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1.
EMBO Rep ; 20(4)2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30872316

RESUMO

Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.

2.
Micromachines (Basel) ; 9(6)2018 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-30424207

RESUMO

The detection of small molecules has increasingly attracted the attention of researchers because of its important physiological function. In this manuscript, we propose a novel optical sensor which uses an optofluidic microbubble resonator (OFMBR) for the highly sensitive detection of small molecules. This paper demonstrates the binding of the small molecule biotin to surface-immobilized streptavidin with a detection limit reduced to 0.41 pM. Furthermore, binding specificity of four additional small molecules to surface-immobilized streptavidin is shown. A label-free OFMBR-based optical sensor has great potential in small molecule detection and drug screening because of its high sensitivity, low detection limit, and minimal sample consumption.

3.
Nature ; 563(7729): 131-136, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30356214

RESUMO

Accurate repair of DNA double-stranded breaks by homologous recombination preserves genome integrity and inhibits tumorigenesis. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that activates innate immunity by initiating the STING-IRF3-type I IFN signalling cascade1,2. Recognition of ruptured micronuclei by cGAS links genome instability to the innate immune response3,4, but the potential involvement of cGAS in DNA repair remains unknown. Here we demonstrate that cGAS inhibits homologous recombination in mouse and human models. DNA damage induces nuclear translocation of cGAS in a manner that is dependent on importin-α, and the phosphorylation of cGAS at tyrosine 215-mediated by B-lymphoid tyrosine kinase-facilitates the cytosolic retention of cGAS. In the nucleus, cGAS is recruited to double-stranded breaks and interacts with PARP1 via poly(ADP-ribose). The cGAS-PARP1 interaction impedes the formation of the PARP1-Timeless complex, and thereby suppresses homologous recombination. We show that knockdown of cGAS suppresses DNA damage and inhibits tumour growth both in vitro and in vivo. We conclude that nuclear cGAS suppresses homologous-recombination-mediated repair and promotes tumour growth, and that cGAS therefore represents a potential target for cancer prevention and therapy.

4.
Sensors (Basel) ; 18(2)2018 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-29425166

RESUMO

Total internal reflection (TIR) is useful for interrogating physical and chemical processes that occur at the interface between two transparent media. Yet prism-coupled TIR imaging microscopes suffer from limited sensing areas due to the fact that the interface (the object plane) is not perpendicular to the optical axis of the microscope. In this paper, we show that an electrically tunable lens can be used to rapidly and reproducibly correct the focal length of an oblique-incidence scanning microscope (OI-RD) in a prism-coupled TIR geometry. We demonstrate the performance of such a correction by acquiring an image of a protein microarray over a scan area of 4 cm² with an effective resolution of less than 20 microns. The electronic focal length tuning eliminates the mechanical movement of the illumination lens in the scanning microscope and in turn the noise and background drift associated with the motion.

5.
J Biophotonics ; 11(5): e201700245, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29205885

RESUMO

The label-free imaging and spectroscopy method was studied on cervical unstained tissue sections obtained from 36 patients. The native fluorescence spectra of tissues are analyzed by the optical redox ratio (ORR), which is defined as fluorescence intensity ratio between NADH and FAD, and indicates the metabolism change with the cancer development. The ORRs of normal tissues are consistently higher than those of precancer or cancerous tissues. A criterion line of ORR at 5.0 can be used to discriminate cervical precancer/cancer from normal tissues. The sensitivity and specificity of the native fluorescence spectroscopy method for cervical cancer diagnosis are determined as 100% and 91%. Moreover, the native fluorescence spectroscopy study is much more sensitive on the healthy region of cervical precancer/cancer patients compared with the traditional clinical staining method. The results suggest label-free imaging and spectroscopy is a fast, highly sensitive and specific method on the detection of cervical cancer.

6.
Nanomaterials (Basel) ; 7(10)2017 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-29053580

RESUMO

TiO2 nanoparticles modified with phthalocyanines (Pc) have been proven to be a potential photosensitizer in the application of photodynamic therapy (PDT). However, the generation of reactive oxygen species (ROS) by TiO2 nanoparticles modified with Pc has not been demonstrated clearly. In this study, nitrogen-doped TiO2 conjugated with Pc (N-TiO2-Pc) were studied by means of monitoring the generation of ROS. The absorbance and photokilling effect on HeLa cells upon visible light of different regions were also studied and compared with non-doped TiO2-Pc and Pc. Both N-TiO2-Pc and TiO2-Pc can be activated by visible light and exhibited much higher photokilling effect on HeLa cells than Pc. In addition, nitrogen-doping can greatly enhance the formation of ¹O2 and •O2-, while it suppresses the generation of OH•. This resulted in significant photodynamic activity. Therefore, N-TiO2-Pc can be an excellent candidate for a photosensitizer in PDT with wide-spectrum visible irradiation.

