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PubMed1998

Analysis of the t(2;5) (p23;q35) translocation in CD30+ primary cutaneous lymphoproliferative disorders and Hodgkin's disease.

The t(2;5) (p23;q35) chromosomal translocation is found in about 40% of lymph node-based CD30+ anaplastic large cell lymphomas of T-cell or null-cell lineage. This translocation results in the expression of a fusion protein containing the catalytic domain of anaplastic lymphoma kinase (ALK) under the control of the promoter for nucleophosmin (NPM), a nucleolar phosphoprotein. Expression of ALK activity, normally absent in lymphocytes, is postulated to be involved in the pathogenesis of lymphomas bearing the t(2;5) translocation. Certain primary cutaneous lymphoproliferative disorders and Hodgkin's disease are also known to contain CD30+ large lymphoid cells. In order to determine the role of the t(2;5) translocation in these diseases, several investigators have employed a variety of techniques including cytogenetics, genomic Southern blot analysis, RNA- and DNA-based PCR assays, various forms of in-situ hybridization, and immunostaining for the p80 fusion protein encoded by the chimeric t(2;5) transcripts. These studies included approximately 415 cases of Hodgkin's disease, 65 cases of CD30+ primary cutaneous large cell lymphoma, and 38 cases of lymphomatoid papulosis. The aggregate results of these studies indicate that the t(2;5) translocation or other somatic mutations resulting in inappropriate expression of ALK are involved rarely if at all in the pathogenesis of Hodgkin's disease, but may be present in about 10% of cases of lymphomatoid papulosis and 20% of cases of CD30+ primary cutaneous large cell lymphoma. However, the t(2;5) has not been detected yet in any case involving multiple or secondary CD30+ lymphoproliferative disorders, thereby providing no evidence for a role in tumor clone progression. Additional studies will be needed to determine if t(2;5) status has any clinical significance for patients with CD30+ primary cutaneous lymphoproliferative disorders.

Anaplastic Lymphoma Kinase↗
PubMed2005

Mild recessive epidermolytic hyperkeratosis associated with a novel keratin 10 donor splice-site mutation in a family of Norfolk terrier dogs.

BACKGROUND: Epidermolytic hyperkeratosis in humans is caused by dominant-negative mutations in suprabasal epidermal keratins 1 and 10. However, spontaneous keratin mutations have not been confirmed in a species other than human. OBJECTIVES: To describe an autosomal recessive, mild, nonpalmar/plantar epidermolytic ichthyosis segregating in an extended pedigree of Norfolk terrier dogs due to a splice-site mutation in the gene encoding keratin 10 (KRT10). METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Genomic DNA samples and cDNA from skin RNA were sequenced and defined a mutation in KRT10. Consequences of the mutation were evaluated by assessing protein expression with immunohistochemistry and Western blotting and gene expression with real-time RT-PCR (reverse transcriptase-polymerase chain reaction). RESULTS: Adult dogs with the disease had generalized, pigmented hyperkeratosis with epidermal fragility. Light microscopic examination defined epidermolysis with hyperkeratosis; ultrastructural changes included a decrease in tonofilaments and abnormal filament aggregation in upper spinous and granular layer keratinocytes. Affected dogs were homozygous for a single base GT-->TT change in the consensus donor splice site of intron 5 in KRT10. Keratin 10 protein was not detected with immunoblotting in affected dogs. Heterozygous dogs were normal based on clinical and histological appearance and keratin 10 protein expression. The mutation caused activation of at least three cryptic or alternative splice sites. Use of the cryptic sites resulted in transcripts containing premature termination codons. One transcript could result in shortening of the proximal portion of the 2B domain before the stutter region. Quantitative real-time PCR indicated a significant decrease in KRT10 mRNA levels in affected dogs compared with wild-type dogs. CONCLUSIONS: This disease is the first confirmed spontaneous keratin mutation in a nonhuman species and is the first reported recessive form of epidermolytic hyperkeratosis.

Animals↗
PubMed1998

Ultrastructural localization of interferon-inducible double-stranded RNA-activated enzymes in human cells.

