Laura M. Doyle

Bio: Laura M. Doyle is an academic researcher from University of Kansas. The author has contributed to research in topics: Protein subcellular localization prediction & Organelle. The author has an hindex of 3, co-authored 3 publications receiving 512 citations.

Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis

Abstract: The use of extracellular vesicles, specifically exosomes, as carriers of biomarkers in extracellular spaces has been well demonstrated Despite their promising potential, the use of exosomes in the clinical setting is restricted due to the lack of standardization in exosome isolation and analysis methods The purpose of this review is to not only introduce the different types of extracellular vesicles but also to summarize their differences and similarities, and discuss different methods of exosome isolation and analysis currently used A thorough understanding of the isolation and analysis methods currently being used could lead to some standardization in the field of exosomal research, allowing the use of exosomes in the clinical setting to become a reality

A Mechanistic Understanding of Polysorbate 80 Oxidation in Histidine and Citrate Buffer Systems—Part 2

Abstract: In our previous published work, we reported rapid Polysorbate80 (PS80) oxidation in histidine buffer after brief stainless steel exposure and the ability of citrate and EDTA to prevent this oxidation. The focus of our current study was to mechanistically understand PS80 oxidation by studying the impact of temperature, light, stainless steel and the role of citrate and EDTA. Additionally, PS80 oxidation was studied in three different buffer sytems: histidine, citrate, and phosphate. When the PS80 containing buffers were exposed to elevated temperature of 50 °C in glass containers, no PS80 oxidation was observed in either histidine and citrate buffer systems after 30 days; however, PS80 oxidation was observed in phosphate buffer within 14 days. This study demonstrated that temperature does not initiate PS80 oxidation in histidine or citrate buffer systems, but may be a factor in phosphate buffer. When the 3 buffer systems containing PS80 were exposed to 20, 50, and 100 % ICH Q1B light conditions and subsequently incubated at 50°C (dark), the PS80 in phosphate buffer underwent oxidation within 7 days, whereas the PS80 in histidine and citrate buffer systems showed oxidation products only after 14 and 35 days, respectively. While PS80 in phosphate buffer seemed to be most vulnerable to light, PS80 in both histidine and citrate buffers underwent oxidation to a lesser extent, with the histidine buffer showing rapid oxidation compared to citrate buffer. Finally, the ability of citrate and EDTA to act not only as chelators, but also as a radical quencher/scavengers was demonstrated when metal ions such as Fe (2+) were spiked into histidine buffer containing PS80. While radicals could not be unambiguously identified by NMR or EPR, the observation of PS80 oxidation products indicate their presence.

Minimal Cerebrospinal Fluid Concentration of Miltefosine despite Therapeutic Plasma Levels during the Treatment of Amebic Encephalitis.

Abstract: Miltefosine is an alkylphosphocholine compound that is used primarily for treatment of leishmaniasis and demonstrates in vitro and in vivo antiamebic activity against Acanthamoeba species. Recommendations for treatment of amebic encephalitis generally include miltefosine therapy. Data indicate that treatment with an amebicidal concentration of at least 16 μg/ml of miltefosine is required for most Acanthamoeba species. Although there is a high level of mortality associated with amebic encephalitis, a paucity of data regarding miltefosine levels in plasma and cerebrospinal fluid in vivo exists in the literature. We found that despite aggressive dosing (oral miltefosine 50 mg every 6 h) and therapeutic plasma levels, the miltefosine concentration in cerebrospinal fluid was negligible in a patient with AIDS and Acanthamoeba encephalitis.

Development of a UPLC-MRM-based targeted proteomic method to profile subcellular organelle marker proteins from human liver tissues

Abstract: Abstract Subcellular organelles have long been an interest in biochemical research and drug development as the isolation of those organelles can help to probe protein functions and elucidate drug disposition within the cell. Usually, the purity of isolated subcellular organelle fractions was determined using immunoblot analysis of subcellular organelle marker proteins, which can be labor-intensive and lack reproducibility due to antibody batch-to-batch variability. As such, a higher throughput and more robust method is needed. Here, a UPLC-MRM-based targeted proteomic method was developed for a panel of human organelle marker proteins and used to profile a series of sucrose fractions isolated from the protein extract of human liver tissues. The method was validated by comparing to the traditional immunoblot and determining subcellular localization of three case study proteins (CYP3A4, FcRn, and β2M) pertaining to the disposition of small molecule and biologic drugs. All three case study proteins were co-enriched with their corresponding subcellular protein marker, and complete recoveries were achieved from isolated fractions. This newly developed MRM method for the panel of human organelle marker proteins can potentially accelerate future intracellular drug disposition analysis and facilitate subcellular organelle quality assessment.