7.
Cell Death Discov ; 3: 17034, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28698806

RESUMO

Poly(ADP-ribose) polymerases (PARPs) are ADP-ribosylating enzymes and play important roles in a variety of cellular processes. Most small-molecule PARP inhibitors developed to date have been against PARP1, a poly-ADP-ribose transferase, and suffer from poor selectivity. PARP16, a mono-ADP-ribose transferase, has recently emerged as a potential therapeutic target, but its inhibitor development has trailed behind. Here we newly characterized epigallocatechin-3-gallate (EGCG) as a potential inhibitor of PARP16. We found that EGCG was associated with PARP16 and dramatically inhibited its activity in vitro. Moreover, EGCG suppressed the ER stress-induced phosphorylation of PERK and the transcription of unfolded protein response-related genes, leading to dramatically increase of cancer cells apoptosis under ER stress conditions, which was dependent on PARP16. These findings newly characterized EGCG as a potential inhibitor of PARP16, which can enhance the ER stress-induced cancer cell apoptosis, suggesting that a combination of EGCG and ER stress-induced agents might represent a novel approach for cancer therapy or chemoprevention.

8.
Appl Opt ; 55(33): 9459-9466, 2016 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-27869851

RESUMO

Oblique-incidence reflectivity difference (OI-RD) is a form of polarization-modulation ellipsometry that measures properties of thin films on a solid surface through the change in polarization state of light upon reflection from the surface. The measurement accuracy depends on the precision of the phase modulation amplitude and azimuthal alignments of key polarizing optical elements and, thus, requires careful calibration. In the present work, we describe robust methods of such calibrations that enable precise determination of the modulation amplitude and static retardation of a phase modulator and azimuths of key polarizing optics in an OI-RD system.

9.
Anal Biochem ; 509: 67-72, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27372609

RESUMO

In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies.


Assuntos
Acanthamoeba castellanii/metabolismo , Análise Serial de Proteínas/métodos , Proteínas de Protozoários/metabolismo , Acanthamoeba castellanii/química , Proteínas de Protozoários/química
10.
Colloids Surf B Biointerfaces ; 143: 148-155, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27003465

RESUMO

Titanium dioxide nanoparticles (TiO2 NPs) have a potential in the field of biological application. However, its poor dispersibility in water hampered its applications. In this study, 3-phosphonopropionic acid and 3-aminopropyl-triethoxysilane were respectively used for surface modification on TiO2 NPs with negative and positive surface charges (denoted as TiO2-COOH and TiO2-NH2). Zeta potentials of the prepared samples with high absolute value demonstrate the great improvement in their dispersibility. In terms of viability experiment, both TiO2-COOH and TiO2-NH2 showed low cytotoxicity. The cellular uptake efficiency and the uptake pathways of TiO2-COOH and TiO2-NH2 for cancer cells were studied. The exocytosis of TiO2-NH2 was also observed in the experiment.


Assuntos
Portadores de Fármacos , Nanopartículas Metálicas/química , Compostos Organofosforados/química , Propilaminas/química , Silanos/química , Titânio/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Endocitose , Exocitose , Células HeLa , Humanos , Nanopartículas Metálicas/ultraestrutura , Eletricidade Estática , Propriedades de Superfície , Titânio/farmacologia
11.
Sensors (Basel) ; 16(3)2016 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-26999137

RESUMO

Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in drug discovery, and efficient immobilization of molecular compounds is a pre-requisite for the success of such a platform. On an isocyanate functionalized surface, we studied the dependence of immobilization efficiency on chemical residues on molecular compounds, terminal residues on isocyanate functionalized surface, lengths of spacer molecules, and post-printing treatment conditions, and we identified a set of optimized conditions that enable us to immobilize small molecules with significantly improved efficiencies, particularly for those molecules with carboxylic acid residues that are known to have low isocyanate reactivity. We fabricated microarrays of 3375 bioactive compounds on isocyanate functionalized glass slides under these optimized conditions and confirmed that immobilization percentage is over 73%.