The protein kinase PKR and the 2',5'-oligoadenylate (2-5A) synthetase are two interferon-induced and double-stranded RNA-activated enzymes which are implicated in the mechanism of action of interferon. Their distribution was undertaken here at the ultrastructural level by the immunogold procedure, following the use of specific monoclonal antibodies directed against PKR and 69- and 100-kDa forms of the 2-5A synthetase. These enzymes were detected as a pool of nonaggregated proteins scattered throughout the cell and as aggregates often associated with electron-dense doughnut-like structures showing a similar aspect whatever their subcellular localization: the cytoplasm, the nuclear envelope, and the nucleus. In general, the 2-5A synthetases were present in much more lower amounts than the PKR, probably due to the difficulty of detecting traces of proteins by electron microscopy. To circumvent this, we used a human lymphoblastoid cell line overexpressing the 69-kDa form of the 2-5A synthetase. In such cells, the synthetase was then clearly observed in both the cytoplasm and the nucleus; isolated or small clusters of gold particles were numerous in the cell mainly over the RNP fibrils of the interchromatin space, nucleolus, and ribosomes. Interestingly, gold particles were also found to be associated with the membranes of nuclear envelope and rough endoplasmic reticulum probably due to the myristilated motif of this form of 2-5A synthetase. Finally, intensely labeled electron-opaque dots sometimes associated with the nuclear pore complexes were present in the nucleus and in the cytoplasm of cells which might suggest their transport from the nucleus to the cytoplasm or reciprocally through the nuclear pore complexes. These observations indicate the wider distribution of the dsRNA-activated enzymes in the cell, thus pointing out their potential implication in as yet undetermined physiological function(s) necessary for various cellular metabolic reactions.

2',5'-Oligoadenylate Synthetase↗
PubMed1989

Cooperative DNA binding by Xenopus transcription factor IIIA. Use of a 66-base pair DNA fragment containing the intragenic control region of the 5 S RNA gene to study specific and nonspecific interactions.

The interaction of Xenopus transcription factor IIIA (TFIIIA) with a synthetic 66-base pair DNA fragment containing the intragenic control region (ICR) of the 5 S RNA gene was investigated by mobility shift gel electrophoresis and DNase I protection experiments. Specific EDTA-sensitive protein-DNA complexes were observed only in the presence of high concentrations of either the ICR-containing fragment or an excess of unlabeled nonspecific DNA. This predominant complex was determined to be composed of equal molar ratios of TFIIIA and the 66-base pair ICR-containing fragment, although complexes containing two TFIIIA molecules/ICR and three TFIIIA molecules/ICR were also found. In the absence of excess unlabeled specific or nonspecific DNA, TFIIIA was observed to interact with the labeled 66-mer as an aggregate that remained at the top of the gel matrix. Although the binding affinities of TFIIIA for the 66-base pair specific DNA fragment and the 74-base pair nonspecific DNA fragment were comparable as observed by direct measurement with mobility shift gel electrophoresis, a greater than 500-fold difference was observed by competition experiments at high DNA concentrations. Furthermore, a similar difference was also detected when proteolytically cleaved TFIIIA was used in the binding reaction, which substantially reduced the protein-protein interactions. DNase I digestion patterns of the noncoding strand of the 66-mer in the absence or presence of TFIIIA demonstrated that this region of DNA is structurally equivalent to the ICR contained within the 5 S RNA gene. In addition, the mode of binding observed in the mobility shift gels is identical to that observed by direct footprint analysis. Protein-protein interactions appear, therefore, to be an intrinsic and necessary activity of TFIIIA.

Animals↗
PubMed1987

Mutational analysis of the embryo-specific urease locus of soybean.

By a non-destructive urease screen of M2 soybean (Glycine max [L.] Merr. cv. Williams) seeds, four true-breeding mutants (n4, n6, n7 and n8) were recovered which lack most (n6, n8) or all (n4, n7) embryo-specific urease activity. This trait was due to a single, recessive lesion at the Sun (seed urease-null) locus identified earlier in an exotic germplasm (PI 229324, Itachi). All sun mutants produced normal ubiquitous urease, the low abundance isozyme found in all soybean tissues examined. Tight mutants n4 and n7 accumulated no detectable embryo-specific urease protein or mRNA; n6 and n8 accumulated normal or near normal levels of urease mRNA but had seed urease protein levels approximately 5% and .05%, respectively, of the progenitor. Mutant n8 appeared to produce a low level of fully active urease (approximately 0.7% activity level, approximately 0.5% protein level) while n6 produced a higher level of an altered, nearly inactive urease (.0.09% activity level, approximately 5% protein level). Urease alterations in n6 were manifested by its increased temperature sensitivity and variation in aggregation state and pH preference. Thus, mutations in the Sun locus affected both the level and the nature of the embryo-specific urease gene products indicating that Sun encodes the embryo-specific urease. We reported earlier that the Eul locus, which controls the aggregation state of the embryo-specific urease, is on mep unit from Sun and that the Eul allele is cis to sun is not expressed (Kloth et al. 1987). That the level of urease gene product, its aggregation state and other enzyme properties can be affected by induced sun mutations, suggests that the Eul and sun alleles are at the same locus.

