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Quercetin encapsulated biodegradable plasmonic nanoparticles for photothermal therapy of hepatocellular carcinoma cells
摘要： Photothermal therapy (PTT) is emerging as an effective treatment modality for cancer due to its non-invasive nature. However, the pro-inflammatory necrotic cell death during PTT limits its successful clinical application. Here, we have developed quercetin (QE) loaded biodegradable plasmonic nanoparticles that can specifically induce apoptosis in cancer cells after PTT. We have synthesized gold-coated liposome (LiposAu) and QE loaded gold-coated liposome (QE-LiposAu) nanoparticles by in situ reduction of chloroauric acid with ascorbic acid in the presence of bare liposomes (Lipos) or QE loaded liposomes (QE-Lipos), respectively. The gold coating was confirmed by transmission electron microscopic analysis, dynamic light scattering, and zeta potential measurements. LiposAu and QE-LiposAu nanoparticles showed a similar level of temperature rise upon 750 nm near-infrared (NIR) laser (650 mW, 3 W cm-2) irradiation. The photothermal conversion efficiency of QE-LiposAu nanoparticles was determined to be ~75%. The efficacy of PTT was found to be dependent on the internalization efficiency of LiposAu nanoparticles in cancer cells. Importantly, QE-LiposAu nanoparticles showed increased PTT efficacy over LiposAu nanoparticles in hepatocellular carcinoma cells (Huh-7). Moreover, QE-LiposAu nanoparticles induced apoptosis-mediated cell death after the PTT, and the extent of apoptosis was significantly higher than the LiposAu nanoparticles in Huh-7 cells. Further, QE-LiposAu nanoparticles-mediated PTT depolymerized microtubules network, suppressed Hsp70 expression, and caused DNA damage. QE-LiposAu nanoparticles were also found to be hemocompatible. The results together suggested that biodegradable QE-LiposAu nanoparticles are promising photothermal agents for cancer therapy.
关键词： heat shock protein,liposome,microtubule,apoptosis,gold nanoparticles,DNA damage,Photothermal therapy
更新于2025-09-12 10:27:22
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Photocaged Quinone Methide Cross-linkers for Light-controlled Chemical Cross-linking of Protein-protein and Protein-DNA Complexes
摘要： Small molecule cross-linkers are invaluable for probing biomolecular interactions and for cross-linking mass spectrometry (CXMS) in addressing large protein complexes and intrinsically disordered proteins. Existing chemical cross-linkers target only a small selection of amino acid residues, limiting the number and type of cross-links, while conventional photocross-linkers target virtually all residues non-selectively, complicating data analysis. Here we report photocaged quinone methide (PQM)-based cross-linkers that are able to multitarget nine nucleophilic residues through specific Michael addition. In addition to Asp, Glu, Lys, Ser, Thr, and Tyr, PQM cross-linkers notably cross-linked Gln, Arg, and Asn hitherto untargetable by existing chemical cross-linkers, markedly increasing the number of residues targetable with a single cross-linker. Such multiplicity of cross-links will increase the abundance of cross-linked peptides for CXMS identification and afford ample constraints to facilitate structural deciphering. PQM cross-linkers were used in vitro, in E. coli, and in mammalian cells to cross-link dimeric proteins and endogenous membrane receptors. The cross-linker NHQM could directly cross-link proteins to DNA, for which few cross-linkers exist. The photoactivatable and multitargeting reactivity of these PQM cross-linkers will substantially enhance chemical cross-linking based technologies for studies of protein-protein and protein-DNA networks and for structural biology.
关键词： quinone methide,photo-cross-linking,DNA-protein interaction,cross-linker,protein-protein interaction
更新于2025-09-11 14:15:04
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Simultaneous measurement of p53:Mdm2 and p53:Mdm4 protein-protein interactions in whole cells using fluorescence labelled foci
摘要： In this report we describe the development of a fluorescent protein-protein interaction-visualization (FLUOPPI) to enable the simultaneous measurement of both Mdm2:p53 and Mdm4:p53 interactions in order to assess the relative efficiencies of mimetic molecules of the p53 peptide helix against both PPIs. Mdm2 and Mdm4 overexpression frequently leads to the inactivation of non-mutated p53 in human cancers, via inhibition of its transcriptional activity, enhancing its degradation by the proteasome or by preventing its nuclear import. Development of inhibitors to disrupt the binding of one or both of these protein interactions have been the subject of intensive pharmaceutical development for anti-cancer therapies. Using the bimodal FLUOPPI system we have characterised compounds that were either monospecific for Mdm2 or bispecific for both Mdm2 and Mdm4. We have also demonstrated that the FLUOPPI assay can reliably differentiate between specific and non-specific disruption of these protein complexes via accurate assessment and normalization to the cell population under measurement. We envision that this methodology will increase the efficiency of identifying compounds that are either specific against a single PPI from a closely related family of interactions or compounds that interact across multiple related PPI pairs, depending on which is more desirable.