Assuntos
Descoberta de Drogas , Isocianatos/química , Análise em Microsséries , Bibliotecas de Moléculas Pequenas/química , Vidro/química , Humanos , Bibliotecas de Moléculas Pequenas/farmacologia
12.
Nanomaterials (Basel) ; 6(6)2016 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-28335242

RESUMO

Titanium dioxide (TiO2) has attracted wide attention as a potential photosensitizer (PS) in photodynamic therapy (PDT). However, bare TiO2 can only be excited by ultraviolet illumination, and it lacks specific targeting ligands, which largely impede its application. In our study, we produced nitrogen-doped TiO2 and linked it with an effective cancer cell targeting agent, folic acid (FA), to obtain N-TiO2-FA nanoconjugates. Characterization of N-TiO2-FA included Zeta potential, absorption spectra and thermogravimetric analysis. The results showed that N-TiO2-FA was successfully produced and it possessed better dispersibility in aqueous solution than unmodified TiO2. The N-TiO2-FA was incubated with human nasopharyngeal carcinoma (KB) and human pulmonary adenocarcinoma (A549) cells. The KB cells that overexpress folate receptors (FR) on cell membranes were used as FR-positive cancer cells, while A549 cells were used as FR-negative cells. Laser scanning confocal microscopy results showed that KB cells had a higher uptake efficiency of N-TiO2-FA, which was about twice that of A549 cells. Finally, N-TiO2-FA is of no cytotoxicity, and has a better photokilling effect on KB cells under visible light irradiation. In conclusion, N-TiO2-FA can be as high-value as a PS in cancer targeting PDT.

13.
Biomolecules ; 5(3): 1480-98, 2015 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-26193329

RESUMO

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.


Assuntos
Vírus da Influenza A/metabolismo , Análise em Microsséries/métodos , Dispositivos Ópticos , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , Animais , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Virus da Influenza A Subtipo H5N1/metabolismo , Cinética , Ligantes , Análise em Microsséries/instrumentação , Especificidade da Espécie , Especificidade por Substrato , Termodinâmica
14.
Rev Sci Instrum ; 84(11): 114102, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24289409

RESUMO

A biological state is equilibrium of multiple concurrent biomolecular reactions. The relative importance of these reactions depends on physiological temperature typically between 10 °C and 50 °C. Experimentally the temperature dependence of binding reaction constants reveals thermodynamics and thus details of these biomolecular processes. We developed a variable-temperature opto-fluidic system for real-time measurement of multiple (400-10,000) biomolecular binding reactions on solid supports from 10 °C to 60 °C within ±0.1 °C. We illustrate the performance of this system with investigation of binding reactions of plant lectins (carbohydrate-binding proteins) with 24 synthetic glycans (i.e., carbohydrates). We found that the lectin-glycan reactions in general can be enthalpy-driven, entropy-driven, or both, and water molecules play critical roles in the thermodynamics of these reactions.


Assuntos
Dispositivos Ópticos , Lectinas de Plantas/metabolismo , Polissacarídeos/metabolismo , Temperatura Ambiente , Modelos Moleculares , Lectinas de Plantas/química , Polissacarídeos/química , Ligação Proteica , Conformação Proteica , Termodinâmica , Fatores de Tempo , Titânio/química
15.
Assay Drug Dev Technol ; 11(5): 326-32, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23772553

RESUMO

We present here a label-free microarray-based assay platform that we used to identify inhibitors of vascular endothelial growth factor (VEGF)-kinase-insertion domain receptor (KDR) binding. Supported by a combination of special ellipsometry-based optical detection and small molecule microarrays (SMM), this platform consists of three assays: (1) the first assay detects binding of a target protein with SMM and identifies ligands to the protein as inhibitor candidates; (2) the second assay detects binding of a receptor protein with identical SMM and subsequent binding of the target protein (a sandwich assay) to identify the ligands to the receptor protein that do not interfere with the target-receptor binding; (3) the third assay detects binding of the target protein to the receptor protein in the presence of the ligands of the target protein identified from the first assay, with the receptor protein immobilized to a solid surface through the ligands identified in the second assay, to yield dose-response curves. Using this platform, we screened 7,961 compounds from the National Cancer Institute and found 12 inhibitors to VEGF-KDR (VEGFR2) interactions with IC50 ranging from 0.3 to 60 µM. The inhibitory potency of these inhibitors found in the microarray-based assay was confirmed by their inhibition of VEGF-induced VEGFR2 phosphorylation in a cell-based assay.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Peptídeos/química , Análise Serial de Proteínas/métodos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Desenho de Drogas , Ligantes , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Coloração e Rotulagem
16.
Assay Drug Dev Technol ; 10(3): 250-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22192305