Genes, Plant↗
PubMed2006

Heat shock proteins reduce alpha-synuclein aggregation induced by MPP+ in SK-N-SH cells.

Alpha-synuclein has been implicated in the pathogenesis of Parkinson's disease (PD). Heat shock proteins (HSPs) can reduce protein misfolding and accelerate the degradation of misfolded proteins. 1-methyl-4-phenylpyridinium ion (MPP+) is the compound responsible for the PD-like neurodegeneration caused by MPTP. In this study, we found that MPP+ could increase the expression of alpha-synuclein mRNA but could not elevate proteasome activity sufficiently, leading to alpha-synuclein protein accumulation followed by aggregation. Both HSPs and HDJ-1, a homologue of human Hsp40, can inhibit MPP+-induced alpha-synuclein mRNA expression, promote ubiquitination and elevate proteasome activity. These findings suggest that HSPs may inhibit the MPP+-induced alpha-synuclein expression, accelerate alpha-synuclein degradation, thereby reducing the amount of alpha-synuclein protein and accordingly preventing its aggregation.

1-Methyl-4-phenylpyridinium↗
PubMed2004

Genotoxicity induced by saponified coconut oil surfactant in prokaryote systems.

Surfactants are amphiphilic substances with special properties and chemical structures that allow a reduction in interfacial tension, which permits an increase in molecule solubilization. The critical micelle concentration (CMC) is an important characteristic of surfactants that determines their aggregate state, which is generally related to its functional mechanism. In this work the genotoxic potential of saponified coconut oil (SCO), a surfactant obtained from Cocos nucifera, was analyzed using prokaryote systems. DNA strand breaks were not observed after treatment of a plasmid with SCO. Negative results were also obtained in the SOS Chromotest using Escherichia coli strains PQ35 and PQ37. A moderate toxicity of SCO was observed after treatment of strain CC104 with a concentration above its CMC, in which micelles were found. Nevertheless, this treatment was not cytotoxic to a CC104mutMmutY strain. Furthermore, in this DNA repair-deficient strain treatment with a SCO dose below its CMC, in which only monomers were found, demonstrated the possibility of an antioxidant effect, since a reduction in spontaneous mutagenesis frequency was observed. Finally, in an Ames test without metabolic activation mutagenicity induction was observed in strains TA100 and TA104 with treatment doses below the CMC. The cytotoxic, antioxidant and mutagenic effects of SCO can be influenced by the aggregational state.

Adenosine Triphosphatases↗
PubMed1997

Further evidence for the presence of mitochondrially encoded subunits in cytochrome c oxidase of the trypanosomatid Crithidia fasciculata.

Mitochondrial mRNAs in trypanosomatids are edited by uridylate insertion and deletion. The respiratory chain complexes cytochrome c reductase, cytochrome c oxidase and F0F1-ATPase of the insect trypanosomatid Crithidia fasciculata have been isolated and analysed by peptide microsequencing, but so far, proteins encoded by edited (and unedited) mitochondrial mRNAs have not been found. In this paper, we provide evidence that the mitochondrial mRNAs encoding the three large subunits of cytochrome c oxidase are indeed translated. First, purified holo cytochrome c oxidase turned out to be cysteine-rich, in agreement with the high cysteine codon-content of the sequence of mitochondrial cox subunit mRNAs. Second, in mass spectrometry measurements of cytochrome c oxidase, a protein was detected with the predicted molecular weight of cytochrome c oxidase subunit 2. Finally, an antibody generated against a fusion protein produced in Escherichia coli from constructs containing a segment of cytochrome c oxidase subunit 2 cDNA, specifically recognised protein bands present in cytochrome c oxidase following SDS PAGE. However, these proteins were present in the high molecular weight region of the gel, suggesting that cytochrome c oxidase subunit 2 aggregates in the presence of SDS.

Animals↗
PubMed1990

De novo formation of cytokeratin filaments in calf lens cells and cytoplasts after transfection with cDNAs or microinjection with mRNAs encoding human cytokeratins.