关键词： Mdm4,Mdm2,protein-protein interactions,cancer therapy,FLUOPPI,p53
更新于2025-09-11 14:15:04
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Er:YAG Laser and Cyclosporin A Effect on Cell Cycle Regulation of Human Gingival Fibroblast Cells
摘要： Introduction: Periodontitis is a set of inflammatory disorders characterized by periodontal attachment loss and alveolar bone resorption. Because of deficiency in periodontitis mechanical therapy, this study was aimed to explore the molecular influence of the erbium-doped: yttrium aluminum garnet (Er:YAG) laser and cyclosporin A (CsA) on human gingival fibroblasts (HGFs) for improvement in periodontal diseases therapy. Methods: We focused on articles that studied the proteome profiles of HGFs after treatment with laser irradiation and application of CsA. The topological features of differentially expressed proteins were analyzed using Cytoscape Version 3.4.0 followed by module selection from the protein-protein interaction (PPI) network using Cluster ONE plugin. In addition, we performed gene ontology (GO) enrichment analysis for the densely connected region and key proteins in both PPI networks. Results: Analysis of PPI network of Er:YAG laser irradiation on HGFs lead to introducing YWHAZ, VCP, HNRNPU, YWHAE, UBA52, CLTC, FUS and IGHG1 as key proteins while similar analysis revealed that ACAT1, CTSD, ALDOA, ANXA2, PRDX1, LGALS3, ARHGDI and EEF1A1 are the crucial proteins related to the effect of drug. GO enrichment analysis of hub-bottleneck proteins of the 2 networks showed the different significant biological processes and cellular components. The functional enrichments of module of Er:YAG laser network are included as fatty acid transmembrane transport, cytokinesis, regulation of RNA splicing and asymmetric protein localization. There are not any significant clusters in network of HGF treated by CsA. Conclusion: The results indicate that there are 2 separate biomarker panels for the 2 treatment methods.
关键词： cyclosporin A (CsA),human fibroblast cell,Er:YAG laser,Protein-protein interaction (PPI) network analysis,Gene ontology
更新于2025-09-11 14:15:04
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Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism
摘要： Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signaling processes. These proteins often gain structure upon binding to their target proteins in multistep reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain and the disordered interaction domain from activator of thyroid hormone and retinoid receptors. We demonstrate that helix formation in a rapid binding reaction can be followed by stopped-flow synchrotron-radiation circular dichroism (CD) spectroscopy and describe the design of such a beamline. Fluorescence-monitored binding experiments of activator of thyroid hormone and retinoid receptors and nuclear coactivator binding domain display several kinetic phases, including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time-resolved CD experiments show that almost all helicity is formed upon initial association of the proteins or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron-radiation CD for mechanistic studies of protein-protein interactions.
关键词： coupled folding and binding,synchrotron-radiation circular dichroism,protein-protein interactions,Intrinsically disordered proteins,helix formation
更新于2025-09-11 14:15:04
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Quaternary Solar Cells with 12.5% Efficiency Enabled with Non‐Fullerene and Fullerene Acceptor Guests to Improve Open Circuit Voltage and Film Morphology
摘要： Vaccines and antivirals to combat dengue, Zika, and other flavivirus pathogens present a major, unmet medical need. Vaccine development has been severely challenged by the antigenic diversity of these viruses and the propensity of non-neutralizing, cross-reactive antibodies to facilitate cellular infection and increase disease severity. As an alternative, direct-acting antivirals targeting the flavivirus envelope protein, E, have the potential to act via an analogous mode of action without the risk of antibody-dependent enhancement of infection and disease. We previously discovered that structurally diverse small molecule inhibitors of the dengue virus E protein exhibit varying levels of antiviral activity against other flaviviruses in cell culture. Here, we demonstrate that the broad-spectrum activity of several cyanohydrazones against dengue, Zika, and Japanese encephalitis viruses is due to specific inhibition of E-mediated membrane fusion during viral entry and provide proof of concept for pharmacological inhibition of E as an antiviral strategy in vivo.