RESUMO

Fluorescence-based endpoint detection of microarrays with 10,000 or more molecular targets is a most useful tool for high-throughput profiling of biomolecular interactions, including screening large molecular libraries for novel protein ligands. However, endpoint fluorescence data such as images of reacted microarrays contain little information on kinetic rate constants, and the reliability of endpoint data as measures of binding affinity depends on reaction conditions and postreaction processing. We here report a simultaneous measurement of binding curves of a protein probe with 10,000 molecular targets in a microarray with an ellipsometry-based (label-free) optical scanner. The reaction rate constants extracted from these curves (k(on), k(off), and k(a)=k(on)/k(off)) are used to characterize the probe-target interactions instead of the endpoints. This work advances the microarray technology to a new milestone, namely, from an endpoint assay to a kinetic constant assay platform. The throughput of this binding curve assay platform is comparable to those at the National Institutes of Health Molecular Library Screening Centers, making it a practical method in screening compound libraries for novel ligands and for system-wide affinity profiling of proteins, viruses, or whole cells against diverse molecular targets.


Assuntos
Algoritmos , Modelos Químicos , Análise Serial de Proteínas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Simulação por Computador , Cinética , Ligantes
17.
Mol Biosyst ; 7(12): 3343-52, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22009201

RESUMO

Interactions of glycan-binding proteins (GBPs) with glycans are essential in cell adhesion, bacterial/viral infection, and cellular signaling pathways. Experimental characterization of these interactions based on glycan microarrays typically involves (1) labeling GBPs directly with fluorescent reagents before incubation with the microarrays, or (2) labeling GBPs with biotin before the incubation and detecting the captured GBPs after the incubation using fluorescently labeled streptavidin, or (3) detecting the captured GBPs after the incubation using fluorescently labeled antibodies raised against the GBPs. The fluorescent signal is mostly measured ex situ after excess fluorescent materials are washed off. In this study, by using a label-free optical scanner for glycan microarray detection, we measured binding curves of 7 plant lectins to 24 glycans: four ß1-4-linked galactosides, three ß1-3-linked galactosides, one ß-linked galactoside, one α-linked N-acetylgalactosaminide, eight α2-3-linked sialosides, and seven α2-6-linked sialosides. From association and dissociation constants deduced by global-fitting the binding curves, we found that (1) labeling lectins directly with fluorescent agents change binding profiles of lectins, in some cases by orders of magnitude; (2) those lectin-glycan binding reactions characterized with large dissociation rates, though biologically relevant, are easily missed or deemed insignificant in ex situ fluorescence-based assays as most captured lectins are washed off before detection. This study highlights the importance of label-free real-time detection of protein-ligand interactions and the potential pitfall in interpreting fluorescence-based assays for characterization of protein-glycan interactions.


Assuntos
Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Lectinas de Plantas/metabolismo , Polissacarídeos/metabolismo , Ligantes , Lectinas de Plantas/química , Análise Serial de Proteínas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Coloração e Rotulagem
18.
Int Drug Discov ; : 8-13, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22306883

RESUMO

Small-molecule compounds remain the major source of therapeutic and preventative drugs. Developing new drugs against a protein target often requires screening large collections of compounds with diverse structures for ligands or ligand fragments that exhibit sufficiently affinity and desirable inhibition effect on the target before further optimization and development. Since the number of small molecule compounds is large, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a suitable binding assay form a viable HTS platform. We demonstrate that by combining an oblique-incidence reflectivity difference optical scanner with SMM we can screen 10,000 small-molecule compounds on a single glass slide for protein ligands without fluorescence labeling. Furthermore using such a label-free assay platform we can simultaneously acquire binding curves of a solution-phase protein to over 10,000 immobilized compounds, thus enabling full characterization of protein-ligand interactions over a wide range of affinity constants.

19.
J Biomed Opt ; 15(1): 016018, 2010 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20210464

RESUMO

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm x 4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Microscopia de Fluorescência/métodos , Análise Serial de Proteínas/métodos , Animais , Biotina/metabolismo , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Imunoglobulina G/metabolismo , Células Jurkat , Camundongos , Bibliotecas de Moléculas Pequenas/metabolismo , Estreptavidina/metabolismo
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