos Animais de Doenças , Encefalite/patologia , Masculino , Camundongos , Imagem Molecular/métodos , Sondas Moleculares/química , Técnicas Fotoacústicas/instrumentação
7.
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
8.
Arch Biochem Biophys ; 699: 108748, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33444627

RESUMO

ApoA-I is the main protein of HDL which has anti-atherogenic properties attributed to reverse cholesterol transport. It shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and inter-molecularly, providing structural stabilization by a complex self-association mechanism. In this study, we employed a multi-parametric fluorescent probe to study the self-association of apoA-I. We constructed six single cysteine mutants spanning positions along three helices: F104C, K107C (H4), K133C, L137C (H5), F225C and K226C (H10); and labelled them with N-Maleimide Pyrene. Taking advantage of its spectral properties, namely formation of an excited dimer (excimer) and polarity-dependent changes in its fluorescence fine structure (P-value), we monitored the apoA-I self-association in its lipid-free form as a function of its concentration. Interactions in helices H5 (K133C) and H10 (F225C and K226C) were highlighted by excimer emission; while polarity changes were reported in helix H4 (K107C), as well as in helices H5 and H10. Mathematical models were developed to enrich data analysis and estimate association constants (KA) and oligomeric species distribution. Furthermore, we briefly discuss the usefulness of the multi-parametric fluorescent probe to monitor different equilibria, even at a single labelling position. Results suggest that apoA-I self-association must be considered to fully understand its physiological roles. Particularly, some contacts that stabilize discoidal HDL particles seem to be already present in the lipid-free apoA-I oligomers.


Assuntos
Apolipoproteína A-I/química , Corantes Fluorescentes/química , Sondas Moleculares/química , Multimerização Proteica , Pirenos/química , Apolipoproteína A-I/genética , Cisteína/química , Humanos , Mutação , Espectrometria de Fluorescência
9.
Anal Chem ; 93(4): 2226-2234, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33417427

RESUMO

Real-time imaging of multiple low-abundance microRNAs (miRNAs) simultaneously in living cells with high sensitivity is of vital importance for accurate cancer clinical diagnosis and prognosis studies. Maintaining stability of nanoprobes resistant to enzyme degradation and enabling effective signal amplification is highly needed for in vivo imaging studies. Herein, a rationally designed one-pot assembled multicolor tetrahedral DNA frameworks (TDFs) by encoding multicomponent nucleic acid enzymes (MNAzymes) was developed for signal-amplified multiple miRNAs imaging in living cells with high sensitivity and selectivity. TDFs could enter cells via self-delivery with good biocompatibility and stability. Two kinds of MNAzymes specific for miRNA-21 and miRNA-155 with fluorescein labeling were encoded in the structure of TDFs respectively through one-step thermal annealing. In the intracellular environment, the TDFs could be specifically bound with its specific miRNA target and form an active DNAzyme structure. The cleavage of the active site would trigger the release of target miRNA and circular fluorescence signal amplification, which enabled accurate diagnosis on miRNA identifications of different cell lines with high sensitivity. Meanwhile, with the specific AS1411 aptamer targeting for nucleolin overexpressed on the surface of the carcinoma cells, this well-designed TDFs nanoprobe exhibited good discrimination between cancer cells and normal cells. The strategy provides an efficient tool for understanding the biological function of miRNAs in cancer pathogenesis and therapeutic applications.


Assuntos
DNA/química , MicroRNAs/química , Imagem Molecular/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Espaço Intracelular/metabolismo , Microscopia de Força Atômica , Sondas Moleculares/química , Nanotecnologia/métodos , Conformação de Ácido Nucleico
10.
Nature ; 589(7843): 630-632, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33500572

Assuntos
Anticorpos/uso terapêutico , Biologia Celular , Biologia do Desenvolvimento , Nariz Eletrônico , Espectrometria de Massas/instrumentação , Neurociências , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/imunologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Bioimpressão/tendências , /imunologia , /química , /provisão & distribução , Biologia Celular/instrumentação , Biologia Celular/tendências , Biologia do Desenvolvimento/métodos , Biologia do Desenvolvimento/tendências , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Holografia/tendências , Humanos , Imunoglobulina E/química , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina E/uso terapêutico , Canais Iônicos/metabolismo , Espectrometria de Massas/métodos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/efeitos da radiação , Camundongos , Microscopia/instrumentação , Microscopia/tendências , Sondas Moleculares/análise , Neoplasias/tratamento farmacológico , Neurociências/métodos , Neurociências/tendências , Optogenética/tendências , Análise de Célula Única , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
Nat Commun ; 12(1): 109, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397937