关键词： viral entry inhibitor,envelope protein inhibitor,dengue virus,antiviral,fusion inhibitor,flavivirus
更新于2025-09-11 14:15:04
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Simultaneous Unlocking Optoelectronic and Interfacial Properties of C <sub/>60</sub> for Ultrasensitive Immunosensing by Coupling to Metal?Organic Framework
摘要： Due to exceptional electron-accepting ability, light-absorption and delocalized conjugated structure, buckminsterfullerene (C60) has attracted fascinating interests in the field of organic solar cells. However, poor delocalization and accumulation of electrons for pristine C60 in physiological aqueous solution and difficulties in conjugation with biomolecules limit its extended photovoltaic applications in bioassay. Herein, we reported the non-covalent coupling of C60 to an electronically complementary porphyrin-derived metal-organic framework (PCN-224) with carboxyl-group terminals. Such assembly not only offered a friendly interface for bioconjugation but also resulted in a long-range ordering C60@PCN-224 donor-acceptor system that demonstrated an unprecedented photocurrent enhancement up to 10 times with respect to each component. As an example, by further cooperating with Nanobodies, the as-prepared C60@PCN-224 was applied to a photoelectrochemical (PEC) immunosensor for S100 calcium-binding protein B with by far the most promising detection activities. This work may open a new venue to unlock the great potential of C60 in PEC biosensing with excellent performances.
关键词： photoelectrochemical immunosensor,S100 calcium-binding protein B,Nanobodies,metal-organic framework,C60
更新于2025-09-11 14:15:04
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A Plasmonic Approach to Study Protein Interaction Kinetics through the Dimerization of Functionalized Ag Nanoparticles
摘要： Understanding the kinetics of protein interactions plays a key role in biology with significant implications for the design of analytical methods for disease monitoring and diagnosis in medical care, research and industrial applications. Herein, we introduce a novel plasmonic approach to study the binding kinetics of protein-ligand interactions following the formation of silver nanoparticles (Ag nps) dimers by UV-Vis spectroscopy that can be used as probes for antigen detection and quantification. to illustrate and test the method, the kinetics of the prototype biotin-streptavidin (Biot-StV) pair interaction was studied. controlled aggregates (dimers) of StV functionalized Ag nps were produced by adding stoichiometric quantities of gliadin-specific biotinylated antibodies (IgG-Biot). The dimerization kinetics was studied in a systematic way as a function of Ag NPs size and at different concentrations of IgG-Biot. The kinetics data have shown to be consistent with a complex reaction mechanism in which only the Ag NPs attached to the IgG-Biot located in a specific STV site are able to form dimers. These results help in elucidating a complex reaction mechanism involved in the dimerization kinetics of functionalized Ag nps, which can serve as probes in surface plasmon resonance-based bioassays for the detection and quantification of different biomarkers or analytes of interest.
关键词： biotin-streptavidin interaction,silver nanoparticles,dimerization,protein interaction kinetics,plasmonic approach,surface plasmon resonance,UV-Vis spectroscopy
更新于2025-09-11 14:15:04
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Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy
摘要： In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10–11 mM of the surfactant n-tetradecyl-β-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan’s GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan’s REES values are high (9.3–18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4–5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan’s excited state dipole moment, and “solvent” reorientation around Laurdan’s chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan’s lifetime.
关键词： glycerol dialkyl glycerol tetraether (GDGT),liposomes,intrinsic protein fluorescence,microvesicles,red edge excitation shift (REES),glycerol dialkyl calditol tetraether (GDNT),generalized polarization (GP),thermoacidophilic archaea,membrane probe,Laurdan
更新于2025-09-11 14:15:04
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<i>ATF6</i> Is Mutated in Early Onset Photoreceptor Degeneration With Macular Involvement
摘要： PURPOSE. Photoreceptor degeneration (PRD) is a genetically heterogeneous retinal disorder. Although a number of genes involved in PRD have been identi?ed, their genetic basis remains unknown in a signi?cant number of patients. In this study, we aimed to identify novel disease-causing genes of PRD. METHODS. Comprehensive ocular examinations were performed in a 2-year-old patient diagnosed with early onset PRD. Retinal capture sequencing was performed to screen causative mutations in known retinal disease-causing loci. Whole-exome sequencing (WES) and a series of variant-?ltering strategies were applied for identifying novel disease-causing genes. Retina ATF6 expression was con?rmed by immunohistochemistry. RT-PCR was performed to identify ATF6 mRNA in the patient. RESULTS. The patient showed typical PRD features, with macular involvement and ellipsoid zone irregularities. Results of retinal capture sequencing were negative. WES data led to identi?cation of biallelic loss-of-function mutations in the ATF6 gene. The ?rst variant generates a premature stop codon (NCBI accession no. NM_007348: c.1126C>T, p.R376*) and the second variant affects a splicing donor site (NM_007348: c.1533t1G>C). Sanger sequencing con?rmed the 2 alleles are from 1 parent each. Both of the variants are extremely rare in the population. The splicing variant causes either intron inclusion or exon skipping in the patient, thus severely disrupting ATF6 functional domains. ATF6 is expressed in three neuronal cell layers of mouse retina. CONCLUSIONS. Our results support ATF6 as a novel disease-causing gene for PRD and suggest that disrupted protein quality control mechanisms may be a novel pathological mechanism underlying human retinal degeneration.
关键词： unfolded protein response,next-generation sequencing,ATF6,photoreceptor degeneration
更新于2025-09-11 14:15:04