Biomolecular Approaches Research Articles

Advantage of Nanotechnology-Based Genome Editing System and Its Application in Crop Improvement.

Agriculture is an important source of human food. However, current agricultural practices need modernizing and strengthening to fulfill the increasing food requirements of the growing worldwide population. Genome editing (GE) technology has been used to produce plants with improved yields and nutritional value as well as with higher resilience to herbicides, insects, and diseases. Several GE tools have been developed recently, including clustered regularly interspaced short palindromic repeats (CRISPR) with nucleases, a customizable and successful method. The main steps of the GE process involve introducing transgenes or CRISPR into plants via specific gene delivery systems. However, GE tools have certain limitations, including time-consuming and complicated protocols, potential tissue damage, DNA incorporation in the host genome, and low transformation efficiency. To overcome these issues, nanotechnology has emerged as a groundbreaking and modern technique. Nanoparticle-mediated gene delivery is superior to conventional biomolecular approaches because it enhances the transformation efficiency for both temporal (transient) and permanent (stable) genetic modifications in various plant species. However, with the discoveries of various advanced technologies, certain challenges in developing a short-term breeding strategy in plants remain. Thus, in this review, nanobased delivery systems and plant genetic engineering challenges are discussed in detail. Moreover, we have suggested an effective method to hasten crop improvement programs by combining current technologies, such as speed breeding and CRISPR/Cas, with nanotechnology. The overall aim of this review is to provide a detailed overview of nanotechnology-based CRISPR techniques for plant transformation and suggest applications for possible crop enhancement.

Comparing the Use of Magnetic Beads with Ultrafiltration for Ancient Dental Calculus Proteomics.

Over the past two decades, proteomic analysis has greatly developed in application to the field of biomolecular archaeology, coinciding with advancements in LC-MS/MS instrumentation sensitivity and improvements in sample preparation methods. Recently, human dental calculus has received much attention for its well-preserved proteomes locked in mineralized dental plaque which stores information on human diets and the oral microbiome otherwise invisible to other biomolecular approaches. Maximizing proteome recovery in ancient dental calculus, available only in minute quantities and irreplaceable after destructive analysis, is of paramount importance. Here, we compare the more traditional ultrafiltration-based and acetone precipitation approaches with the newer paramagnetic bead approach in order to test the influence of demineralization acid on recovered proteome complexity obtained from specimens as well as the sequence coverages matched for significant proteins. We found that a protocol utilizing EDTA combined with paramagnetic beads increased proteome complexity, in some cases doubling the number of unique peptides and number of proteins matched, compared to protocols involving the use of HCl and either acetone precipitation or ultrafiltration. Although the increase in the number of proteins was almost exclusively of bacterial origin, a development that has implications for the study of diseases within these ancient populations, an increase in the peptide number for the dairy proteins β-lactoglobulin and casein was also observed reflecting an increase in sequence coverage for these dietary proteins of interest. We also consider structural explanations for the discrepancies observed between these two key dietary proteins preserved in archaeological dental calculus.

Isolation, culture, and characterization of chicken intestinal epithelial cells

BackgroundEnterocytes exert an absorptive and protective function in the intestine, and they encounter many different challenging factors such as feed, bacteria, and parasites. An intestinal epithelial in vitro model can help to understand how enterocytes are affected by these factors and contribute to the development of strategies against pathogens.ResultsThe present study describes a novel method to culture and maintain primary chicken enterocytes and their characterization by immunofluorescence and biomolecular approaches. Starting from 19-day-old chicken embryos it was possible to isolate viable intestinal cell aggregates that can expand and produce a self-maintaining intestinal epithelial cell population that survives until 12 days in culture. These cells resulted positive in immunofluorescence to Cytokeratin 18, Zonula occludens 1, Villin, and Occludin that are common intestinal epithelial markers, and negative to Vimentin that is expressed by endothelial cells. Cells were cultured also on Transwell® permeable supports and trans-epithelial electrical resistance, was measured. This value gradually increased reaching 64 Ω*cm2 7 days after seeding and it remained stable until day 12.ConclusionsBased on these results it was confirmed that it is possible to isolate and maintain chicken intestinal epithelial cells in culture and that they can be suitable as in vitro intestinal model for further studies.