We have studied the formation of new intermediate-sized filaments (IFs) by human cytokeratins (CKs) 8 and/or 19 in cultured bovine lens cells stably transfected with the corresponding cDNAs under SV40 promoter control. In the transfected cells, polypeptides of both type I and type II CKs were synthesized to near-equimolar amounts, formed heterotypic complexes and assembled into IFs with a peculiar tendency to accumulate into variously sized, often roundish aggregates in the juxtanuclear region, usually one per cell. Electron microscopy of these large CK IF aggregates revealed typical 7 to 12-nm IFs, tightly packed together in an apparently haphazard mode. By immunoelectron microscopy, the CK IFs could be readily distinguished from the vimentin IFs which were abundant in these cells. Electron microscopy also showed that many of the CK IF aggregates were located in the vicinity of the nucleus but did not have direct contact with the nuclear envelope; moreover, their location did not regularly correspond to those of the centrosomes and the Golgi apparatus. During enucleation of transfected cells in the presence of cytochalasin B, the CK aggregates were often retained in the cytoplast. After microinjection of CK 8 and 19 mRNAs, synthesized in vitro from cDNA molecules, into enucleated cytoplasts prepared from untransfected cells, CK IFs similar to those observed in microinjected whole cells were formed but often showed a wider cytoplasmic distribution. Our observations indicate that typical CK IFs can form, in vivo, in the absence of any nuclear structures. We discuss possible reasons for the tendency of the CK IFs to accumulate, in this cell line, into a juxtanuclear aggregate, in relation to similar CK-IF aggregates formed in certain normal cell types and upon toxic damages.

Animals↗
PubMed2004

The ultrastructure of the developing replication site in foot-and-mouth disease virus-infected BHK-38 cells.

Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus of the Picornaviridae: Infection by picornaviruses results in a major rearrangement of the host cell membranes to create vesicular structures where virus genome replication takes place. In this report, using fluorescence and electron microscopy, membrane rearrangements in the cytoplasm of FMDV-infected BHK-38 cells are documented. At 1.5-2.0 h post-infection, free ribosomes, fragmented rough endoplasmic reticulum, Golgi and smooth membrane-bound vesicles accumulated on one side of the nucleus. Newly synthesized viral RNA was localized to this region of the cell. The changes seen in FMDV-infected cells distinguish this virus from other members of the Picornaviridae, such as poliovirus. Firstly, the collapse of cellular organelles to one side of the cell has not previously been observed for other picornaviruses. Secondly, the membrane vesicles, induced by FMDV, appear distinct from those induced by other picornaviruses such as poliovirus and echovirus 11 since they are relatively few in number and do not aggregate into densely packed clusters. Additionally, the proportion of vesicles with double membranes is considerably lower in FMDV-infected cells. These differences did not result from the use of BHK-38 cells in this study, as infection of these cells by another picornavirus, bovine enterovirus (a close relative of poliovirus), resulted in morphological changes similar to those reported for poliovirus-infected cells. With conventional fixation, FMDV particles were not seen; however, following high-pressure freezing and freeze-substitution, many clusters of virus-like particles were seen.

Animals↗
PubMedDate not supplied

Molecular characterization of a dominantly inherited bleeding disorder with impaired platelet responses to thromboxane A2.

Thromboxane A2 (TXA2) is a major arachidonic acid metabolite of platelets and induces platelet functions by binding to specific receptors on the membrane. We have found patients with hemostatic defects due to impaired platelet responses to TXA2, and molecular characterization of the patients has been carried out. Platelets from these two unrelated patients showed impaired aggregation responses to TXA2 and its analogues despite the normal response to thrombin. Although the patients' platelets exhibited normal binding activities to TXA2 analogues, they showed decreased GTPase activity and second messenger formation when stimulated by STA2, a stable TXA2 agonist. To understand the molecular basis of this abnormality, we determined the cDNA sequence of the TXA2 receptor by reverse transcription-polymerase chain reaction (RT-PCR) from the patient's platelet RNA, and identified a single amino acid substitution (Arg60 for Leu) in the first cytoplasmic loop of the receptor. This mutation was found in both isoforms of the platelet TXA2 receptor which we have recently found: TXR alpha with the same structure as the placental TXA2 receptor and TXR beta with the same structure as the endothelial TXA2 receptor, and was detected exclusively in affected members of two unrelated families with the disorder. The mutant TXR alpha and TXR beta expressed in COS-m6 cells showed decreased agonist-induced phospholipase C activation despite their normal ligand binding affinities. These results suggest that the Arg60 for Leu mutation is responsible for the disorder and imply a critical role for the first cytoplasmic loop in the interaction of the TXA2 receptor with the G protein.