RESUMO

Zn2+ plays important roles in metabolism and signaling regulation. Subcellular Zn2+ compartmentalization is essential for organelle functions and cell biology, but there is currently no method to determine Zn2+ signaling relationships among more than two different organelles with one probe. Here, we report simultaneous Zn2+ tracking in multiple organelles (Zn-STIMO), a method that uses structured illumination microscopy (SIM) and a single Zn2+ fluorescent probe, allowing super-resolution morphology-correlated organelle identification in living cells. To guarantee SIM imaging quality for organelle identification, we develop a new turn-on Zn2+ fluorescent probe, NapBu-BPEA, by regulating the lipophilicity of naphthalimide-derived Zn2+ probes to make it accumulate in multiple organelles except the nucleus. Zn-STIMO with this probe shows that CCCP-induced mitophagy in HeLa cells is associated with labile Zn2+ enhancement. Therefore, direct organelle identification supported by SIM imaging makes Zn-STIMO a reliable method to determine labile Zn2+ dynamics in various organelles with one probe. Finally, SIM imaging of pluripotent stem cell-derived organoids with NapBu-BPEA demonstrates the potential of super-resolution morphology-correlated organelle identification to track biospecies and events in specific organelles within organoids.


Assuntos
Rastreamento de Células , Organelas/metabolismo , Zinco/metabolismo , Autofagossomos/metabolismo , Autofagia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Sobrevivência Celular , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Espaço Intracelular/metabolismo , Lisossomos/metabolismo , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Naftalimidas/metabolismo , Organoides/metabolismo , Espectrometria de Fluorescência
12.
Nat Commun ; 12(1): 717, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514717

RESUMO

The Neisseria meningitidis protein FrpC contains a self-processing module (SPM) undergoing autoproteolysis via an aspartic anhydride. Herein, we establish NeissLock, using a binding protein genetically fused to SPM. Upon calcium triggering of SPM, the anhydride at the C-terminus of the binding protein allows nucleophilic attack by its target protein, ligating the complex. We establish a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We optimize NeissLock using the Ornithine Decarboxylase/Antizyme complex. Various sites on the target (α-amine or ε-amines) react with the anhydride, but reaction is blocked if the partner does not dock. Ligation is efficient at pH 7.0, with half-time less than 2 min. We arm Transforming Growth Factor-α with SPM, enabling specific covalent coupling to Epidermal Growth Factor Receptor at the cell-surface. NeissLock harnesses distinctive protein chemistry for high-yield covalent targeting of endogenous proteins, advancing the possibilities for molecular engineering.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Sondas Moleculares/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos , Anidridos/metabolismo , Animais , Imagem Molecular/métodos , Sondas Moleculares/química , Sondas Moleculares/genética , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
13.
Methods Mol Biol ; 2217: 71-81, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33215378

RESUMO

The in situ proximity ligation assay (PLA) is capable of detecting single protein events such as protein protein-interactions and posttranslational modifications (e.g., protein phosphorylation) in tissue and cell samples prepared for analysis by immunofluorescent or immunohistochemical microscopy. The targets are detected using two primary antibodies which must be from different host species. A pair of secondary antibodies (PLA probes) conjugated to complementary oligonucleotides is applied to the sample, and a signal is generated only when the two PLA probes are in close proximity by their binding to the two primary antibodies that have bound to their targets in close proximity. The signal from each pair of PLA probes is visualized as an individual fluorescent spot. These PLA signals can be quantified (counted) using image analysis software (ImageJ), and also assigned to a specific subcellular location based on microscopy image overlays. In principle, in situ PLA offers a relatively simple and sensitive technique to analyze interactions among any proteins for which suitable antibodies are available. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes consisting of more than 150 proteins, also known as the integrin adhesome, which link the extracellular matrix (ECM) to the actin cytoskeleton and regulate the functioning of mechanosignaling pathways. The in situ PLA approach is well suited for examining the spatiotemporal aspects of protein posttranslational modifications and protein interactions occurring in dynamic multiprotein complexes such as integrin mediated focal adhesions.