Volume and compressibility differences between protein conformations revealed by high-pressure NMR

Proteins often interconvert between different conformations in ways critical to their function. Although manipulating such equilibria for biophysical study is often challenging, the application of pressure is a potential route to achieve such control by favoring the population of lower volume states. Here, we use this feature to study the interconversion of ARNT PAS-B Y456T, which undergoes a dramatic +3 slip in the β-strand register as it switches between two stably folded conformations. Using high-pressure biomolecular NMR approaches, we obtained the first, to our knowledge, quantitative data testing two key hypotheses of this process: the slipped conformation is both smaller and less compressible than the wild-type equivalent, and the interconversion proceeds through a chiefly unfolded intermediate state. Data collected in steady-state pressure and time-resolved pressure-jump modes, including observed pressure-dependent changes in the populations of the two conformers and increased rate of interconversion between conformers, support both hypotheses. Our work exemplifies how these approaches, which can be generally applied to protein conformational switches, can provide unique information that is not easily accessible through other techniques.

The SARS-CoV-2 B.1.618 variant slightly alters the spike RBD-ACE2 binding affinity and is an antibody escaping variant: a computational structural perspective.

Continuing reports of new SARS-CoV-2 variants have caused worldwide concern and created a challenging situation for clinicians. The recently reported variant B.1.618, which possesses the E484K mutation specific to the receptor-binding domain (RBD), as well as two deletions of Tyr145 and His146 at the N-terminal binding domain (NTD) of the spike protein, must be studied in depth to devise new therapeutic options. Structural variants reported in the RBD and NTD may play essential roles in the increased pathogenicity of this SARS-CoV-2 new variant. We explored the binding differences and structural-dynamic features of the B.1.618 variant using structural and biomolecular simulation approaches. Our results revealed that the E484K mutation in the RBD slightly altered the binding affinity through affecting the hydrogen bonding network. We also observed that the flexibility of three important loops in the RBD required for binding was increased, which may improve the conformational optimization and consequently binding of the new variant. Furthermore, we found that deletions of Tyr145 and His146 at the NTD reduced the binding affinity of the monoclonal antibody (mAb) 4A8, and that the hydrogen bonding network was significantly affected consequently. This data show that the new B.1.618 variant is an antibody-escaping variant with slightly altered ACE2–RBD affinity. Moreover, we provide insights into the binding and structural-dynamics changes resulting from novel mutations in the RBD and NTD. Our results suggest the need for further in vitro and in vivo studies that will facilitate the development of possible therapies for new variants such as B.1.618.

Relative Protein Abundances and Biological Ageing in Whole Skeletal Elements.

Establishing biological age is an integral part of forensic investigations, currently achieved through morphological methods with varying degrees of accuracy. Furthermore, biological ageing is much easier in juveniles than in adults, at which point traditional ageing methods struggle. Therefore, biomolecular approaches are considered of great interest, with several protein markers already recognized for their potential forensic significance. However, previous studies have typically relied on subsampling different parts of skeletal elements. Here, we attempt to evaluate the proteome of complete elements using a rat model. In the analysis of specimens spanning beyond adulthood (1 week to 1.5 years), we observed 729 unique proteins across 33 samples (three for each sex for each of the five (female) or six (male)), five of which represent newly identified proteins in relation to age estimation: vimentin, osteopontin, matrilin-1, apolipoprotein A-I, and prothrombin. Most of these follow the trend of decreasing abundance through age, with the exception of prothrombin that increases. We consider the combined use of these relative abundances, along with those of previously noted fetuin-A, biglycan, albumin, and chromogranin-A signatures, as being of potential value to the development of an age estimation tool worthy of further evaluation in forensic contexts.

Biomolecular approaches to understanding metal tolerance and hyperaccumulation in plants.

