Browsing by Author "Jiang, Bo"

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    Deconvoluting binding sites in amyloid nanofibrils using time-resolved spectroscopy
    (Royal Society of Chemisty, 2023) Jiang, Bo; Umezaki, Utana; Augustine, Andrea; Jayasinghe-Arachchige, Vindi M.; Serafim, Leonardo F.; He, Zhi Mei Sonia; Wyss, Kevin M.; Prabhakar, Rajeev; Martí, Angel A.
    Steady-state fluorescence spectroscopy has a central role not only for sensing applications, but also in biophysics and imaging. Light switching probes, such as ruthenium dipyridophenazine complexes, have been used to study complex systems such as DNA, RNA, and amyloid fibrils. Nonetheless, steady-state spectroscopy is limited in the kind of information it can provide. In this paper, we use time-resolved spectroscopy for studying binding interactions between amyloid-β fibrillar structures and photoluminescent ligands. Using time-resolved spectroscopy, we demonstrate that ruthenium complexes with a pyrazino phenanthroline derivative can bind to two distinct binding sites on the surface of fibrillar amyloid-β, in contrast with previous studies using steady-state photoluminescence spectroscopy, which only identified one binding site for similar compounds. The second elusive binding site is revealed when deconvoluting the signals from the time-resolved decay traces, allowing the determination of dissociation constants of 3 and 2.2 μM. Molecular dynamic simulations agree with two binding sites on the surface of amyloid-β fibrils. Time-resolved spectroscopy was also used to monitor the aggregation of amyloid-β in real-time. In addition, we show that common polypyridine complexes can bind to amyloid-β also at two different binding sites. Information on how molecules bind to amyloid proteins is important to understand their toxicity and to design potential drugs that bind and quench their deleterious effects. The additional information contained in time-resolved spectroscopy provides a powerful tool not only for studying excited state dynamics but also for sensing and revealing important information about the system including hidden binding sites.
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    Intratumoral Heterogeneity and Clonal Evolution Induced by HPV Integration
    (AACR, 2023) Akagi, Keiko; Symer, David E.; Mahmoud, Medhat; Jiang, Bo; Goodwin, Sara; Wangsa, Darawalee; Li, Zhengke; Xiao, Weihong; Dan Dunn, Joe; Ried, Thomas; Coombes, Kevin R.; Sedlazeck, Fritz J.; Gillison, Maura L.
    The human papillomavirus (HPV) genome is integrated into host DNA in most HPV-positive cancers, but the consequences for chromosomal integrity are unknown. Continuous long-read sequencing of oropharyngeal cancers and cancer cell lines identified a previously undescribed form of structural variation, “heterocateny,” characterized by diverse, interrelated, and repetitive patterns of concatemerized virus and host DNA segments within a cancer. Unique breakpoints shared across structural variants facilitated stepwise reconstruction of their evolution from a common molecular ancestor. This analysis revealed that virus and virus–host concatemers are unstable and, upon insertion into and excision from chromosomes, facilitate capture, amplification, and recombination of host DNA and chromosomal rearrangements. Evidence of heterocateny was detected in extrachromosomal and intrachromosomal DNA. These findings indicate that heterocateny is driven by the dynamic, aberrant replication and recombination of an oncogenic DNA virus, thereby extending known consequences of HPV integration to include promotion of intratumoral heterogeneity and clonal evolution.Long-read sequencing of HPV-positive cancers revealed “heterocateny,” a previously unreported form of genomic structural variation characterized by heterogeneous, interrelated, and repetitive genomic rearrangements within a tumor. Heterocateny is driven by unstable concatemerized HPV genomes, which facilitate capture, rearrangement, and amplification of host DNA, and promotes intratumoral heterogeneity and clonal evolution.See related commentary by McBride and White, p. 814.This article is highlighted in the In This Issue feature, p. 799