The exosome journey: from biogenesis to uptake and intracellular signalling.

Abstract: The use of exosomes in clinical settings is progressively becoming a reality, as clinical trials testing exosomes for diagnostic and therapeutic applications are generating remarkable interest from the scientific community and investors. Exosomes are small extracellular vesicles secreted by all cell types playing intercellular communication roles in health and disease by transferring cellular cargoes such as functional proteins, metabolites and nucleic acids to recipient cells. An in-depth understanding of exosome biology is therefore essential to ensure clinical development of exosome based investigational therapeutic products. Here we summarise the most up-to-date knowkedge about the complex biological journey of exosomes from biogenesis and secretion, transport and uptake to their intracellular signalling. We delineate the major pathways and molecular players that influence each step of exosome physiology, highlighting the routes of interest, which will be of benefit to exosome manipulation and engineering. We highlight the main controversies in the field of exosome research: their adequate definition, characterisation and biogenesis at plasma membrane. We also delineate the most common identified pitfalls affecting exosome research and development. Unravelling exosome physiology is key to their ultimate progression towards clinical applications. Video Abstract.

Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics.

Abstract: Exosomes are small extracellular vesicles with diameters of 30-150 nm. In both physiological and pathological conditions, nearly all types of cells can release exosomes, which play important roles in cell communication and epigenetic regulation by transporting crucial protein and genetic materials such as miRNA, mRNA, and DNA. Consequently, exosome-based disease diagnosis and therapeutic methods have been intensively investigated. However, as in any natural science field, the in-depth investigation of exosomes relies heavily on technological advances. Historically, the two main technical hindrances that have restricted the basic and applied researches of exosomes include, first, how to simplify the extraction and improve the yield of exosomes and, second, how to effectively distinguish exosomes from other extracellular vesicles, especially functional microvesicles. Over the past few decades, although a standardized exosome isolation method has still not become available, a number of techniques have been established through exploration of the biochemical and physicochemical features of exosomes. In this work, by comprehensively analyzing the progresses in exosome separation strategies, we provide a panoramic view of current exosome isolation techniques, providing perspectives toward the development of novel approaches for high-efficient exosome isolation from various types of biological matrices. In addition, from the perspective of exosome-based diagnosis and therapeutics, we emphasize the issue of quantitative exosome and microvesicle separation.

Mesenchymal stem cell derived-exosomes: a modern approach in translational medicine.

Abstract: Mesenchymal stem cells (MSCs) have captured great attention in regenerative medicine for over a few decades by virtue of their differentiation capacity, potent immunomodulatory properties, and their ability to be favorably cultured and manipulated. Recent investigations implied that the pleiotropic effects of MSCs is not associated to their ability of differentiation, but rather is mediated by the secretion of soluble paracrine factors. Exosomes, nanoscale extracellular vesicles, are one of these paracrine mediators. Exosomes transfer functional cargos like miRNA and mRNA molecules, peptides, proteins, cytokines and lipids from MSCs to the recipient cells. Exosomes participate in intercellular communication events and contribute to the healing of injured or diseased tissues and organs. Studies reported that exosomes alone are responsible for the therapeutic effects of MSCs in numerous experimental models. Therefore, MSC-derived exosomes can be manipulated and applied to establish a novel cell-free therapeutic approach for treatment of a variety of diseases including heart, kidney, liver, immune and neurological diseases, and cutaneous wound healing. In comparison with their donor cells, MSC-derived exosomes offer more stable entities and diminished safety risks regarding the administration of live cells, e.g. microvasculature occlusion risk. This review discusses the exosome isolation methods invented and utilized in the clinical setting thus far and presents a summary of current information on MSC exosomes in translational medicine.