Trace metal elements are essential for plant growth but become toxic at high concentrations, while some non-essential elements, such as Cd and As, show toxicity even in traces. Thus, metal homeostasis is tightly regulated in plants. Plant species colonising metalliferous soils have evolved mechanisms to hypertolerate metals and, in rare cases, can hyperaccumulate them in excess amounts in their shoots. The molecular mechanisms of metal hypertolerance and hyperaccumulation are likely derived from alterations in the basic mechanisms involved in general metal homeostasis. Genes involved in metal transport, synthesis of metal chelators and oxidative stress responses are constitutively and highly expressed in metal hypertolerant and hyperaccumulator species. Plant specialized metabolites and cell wall components have been proposed as major players in these mechanisms. In addition, the high intra-specific natural variation of metal hypertolerance and hyperaccumulation suggests that various molecular mechanisms might be involved in the evolution of these traits. To date, the potential of wild plant populations as systems to study metal tolerance and hyperaccumulation has not been fully exploited. The advent of next-generation sequencing (NGS) has enabled the study of non-model species, providing an opportunity to study natural populations and new tolerant and/or hyperaccumulating species, and will provide new insights into metal tolerance and hyperaccumulation. In this review we highlight background knowledge about metal tolerance and hyperaccumulation in plants and the current state-of-the-art techniques to study and identify the underlying mechanisms of metal hypertolerance and hyperaccumulation. We also outline for the reader the importance of the multidisciplinarity of this research field and how the integration of multiomic approaches will benefit facing the future scientific challenges.

Different continuous training modalities result in distinctive effects on muscle structure, plasticity and function.

The effects of training on muscle structure are dependent on adaptive changes induced by different intensities of physical exercise. Evidence has shown that aerobic training is able to induce adaptive changes to muscle structure based on intensity. The aim of the present study was to investigate the effects of different methods of continuous aerobic training in mice using functional, morphological and biomolecular approaches. The continuous aerobic training methods used in the present study were uniform continuous training (UC), varying continuous training (VC) and progressive continuous training (PC). Mice were made to run 3 times a week for 12 weeks on a motorized RotaRod, following one of the three different training methods at different speeds. The results of the present study demonstrated that the various training methods had different effects on sarcomere length. Ultrastructural analysis demonstrated that UC training resulted in a shortening of sarcomere length, PC training resulted in an elongation of sarcomere length and VC training showed similar sarcomere length when compared with the control sedentary group. Additionally, succinate dehydrogenase complex flavoprotein subunit A levels in muscle tissue following VC training were higher compared with UC and PC training. Overall, the present study showed that varying exercise methods resulted in different types of muscle plasticity, and that the VC protocol resulted in increased coordination and strength endurance in the functional tests, in agreement with the ultrastructural and biochemical profile. These observations support the view that VC training may be more efficient in increasing performance and may thus form the basis of training regimens when an improvement of motor efficiency is required.

Imaging unlabeled proteins on DNA with super-resolution.

Fluorescence microscopy is invaluable to a range of biomolecular analysis approaches. The required labeling of proteins of interest, however, can be challenging and potentially perturb biomolecular functionality as well as cause imaging artefacts and photo bleaching issues. Here, we introduce inverse (super-resolution) imaging of unlabeled proteins bound to DNA. In this new method, we use DNA-binding fluorophores that transiently label bare DNA but not protein-bound DNA. In addition to demonstrating diffraction-limited inverse imaging, we show that inverse Binding-Activated Localization Microscopy or ‘iBALM’ can resolve biomolecular features smaller than the diffraction limit. The current detection limit is estimated to lie at features between 5 and 15 nm in size. Although the current image-acquisition times preclude super-resolving fast dynamics, we show that diffraction-limited inverse imaging can reveal molecular mobility at ∼0.2 s temporal resolution and that the method works both with DNA-intercalating and non-intercalating dyes. Our experiments show that such inverse imaging approaches are valuable additions to the single-molecule toolkit that relieve potential limitations posed by labeling.