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    Investigating the Amyloid-beta aggregation and oxidation using metal complexes
    (2018-03-22) Jiang, Bo; Marti, Angel
    Amyloid-β (Aβ), a short peptide which self-assembles into large aggregates, was observed in the gray matter of Alzheimer’s patients. The aggregates in oligomeric and fibrillar forms were proven to be toxic to our brain. Based on this, many groups have taken on the task of investigating the aggregation process of Aβ. Previous work in our lab has shown that metal complexes can be used as a new family of photoluminescence probes for the detection of Aβ aggregates. More specifically, Dr. Amir Aliyan in our research group showed that [Re(CO)3(dppz)(Py)]+ exhibits a secondary light-switching response in the presence of Aβ fibrils upon UV irradiation, and at the same time performs oxidation on Aβ fibrils. This thesis focuses on investigating the interactions between Aβ and rhenium complexes and also probing Aβ oligomerization using ruthenium complexes. Chapter 1 is an introduction of the photoluminescence probes for the detection of Aβ aggregates, and the previous work of our lab on the metal complexes. Chapter 2 details the interactions between Aβ fibrils and [Re(CO)3(dppz)(Py)]+. Job plot and binding assay were used to determine the dissociation constant Kd as 4.2 ± 0.6 μM. Molecular dynamics simulations were used to propose a binding site for [Re(CO)3(dppz)(Py)]+ on Aβ fibrils is at a hydrophobic cleft between Val18 and Phe20. Due to the fact that Aβ fibrils are oxidized by [Re(CO)3(dppz)(Py)]+ after UV irradiation, the binding site was studied using the oxidation site as a chemical footprint. In addition, the study of the photooxidation of Aβ monomers showed that after UV irradiation His13 and Tyr10 are also prone to be oxidized by [Re(CO)3(dppz)(Py)]+. In order to further study the secondary light-switching behavior of [Re(CO)3(dppz)(Py)]+, functional groups were used to simulate the amino acids of Aβ and we found the photoluminescence of [Re(CO)3(dppz)(Py)]+ was enhanced in the presence of imidazole and dimethyl sulfide in SDS solutions but not in buffer. In addition, the quantum yield of singlet oxygen produced by [Re(CO)3(dppz)(Py)]+ upon UV irradiation, power flux of the irradiation source, and the quantum yields of photooxidation were determined. In chapter 3, I reported using photoluminescence anisotropy of [Ru(bpy)2(dpqp)]2+ for the detection of Aβ oligomerization. Aβ oligomers are believed to form immediately following monomers, however they are invisible to fluorescence sensors, such as Thioflavin T. Given that photoluminescence anisotropy is sensitive to the rotational correlation time of molecules, it is useful for monitoring the formation of biomolecule aggregates. We found that Aβ oligomers start to form from time zero with a steady increase in anisotropy that plateaus after 48 hours. The real-time monitoring of Aβ oligomers is of great importance for understanding the kinetics of aggregation, the forces that bring peptides together and study their inhibition. The formation of Aβ oligomers was supported by Western Blot analysis.
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    Investigation of the amyloid-beta aggregation and oxidation using photoluminescent metal complexes
    (2021-12-03) Jiang, Bo; Marti, Angel A.