Base Sequence↗
PubMed1992

Expression of neurotrophins and the low-affinity NGF receptor in septal and hippocampal reaggregate cultures: local physiologic effects of NGF synthesized in the septal region.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of a family of trophic factors designated the neurotrophins, each of which can bind to the low-affinity NGF receptor (LNGFR). To investigate the mechanisms that regulate the expression of the neurotrophins and the LNGFR in the developing brain, we grew cells from the embryonic mouse septum and hippocampus in reaggregating cell culture and compared neurotrophin and LNGFR expression in developing reaggregates with that seen in the developing septum and hippocampus in situ. NGF, BDNF, NT-3 and LNGFR were each expressed in septal and hippocampal reaggregates as well as the native septum and hippocampus. Additionally, the temporal expression profiles observed in reaggregates were generally similar to those seen in the respective brain regions in situ. In order to determine whether NGF can modulate neurotrophin or LNGFR expression, reaggregates were cultured in the continual presence of either exogenous NGF or anti-NGF antibodies. NGF-treated septal cultures expressed twice the level of LNGFR mRNA as was seen in untreated septal cultures; on the other hand, septal cultures grown in the presence of anti-NGF antibodies, to neutralize endogenously synthesized NGF, displayed a 3-fold decrease in LNGFR mRNA expression compared to untreated cultures. No effects of NGF or anti-NGF were observed on LNGFR expression in hippocampal reaggregates, or on neurotrophin mRNA expression in either reaggregate type. These results suggest that regulatory mechanisms intrinsic to the septal and hippocampal regions control neurotrophin and LNGFR expression. NGF is likely to be one of these regulatory cues since it acts locally in septal reaggregates to control the developmental expression of LNGFR mRNA. The possible roles of locally synthesized NGF and other neurotrophins in the development of septal neurons are discussed.

Aging↗
PubMed2005

Generation of embryoid bodies from mouse embryonic stem cells cultured on STO feeder cells.

Embryoid bodies, which are similar to post-implantation egg-cylinder stage embryos, provide a model for the study of embryo development and stem cell differentiation. We describe here a novel method for generating embryoid bodies from murine embryonic stem (ES) cells cultured on the STO feeder layer. The ES cells grew into compact aggregates in the first 3 days of coculture, then became simple embryoid bodies (EBs) possessing primitive endoderm on the outer layer. They finally turned into cystic embryoid bodies after being transferred to Petri dishes for 1-3 days. Evaluation of the EBs in terms of morphology and differentiating potential indicates that they were typical in structure and could generate cells derived from the three germ layers. The results show that embryoid bodies can form not only in suspension culture but also directly from ES cells cultured on the STO feeder layer.

Animals↗
PubMedDate not supplied

Pharmacologic preconditioning effects: prostaglandin E1 induces heat-shock proteins immediately after ischemia/reperfusion of the mouse liver.

Prostaglandin E1 (PGE1) has several potential therapeutic effects, including cytoprotection, vasodilation, and inhibition of platelet aggregation. This study investigates the protective action of PGE1 against hepatic ischemia/reperfusion injury in vivo using a complementary DNA microarray. PGE1 or saline was continuously administered intravenously to mice in which the left lobe of the liver was made ischemic for 30 minutes and then reperfused. Livers were harvested 0, 10, and 30 minutes postreperfusion. Messenger RNA was extracted, and the samples were labeled with two different fluorescent dyes and hybridized to the RIKEN set of 18,816 full-length enriched mouse complementary DNA microarrays. Serum alanine aminotransferase and aspartate aminotransferase levels at 180 minutes postreperfusion were significantly lower in the PGE1-treated group than in the saline-treated group. The cDNA microarray analysis revealed that the genes encoding heat-shock protein (HSP) 70, glucose-regulated protein 78, HSP86, and glutathione S-transferase were upregulated at the end of the ischemic period (0 minutes postreperfusion) in the PGE1 group. Our results suggested that PGE1 induces HSPs immediately after ischemia reperfusion. HSPs might therefore play an important role in the protective effects of PGE1 against ischemia/reperfusion injury of the liver.

Alprostadil↗
PubMed2005

Three-dimensional growth of extravillous cytotrophoblasts promotes differentiation and invasion.