Effect of nano metal oxides on heme molecule: molecular and biomolecular approaches

Interaction of components of living cells with various nanomaterials in the gas phase has been one of extensive concern since they become intensively utilized in various life aspects. This work is carried out to investigate the interaction between heme molecule, as the main component of hemoglobin, with several familiar and non-familiar divalent structures such as O2, CO2, CO, MgO, CoO, NiO, CuO and ZnO. Geometry optimization processes as well as QSAR descriptors are conducted using semiemprical quantum mechanical calculations at PM6 level. Results illustrate that adsorbing O2 and CO on heme lowers their TDM helping heme in performing its transportation function and not interacting with other species. On the other hand, when CoO and ZnO interacting with heme the TDM of the resultant structures increase greatly reflecting high reactivity which may interact with other species more than performing its function. Therefore, interacting species other than O2 may disturb the transportation function of heme structure. QSAR data of IP regarding interaction of O2 with heme ensure the TDM result that reflects lowering its activity. IP of H-CO adsorbed is the lowest indicating high reactivity while those of H-O2, H-CO2, H-MgO and H-NiO in the complex form are the highest values indicating that it is difficult to form a complex structure with them. Therefore, heme interactions with structures rather than O2 and CO2 affects negatively its function as gas transporter.

Approaching ancient disease from a One Health perspective: Interdisciplinary review for the investigation of zoonotic brucellosis

AbstractToday, brucellosis is the most common global bacterial zoonosis, bringing with it a range of significant health and economic consequences, yet it is rarely identified from the archaeological record. Detection and understanding of past zoonoses could be improved by triangulating evidence and proxies generated through different approaches. The complex socioecological systems that support zoonoses involve humans, animals, and pathogens interacting within specific environmental and cultural contexts, and as such, there is a diversity of potential datasets that can be targeted. To capture this, in this paper, we consider how to approach the study of zoonotic brucellosis in the past from a One Health perspective, one which explicitly acknowledges the health link between people, animals, and environments (both physical and cultural). One Health research is explicitly interdisciplinary and conceptually moves away from an anthropocentric approach, allowing the component parts to be considered in holistic and integrated ways to deliver more comprehensive understanding. To this end, in this paper, we review the methods, selected evidence, and potential for past brucellosis identification and understanding, focussing on osteological markers in humans and animals, historical, biomolecular, and epidemiological approaches. We also present an agenda and potential for future research.

Evaluation of salt tolerant mungbean (Vigna radiata L.) Genotypes on growth through bio-molecular approaches

This study was conducted to obtain saline tolerant mungbean genotypes through evaluating growth, biochemical and molecular parameters, and possible salt tolerant mechanisms were studied in different salt sensitive genotypes. Thirteen prescreened mungbean genotypes were grown on 0, 40, 80 and 120 mMNaCl induced salinity and evaluated by germination percentage, shoot and root length, superoxide (O2•-) generation rate, concentration of H2O2, lipid peroxidation (as malondialdehyde, MDA), methylglyoxal (MG), K+/Na+ and proline content in leaves. Based on these parameters, genotypes BD-10588, BD-6894 and IR-01 were selected as tolerant genotypes. For studying oxidative stress tolerance mechanism, BD-10588 and IR-01 were used as tolerant and BD-6887 and BD-10741 as susceptible genotypes, and comparative ROS (O2•- and H2O2), and MDA as well as LOX activity between the two groups were determined. Analysis of activities of ROS metabolizing antioxidant enzymes strongly suggested that superoxide dismutase in tolerant genotypes provided first line protection from salt induced O2•-. Higher catalase (CAT) and ascorbate peroxidase (APX) played major role in H2O2 metabolism in tolerant genotypes. Both specific and in-gel activities of glutathione peroxidase (GPX) strongly proved the H2O2 metabolism for reducing oxidative damage in both tolerant and susceptible genotypes. However, higher peroxidase activity was important for mitigating salt stress in susceptible mungbean genotypes. Therefore, SOD, APX and GPX are very important for protecting salt mediated oxidative damage in mungbean genotype.
 Bangladesh J. Agril. Res. 44(3): 469-492, September 2019

Oral Cannabidiol Prevents Allodynia and Neurological Dysfunctions in a Mouse Model of Mild Traumatic Brain Injury.