    Amyloid-β (Aβ), a short peptide of 39 to 42 amino acids, is formed by the cleavage of the amyloid precursor protein by proteases in the membrane of neurons, and self-assembles into aggregated species. These aggregates take oligomeric (soluble) and fibrillar (insoluble) forms, which have been proven to be toxic to our brain, playing a key role of the onset of Alzheimer’s disease. In the past few decades, many groups have taken on the task of investigating the aggregation process of Aβ. Previous work in our lab has shown that metal complexes can be used as a new family of photoluminescent probes for the detection of Aβ aggregates. More specifically, we have shown that [Re(CO)3(dppz)(Py)]+ can photooxidize Aβ, providing a new way to investigate the interaction between metal complexes and these species. This thesis covers different topics related to the interaction of Aβ and metal complexes including probing Aβ oligomerization using ruthenium complexes, investigating the interactions between Aβ species and rhenium complexes, and exploring the inhibition effect and degradation of Aβ aggregates. Chapter 1 is a review of the photoluminescent metal complexes that have been developed for detection of Aβ aggregates. In the last few years this area of research has exploded making available photoluminescent metal complexes of ruthenium, iridium, rhenium and platinum for the study of Aβ aggregates. In chapter 2, I reported using the photoluminescence anisotropy of [Ru(bpy)2(dpqp)]2+ for the detection in real time of Aβ oligomers. Aβ oligomers are believed to form immediately following monomers, however they are invisible to fluorescence sensors such as Thioflavin T. Given that photoluminescence anisotropy is sensitive to the rotational correlation time of molecules, it is useful for monitoring the formation of biomolecule aggregates. We found that Aβ oligomers start to form from time zero with a steady increase in anisotropy that plateaus after 48 hours. The real-time monitoring of Aβ oligomers is of great importance for understanding the kinetics of aggregation, the forces that bring peptides together and study their inhibition. The formation of Aβ oligomers was supported by various characterization techniques including Western Blot analysis, SDS-PAGE analysis, dynamic light scattering analysis, transmittance electron microscopy and atomic force microscopy. Chapter 3 details the interactions between Aβ fibrils and [Re(CO)3(dppz)(Py)]+. Job plot and binding assay were used to determine the dissociation constant (Kd) as 4.2 ± 0.6 μM. Molecular dynamics simulations were used to propose a binding site for [Re(CO)3(dppz)(Py)]+ on Aβ fibrils at a hydrophobic cleft between Val18 and Phe20. Due to the fact that Aβ fibrils are oxidized by [Re(CO)3(dppz)(Py)]+ after UV irradiation, the binding site was studied using the oxidation site as a chemical footprint. In addition, the study of the photooxidation of Aβ monomers showed that after UV irradiation His14 is the most likely oxidized residue by [Re(CO)3(dppz)(Py)]+. In order to further study the secondary light-switching behavior of [Re(CO)3(dppz)(Py)]+, functional groups were used to simulate the amino acids of Aβ. We found that the photoluminescence of [Re(CO)3(dppz)(Py)]+ was enhanced in the presence of imidazole and dimethyl sulfide, indicating potential photochemical reaction was occurred . In addition, the quantum yield of singlet oxygen produced by [Re(CO)3(dppz)(Py)]+ upon UV irradiation, power flux of the irradiation source, and the quantum yields of photooxidation were determined. In chapter 4, we used [Ru(bpy)2(dpqp)]2+ in conjunction with time-resolved photoluminescence spectroscopy to assess Aβ aggregation. The added information available in the time-decay curves can be mathematically deconvoluted to obtain specific information about [Ru(bpy)2(dpqp)]2+ bound to Aβ. By considering a two sites non-cooperative binding model, the existence of two different binding sites on Aβ was discovered: one that affects the lifetime of [Ru(bpy)2(dpqp)]2+ and one that shows not affect its lifetime. These binding sites were further studied using MD simulations. The formation of Aβ aggregates was monitored in real-time using time-resolved photoluminescence spectrosocopy and confirmed using AFM. Chapter 5 investigated the inhibition of Aβ aggregation using a real-time assay. The results indicate that upon UV irradiation of [Re(CO)3(dppz)(Py)]+ with Aβ monomers, fibrillar aggregates are not produced. Further studies indicated that the photooxidized Aβ monomers play an important role for the inhibition effect. More investigations including MD simulation and other characterization are needed to explore the mechanism of this inhibition effect which could provide a unique method for the therapeutics of AD. In addition, we studied the photo-degradation of Aβ fibrils using rhenium complexes upon UV irradiation. The spectroscopic results and AFM images confirmed degradation of fibrillar species into small fragments and oligomers. This project is not yet done and needs more detailed and fundamental studies, but will be extremely helpful for the development of therapeutic strategies of AD.