Assuntos
Adesões Focais/metabolismo , Imuno-Histoquímica/métodos , Cadeias alfa de Integrinas/metabolismo , Integrina beta1/metabolismo , Complexos Multiproteicos/metabolismo , Oligonucleotídeos/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Anticorpos/química , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Adesões Focais/ultraestrutura , Mucosa Gástrica/metabolismo , Mucosa Gástrica/ultraestrutura , Humanos , Processamento de Imagem Assistida por Computador , Cadeias alfa de Integrinas/química , Integrina beta1/química , Microscopia de Fluorescência , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Complexos Multiproteicos/química , Músculo Liso/metabolismo , Músculo Liso/ultraestrutura , Oligonucleotídeos/síntese química , Ligação Proteica
14.
J Med Chem ; 64(1): 298-325, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33356214

RESUMO

Elevated levels of reactive oxygen species (ROS) have commonly been implicated in a variety of diseases, including cancer, inflammation, and neurodegenerative diseases. In light of significant differences in ROS levels between the nonpathogenic and pathological tissues, an increasing number of ROS-responsive prodrugs, probes, and theranostic prodrugs have been developed for the targeted treatment and precise diagnosis of ROS-related diseases. This review will summarize and provide insight into recent advances in ROS-responsive prodrugs, fluorescent probes, and theranostic prodrugs, with applications to different ROS-related diseases and various subcellular organelle-targetable and disease-targetable features. The ROS-responsive moieties, the self-immolative linkers, and the typical activation mechanism for the ROS-responsive release are also summarized and discussed.


Assuntos
Sondas Moleculares/metabolismo , Medicina de Precisão , Pró-Fármacos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Humanos
15.
Methods Mol Biol ; 2183: 9-18, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959237

RESUMO

The immunoglobulin capture assay (ICA) enables the enrichment for pathogen-specific plasmablasts from individuals with a confirmed adaptive immune response to vaccination or disseminated infection. Only single recombinant antigens have been used previously as probes in this ICA and it was unclear whether the method was applicable to complex probes such as whole bacterial cells. Here, we describe the enrichment of plasmablasts specific for polysaccharide and protein antigens of both Streptococcus pneumoniae and Neisseria meningitidis using whole formalin-fixed bacterial cells as probes. The modified ICA protocol described here allowed for a pathogen-specific hmAb cloning efficiency of >80%.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos/imunologia , Bactérias/imunologia , Imunoensaio/métodos , Sondas Moleculares , Anticorpos Antibacterianos/imunologia , Afinidade de Anticorpos , Formação de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunoglobulina G/imunologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Streptococcus pneumoniae/imunologia
16.
Methods Mol Biol ; 2183: 19-28, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959238

RESUMO

Chlamydia trachomatis is one of the most prevalent sexually transmitted infectious agents in the world and the leading cause of infectious blindness. The role of antibodies in the prevention and clearance of infection is still not fully understood, but the analysis of the immunoglobulin response to novel vaccine candidates is an important part of many of these studies. In this chapter, we describe a novel method to identify and isolate Chlamydia-specific memory B cells by fluorescence-activated cell sorting (FACS) using fluorescently labeled whole bacteria from cryopreserved human PBMC samples. This method allows for live single cells to be sorted for cell culture, in vitro assays, single-cell RNA sequencing, and cloning of paired heavy and light chains for recombinant monoclonal antibody production.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Chlamydia/imunologia , Sondas Moleculares , Anticorpos Antibacterianos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Vacinas Bacterianas/imunologia , Criopreservação , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Memória Imunológica , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo
17.
Angew Chem Int Ed Engl ; 60(12): 6799-6806, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33350010

RESUMO

Activity-based probes are valuable tools for chemical biology. However, finding probes that specifically target the active site of an enzyme remains a challenging task. Herein, we present a ligand selection strategy that allows to rapidly tailor electrophilic probes to a target of choice and showcase its application for the two cysteine proteases of SARS-CoV-2 as proof of concept. The resulting probes were specific for the active site labeling of 3CLpro and PLpro with sufficient selectivity in a live cell model as well as in the background of a native human proteome. Exploiting the probes as tools for competitive profiling of a natural product library identified salvianolic acid derivatives as promising 3CLpro inhibitors. We anticipate that our ligand selection strategy will be useful to rapidly develop customized probes and discover inhibitors for a wide range of target proteins also beyond corona virus proteases.