Neurological dysfunctions are the most impactful and persistent consequences of traumatic brain injury (TBI). Indeed, previous reports suggest that an association between TBI and chronic pain syndromes, as well anxio-depressive behaviors, tends to be more common in patients with mild forms of TBI. At present, no effective treatment options are available for these symptoms. In the present study, we used a weight drop mild TBI mouse model to investigate the effect of a commercially available 10% Cannabidiol (CBD) oil on both the sensorial and neuropsychiatric dysfunctions associated with mild TBI through behavioral and biomolecular approaches. TBI mice developed chronic pain associated with anxious and aggressive behavior, followed by a late depressive-like behavior and impaired social interaction. Such behaviors were related with specific changes in neurotransmitters release at cortical levels. CBD oral treatment restored the behavioral alterations and partially normalized the cortical biochemical changes. In conclusion, our data show some of the brain modifications probably responsible for the behavioral phenotype associated with TBI and suggest the CBD as a pharmacological tool to improve neurological dysfunctions caused by the trauma.

Fossilised Biomolecules and Biomarkers in Carbonate Concretions from Konservat-Lagerstätten

In the vast majority of fossils, the organic matter is degraded with only an impression or cast of the organism remaining. In rare cases, ideal burial conditions result in a rapid fossilisation with an exceptional preservation of soft tissues and occasionally organic matter. Such deposits are known as Lagerstätten and have been found throughout the geological record. Exceptional preservation is often associated with finely crystalline quartz (e.g., cherts), fine sediments (e.g., muds) or volcanic ashes. Other mechanisms include burial in anoxic/euxinic sediments and in the absence of turbidity or scavenging. Exceptional preservation can also occur when an organism is encapsulated in carbonate cement, forming a concretion. This mechanism involves complex microbial processes, resulting in a supersaturation in carbonate, with microbial sulfate reduction and methane cycling the most commonly suggested processes. In addition, conditions of photic zone euxinia are often found to occur during concretion formation in marine environments. Concretions are ideal for the study of ancient and long-extinct organisms, through both imaging techniques and biomolecular approaches. These studies have provided valuable insights into the evolution of organisms and their environments through the Phanerozoic and have contributed to increasing interest in fields including chemotaxonomy, palaeobiology, palaeoecology and palaeophysiology.

In situ lysis droplet supply to efficiently extract ATP from dust particles for near-real-time bioaerosol monitoring

Simultaneous improvement in detection speed and reliability is critical for bioaerosol monitoring. Recent rapid detection strategies exhibit difficulties with misinterpretation due to signal interference from co-existing nonbiological particles, whereas biomolecular and bioluminescent approaches require long process times (>several tens of minutes) to generate readable values despite their better detection reliability. To overcome these shortcomings, we designed a system to achieve rapid reliable field detection of bioaerosols (>104 relative luminescence units [RLU] per cubic meter of air) in <3 min processing time (equivalent to 24 L sampling air volume) by employing a lysis droplet supply for efficient extraction of adenosine triphosphate (ATP) from particulate matter (PM) and a photomultiplier tube detector for signal amplification of ATP bioluminescence. We also suggested the use of the ratio of RLU (m-3) to total PM (μg m-3), or specific bioluminescence (RLU μg-1), as a measure of the biofraction of PM (i.e., potential biohazards). A correlation between RLU and colony forming unit was also obtained from simultaneous aerosol sampling using an agar-inserted sampler.