Human trophoblast research relies on a combination of in vitro models, including isolated primary cultures, explant cultures, and trophoblast cell lines. In the present study, we have utilized the rotating wall vessel (RWV) bioreactor to generate a three-dimensional (3-D) model of human placentation for the study of cytotrophoblast (CTB) invasion. The RWV supported the growth of the human CTB cell line SGHPL-4 and allowed for the formation of complex, multilayered 3-D aggregates that were morphologically, phenotypically, and functionally distinct from SGHPL-4 monolayers. The cells cultured three-dimensionally differentiated into an aggressively invasive cell population characterized by the upregulation of matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-9 and urokinase-type plasminogen activator (uPA) secretion and activation. Microarray analysis of the 3-D and 2-D cultured cells revealed increased expression in the 3-D cells of various genes that are known mediators of invasion, including MT1-MMP, PECAM-1 and L-selectin, as well as genes not previously associated with CTB differentiation such as MMP-13 and MT5-MMP. These results were verified by quantitative real-time PCR. These findings suggest that when cultured in 3-D, SGHPL-4 cells closely mimic differentiating in utero CTBs, providing a novel approach for the in vitro study of the molecular mechanisms that regulate CTB differentiation and invasion.

Bioreactors↗
PubMed2003

Aggrecan regulates telencephalic neuronal aggregation in culture.

Proteoglycans have been suggested to play roles in pattern formation in the developing central nervous system. In the chick embryo, aggrecan, a chondroitin sulfate proteoglycan, has a regionally-specific and developmentally-regulated expression profile. Telencephalic neuronal cultures, when aggregated, exhibit aggrecan expression patterns comparable to those observed in vivo. The chicken mutation nanomelia produces a truncated aggrecan species that cannot be processed further and is not secreted. Neurons from normal and nanomelic chick embryo telencephalon were scored for aggregate formation and analyzed for distribution of aggrecan protein and expression of aggrecan mRNA. Distinctly different pattern formation, with respect to aggregate size (smaller) and number (fewer) were observed in poly-L-lysine plated neuronal cultures derived from nanomelic embryos when compared to those derived from normal embryos. Significantly, the nanomelic phenotype was subsequently rescued upon addition of the brain-specific form of aggrecan. Modulation of neuronal aggregate formation was mimicked by treatment with chondroitinase ABC but not other glycanases, and was rescued by addition of chondroitin 6-sulfate to the culture media. Lastly, although broad and diffuse distribution of aggrecan among the cell aggregates in the culture paradigm was observed by immunocytochemistry, mRNA in situ hybridization revealed that only a small population of cells in the center of the aggregates was responsible for the production of the secreted aggrecan found associated with neuronal aggregates. These studies suggest a function for aggrecan as a diffusible signal in CNS histomorphogenesis.

Aggrecans↗
PubMed2026

A Rapid Poly(ethylene glycol)-Assisted Magnetic Isolation Approach for High-Throughput Extracellular Vesicle Isolation and Subsequent Biomarker Analysis.

Extracellular vesicles (EVs) are crucial mediators of intercellular communication and have the potential to serve as biomarkers for disease diagnosis and therapeutic monitoring. However, most EV isolation methods often require large sample volumes and specialized instruments or involve trade-offs between purity, yield, cost, and scalability. We developed MagPEG, a workflow that combines poly(ethylene glycol) (PEG)-mediated EV aggregation with magnetic beads to provide a simple, reproducible alternative to ultracentrifugation, size-exclusion chromatography, and commercial precipitation kits. Our optimization experiments clarified the PEG concentration, ionic strength, and bead surface chemistry that collectively influence EV aggregation, capture efficiency, and contaminant coprecipitation, allowing us to define conditions that improve purity while maintaining high recovery. Compared with commonly used methods, MagPEG produced EVs with comparable size distribution, EV markers, and proteomic profiles while relying only on standard laboratory supplies. A key feature of the platform is that EVs and EV-associated DNA, RNA, and proteins can be sequentially extracted from the same bead-bound material, reducing sample loss and hands-on time and enabling multiomic analysis for limited clinical or small animal samples. MagPEG is compatible with downstream applications including proteomics, bead-based assays, and miRNA quantification. When applied to human serum, the method supported high-throughput EV proteomic profiling and enabled the identification of Alzheimer's disease-associated protein signatures, illustrating its utility for biomarker discovery. Overall, our results establish MagPEG as a powerful, rapid, scalable, and high-throughput solution for translational applications in biomarker discovery.

Polyethylene Glycols↗