Assuntos
/química , Inibidores de Cisteína Proteinase/química , Técnicas de Sonda Molecular , Sondas Moleculares/química , Bibliotecas de Moléculas Pequenas/química , Domínio Catalítico , /metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Células Hep G2 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Estudo de Prova de Conceito , Ligação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade
18.
J Med Chem ; 63(24): 15308-15332, 2020 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-33307693

RESUMO

Tuberculosis (TB) remains one of the deadliest infectious diseases and begs the scientific community to up the ante for research and exploration of completely novel therapeutic avenues. Chemical biology-inspired design of tunable chemical tools has aided in clinical diagnosis, facilitated discovery of therapeutics, and begun to enable investigation of virulence mechanisms at the host-pathogen interface of Mycobacterium tuberculosis. This Perspective highlights chemical tools specific to mycobacterial proteins and the cell lipid envelope that have furnished rapid and selective diagnostic strategies and provided unprecedented insights into the function of the mycobacterial proteome and lipidome. We discuss chemical tools that have enabled elucidating otherwise intractable biological processes by leveraging the unique lipid and metabolite repertoire of mycobacterial species. Some of these probes represent exciting starting points with the potential to illuminate poorly understood aspects of mycobacterial pathogenesis, particularly the host membrane-pathogen interactions.


Assuntos
Mycobacterium tuberculosis/metabolismo , Tuberculose/diagnóstico , Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Corantes Fluorescentes/química , Humanos , Lipídeos/química , Sondas Moleculares/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Peptidoglicano/química , Sulfatases/química , Sulfatases/metabolismo , Trealose/química , Tuberculose/microbiologia , Virulência/genética
19.
Cell Rep ; 33(4): 108322, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33091382

RESUMO

Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Sondas Moleculares/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Especificidade de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Biotinilação , Microscopia Crioeletrônica , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo
20.
PLoS One ; 15(10): e0239282, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33095778

RESUMO

OBJECTIVES: To determine if the URO-MCP-1 mouse model for bladder IC/BPS is associated with in vivo bladder hyper-permeability, as measured by contrast-enhanced MRI (CE-MRI), and assess whether molecular-targeted MRI (mt-MRI) can visualize in vivo claudin-2 expression as a result of bladder hyper-permeability. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects primarily women. It is known that permeability plays a substantial role in IC/BPS. Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. Claudin-2 is a molecular marker that is associated with increased hyperpermeability in the urothelium. MATERIALS AND METHODS: CE-MRI was used to measure bladder hyper-permeability in the URO-MCP-1 mice. A claudin-2-specific mt-MRI probe was used to assess in vivo levels of claudin-2. The mt-MRI probe consists of an antibody against claudin-2 conjugated to albumin that had Gd-DTPA (gadolinium diethylenetriamine pentaacetate) and biotin attached. Verification of the presence of the mt-MRI probe was done by targeting the biotin moiety for the probe with streptavidin-horse radish peroxidase (SA-HRP). Trans-epithelial electrical resistance (TEER) was also used to assess bladder permeability. RESULTS: The URO-MCP-1 mouse model for IC/BPS was found to have a significant increase in bladder permeability, following liposaccharide (LPS) exposure, compared to saline-treated controls. mt-MRI- and histologically-detectable levels of the claudin-2 probe were found to increase with LPS -induced bladder urothelial hyper-permeability in the URO-MCP-1 IC mouse model. Levels of protein expression for claudin-2 were confirmed with immunohistochemistry and immunofluorescence imaging. Claudin-2 was also found to highly co-localize with zonula occlidens-1 (ZO-1), a tight junction protein. CONCLUSION: The combination of CE-MRI and TEER approaches were able to demonstrate hyper-permeability, a known feature associated with some IC/BPS patients, in the LPS-exposed URO-MCP-1 mouse model. This MRI approach could be clinically translated to establish which IC/BPS patients have bladder hyper-permeability and help determine therapeutic options. In addition, the in vivo molecular-targeted imaging approach can provide invaluable information to enhance our understanding associated with bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing further therapeutic strategies.


Assuntos
Claudina-2/metabolismo , Cistite Intersticial/patologia , Imagem por Ressonância Magnética/métodos , Sondas Moleculares/química , Bexiga Urinária/fisiopatologia , Animais , Anticorpos/química , Anticorpos/imunologia , Claudina-2/imunologia , Cistite Intersticial/metabolismo , Modelos Animais de Doenças , Gadolínio DTPA/química , Imuno-Histoquímica , Lipopolissacarídeos/toxicidade , Camundongos , Permeabilidade/efeitos dos fármacos , Albumina Sérica/química
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