Sorting the sheep from the goats in the Pastoral Neolithic: morphological and biomolecular approaches at Luxmanda, Tanzania

Large-scale reconstructions of the spread of food production systems require fine-scale analyses of dietary evidence. One current impediment to understanding early African pastoralism is a lack of high-resolution portraits of herd management, specifically with respect to sheep (Ovis aries) and goat (Capra hircus), osteologically similar but behaviorally distinct caprines. In this study, we argue for the anthropological relevance of distinguishing sheep and goat remains in African pastoralist contexts, commenting upon implications for ecological settings and pastoralists’ strategies for production and risk management. We explain why sheep/goat distinction is rare in African zooarchaeological studies, particularly in comparison to Southwest Asia. We then apply three methods to distinguish caprines in an archaeofaunal sample from the Pastoral Neolithic site of Luxmanda, Tanzania, dated to c. 3000 BP: morphological identifications by two independent analysts, collagen-peptide mass fingerprinting (Zooarchaeology by Mass Spectrometry, ZooMS), and carbon stable isotope analyses. A comparative assessment of the results demonstrates the ability of biomolecular methods to improve resolution of faunal records, particularly where preservation is poor. We call for wider application of these methods to legacy collections, in order to refine existing regional models for the spread of herding in Africa, and to better understand ancient pastoralists’ herd-management decisions.

Authenticity and traceability in beverages.

Nowadays, consumers expect to be sure that a food product complies with its label and demand further information regarding the geographical origin of the product. Beverages are among the most likely foods to be the target of food fraud. Adulteration through practices including, among the others, mixing or substituting the original material with other materials or useless parts of the same materials, which do not conform to the official standards and the food label, originate non-authentic beverages. For the determination of beverage authenticity, chemical, biomolecular and isotopic approaches could be applied depending on the level in the structure of the matter for obtaining the target information. This review highlights the recent key studies and related analytical approaches on authentication of beverages including fruit juices, coffee and tea, and wine and other alcoholic beverages.

Reconstruction of human subsistence and husbandry strategies from the Iberian Early Neolithic: A stable isotope approach.

The Early Neolithic involved an important social and economic shift that can be tested not only with the material culture, but also through biomolecular approaches. The Iberian Peninsula presents few Early Neolithic sites where fauna and humans can be analyzed together from an isotopic perspective. Here we present an isotopic study on the site of Cueva de Chaves as an example for understanding the dietary and economical changes that took place during Early Neolithic in Iberia. Here we apply carbon and nitrogen stable isotope analysis to bone collagen from 4 humans and 64 faunal samples from 14 different species. The large dataset belongs to the same unique chrono-cultural context secured by 20 radiocarbon dates. Three direct new radiocarbon dates were carried out on the human remains analyzed. Faunal isotope values show no significant differences between wild and domestic herbivores, although the latter have more homogeneous values. Domestic pigs, potentially considered omnivorous, also show signatures of a herbivore diet. Human isotopic results show a diet mainly based on terrestrial C3 resources and possibly high meat consumption. The only individual found buried with a special funerary treatment presents a slightly different protein intake, when taking into account the long contemporaneous baseline analyzed. Similar values between wild and domestic species could be the result of common feeding resources and/or grazing on the same parts of the landscape. The herbivore diet seen amongst domestic pigs rules out feeding on household leftovers. High meat consumption by humans would support the hypothesis of the existence of a specialized animal husbandry management community in which agriculture was not intensively developed. Our results suggest that the development of agricultural practices and animal husbandry were not necessarily associated together in the early stages of the Western Mediterranean Neolithic.

Birch Bark Tar Production: Experimental and Biomolecular Approaches to the Study of a Common and Widely Used Prehistoric Adhesive

Birch bark tar, the oldest adhesive known in Europe, was widely used during Prehistory. This material, produced by the dry distillation of birch bark, has been identified in various spheres of activities and provides valuable information on the know-how and technical and territorial systems of past societies. This biomaterial can also provide evidence on socio-economic networks and mobility. However, very little is known about the production systems of birch bark tar during Prehistory, including the Neolithic period. The lack of findings in the archaeological record necessitates the development of an approach that combines experimental archaeology and biomolecular chemistry. We present here (1) the results of experiments in which different birch bark tar production systems were tested and (2) the molecular signatures of the birch bark tars produced according to different processes based on the use of ceramic vessels. The key role of bark quality is highlighted for the first time. This study also details direct archaeological inference of the experimental results obtained: a total of 23 samples from the site of Nice-Giribaldi (France, second part of the fifth century BCE) was investigated. Different categories of birch bark tars were identified during Neolithic in the south of France, providing evidence for the existence of complex manufacturing systems and procurement networks.

Abstract PR04: Progress towards development of an exosome-based biomarker assay in Ewing sarcoma

Abstract Background: Ewing sarcoma (EWS) is the second most common primary pediatric bone malignancy in the United States. Despite significant advancements made in the management/treatment of localized EWS, the 5-year overall survival for patients with metastatic or recurrent EWS is less than 30%. Sadly, outside of scans, no clinically relevant predictive biomarkers currently exist; therefore, there is a glaring need for complementary biomolecular approaches to help predict drug resistance, metastasis, and recurrence, with the overall aim of enhancing the management of EWS patients. Recent advancements in the field of biomarker development have demonstrated that nano-sized vesicles, termed exosomes, are actively released by tumor cells and carry content (such as proteins and RNAs) which are representative of their cell of origin. Importantly, these vesicles can be isolated from various biologic fluids including plasma and have the potential to provide a minimally invasive, real-time “shapshot” of the genetic and proteomic content of these tumor cells, thereby providing a rich source of potential diagnostic and predictive biomarkers. We hypothesize that analysis of the proteomic content of EWS-derived exosomes may result in the ability to selectively enrich for EWS-specific exosomes from patient plasma and, upon analysis of the content, provide a novel and clinically relevant source of disease-specific biomarkers. Methods: We isolated exosomes from five EWS cell line derived exosomes with varying EWS-Ets fusions (TC-71, CHLA-258, RD-ES, SK-ES-1, and COG-E-352). We characterized the proteomic and nucleic acid content of these extracellular vesicles using Western blot analysis, Nanoparticle Tracking Analysis, and real-time PCR in order to establish exosome purity and identified already established EWS markers such as the CD99/MIC2 protein and the EWS-Ets fusion transcripts. We next conducted mass spectrometry to obtain a global proteomic assessment of these EWS cell line derived exosomes. Longitudinal pediatric clinical samples collected through an established pediatric sarcoma biobank (16 Ewing sarcoma, 5 rhabdomyosarcoma and 7 osteosarcoma pediatric patients) were utilized for this study. Through the use of this proteomic data set and a portion of the pediatric sarcoma clinical sample set, we have developed a magnetic bead-based immunoprecipitation approach requiring the use of only 250 µL of patient plasma to isolate tumor-derived exosomes in pediatric EWS patients (with localized, metastatic or recurrence of disease). These isolated extracellular vesicles were then evaluated for the presence of EWS markers by Western blot and the EWS-Ets fusion transcripts by nested qPCR. Results: Exosomes from each of the experimental cell lines contained typical exosomal markers (i.e., ALIX and CD81) and had a mean size of 170 nm. EWS cell line derived exosomes demonstrated the presence of EWS-Ets fusion transcripts at both the mRNA level and proteomic level. The analysis of the mass spectrometry data revealed 619 commonly shared proteins. Upon comparison with the content of plasma proteomic and exosomal specific databases, 3 unique proteins of interest were identified. Lastly, exosomes immunoprecipitated from both patient plasma and exosome-spiked plasma displayed EWS markers such as MIC2/CD99 and EWS-FLI1, and contained the EWS-Ets mRNA transcripts. Conclusions: For the first time reported, tumor-derived exosomes from EWS pediatric patients are demonstrated to be isolated by immunoprecipitation techniques and express presence of EWS-specific markers. The future evaluation and analysis of these exosomal EWS markers should provide a simple and cost effective “liquid biopsy” that would allow clinicians to diagnose and monitor tumor burden, detect tumor responsiveness to therapy, monitor changes within the tumor including determining drug sensitivity alterations, and thus enable clinicians to better manage patients with EWS. This abstract is also being presented as Poster A31. Citation Format: Glenson Samuel, Jennifer Crow, Safinur Atay, Andrew K. Godwin. Progress towards development of an exosome-based biomarker assay in Ewing sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR04.