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PubMed2026

3D Proteomics: Structural, Functional, Chemical and Biomarker Discovery Proteomics With LiP-MS.

Protein structural dynamics drive changes in protein function, making the capture of such dynamics essential for interrogating biological systems. Here we review limited proteolysis coupled to mass spectrometry (LiP-MS), a structural and chemical proteomics method that uses changes in susceptibility to protease cleavage to profile proteome-wide protein structural changes within complex biological samples. In the decade since its development, LiP-MS has become a broadly used structural proteomics method, with peptide-level resolution. It has identified drug targets, delineated altered cellular pathways in response to complex perturbations, revealed structural information on otherwise challenging protein targets, and demonstrated the new concept of structural biomarkers of disease. Because LiP-MS simultaneously probes numerous types of molecular events, such as molecular binding, changes in enzyme activity, chemical modifications, allosteric conformational changes, aggregation, and unfolding, it supports a new proteomics workflow which we term 3D proteomics. This workflow enables the detection of specific functional sites within proteins that are altered upon perturbation, thereby guiding the generation of molecular hypotheses. Further, by globally profiling structural in addition to protein abundance changes, LiP-MS has proven able to greatly increase the information content of functional proteomics screens. In sum, LiP-MS has supported the development of a novel conceptual framework for generating, visualizing, and interpreting structural proteomics data with peptide level resolution, thereby comprehensively probing biological systems. Here we survey the applications of LiP-MS, discuss methodological variants developed by us and others, and describe the use of this new type of omics readout for structural, functional, chemical, and biomarker discovery proteomics.

Proteomics↗

PubMed2026

A Parallel Accumulation-Mobility Aligned Fragmentation Strategy Utilizing High-Resolution Ion Mobility for High-Performance Proteomics Analysis.

Here we present a novel data-independent acquisition (DIA) mass spectrometry (MS) operating mode termed parallel accumulation-mobility aligned fragmentation (PAMAF) that offers enhanced speed and sensitivity of ion fragmentation analysis for discovery workflows such as bottom-up proteomics. This mode of operation leverages high-resolution ion mobility (HRIM) separation capabilities of the structures for lossless ion manipulation technology to achieve HRIM-based precursor isolation in place of traditional quadrupole filtering approaches. PAMAF mode increases the number of features that can be identified per MS1/MS2 acquisition cycle by employing mobility-based time alignment to associate fragment ions with their corresponding precursor ions. By using a high-speed, lossless separation technique for precursor isolation instead of the comparatively slow and wasteful quadrupole filtering, ion losses are avoided while simultaneously increasing the rate at which precursor ions are sequentially fragmented and detected. In addition, by accumulating ions while the previous packet of ions is being analyzed, the PAMAF mode achieves ∼100% ion utilization efficiency. Benchmarking results of LC-PAMAF-MS analysis of a whole cell protein digest showed ∼6× more protein group identifications compared to a standard data-dependent acquisition analysis without HRIM on the same QTOF instrument, and >100 x improvement for low-load workflows. Quantitative evaluations demonstrated that PAMAF mode could quantify low abundance peptides, including those undetectable by data-dependent acquisition. In addition, since precursor isolation in PAMAF mode is size-based rather than m/z-based, coeluting isobars and isomers can be resolved prior to fragmentation, eliminating chimeric spectra that compromise identification accuracy. We also explored the benefits of combining HRIM and quadrupole isolation to achieve improved specificity termed DIA-PAMAF mode, which enabled the detection of over 8000 protein groups from a HeLa digest analysis. PAMAF mode brings a powerful new technique to the field of proteomics with the potential to improve the sensitivity and selectivity of mass spectrometry-based proteomics.

Proteomics↗

PubMed2025

Multi-criteria decision making and its application to in silico discovery of vaccine candidates for Toxoplasma gondii.

Vaccine discovery against eukaryotic parasites is not trivial and few exist. Reverse vaccinology is an in silico vaccine discovery approach, designed to identify vaccine candidates from the thousands of protein sequences encoded by a target genome. Previously, we produced the Vacceed bioinformatics pipeline for identification of parasite membrane and excreted/secreted proteins that were likely be exposed to the hosts immune system. More recently, we improved upon machine learning as the final decision-making process to identify parasite proteins that induce a protective response in an animal model. Subsequently, we combined Vacceed with metrics on B and T cell epitope types to produce a new in silico discovery workflow. In this study we extend this in silico workflow to the developability of proteins as vaccines by the incorporation of metrics on the physicochemical properties of proteins. To demonstrate this process, every Toxoplasma gondii protein was ranked in its capacity to provide exposure to the immune system (Vacceed exposure score), presence of epitopes and solubility characteristics by several multicriteria decision making (MCDM) tools (such as TOPSIS, VIKOR and MABAC). A consensus rank was subsequently generated from the results of these tools using a variety of aggregate ranking methods. Levels of uncertainty in the aggregate protein rankings was assessed by conformal interval prediction in association with a machine learning model. Several of the top ranked proteins identified by this approach were novel, uncharacterized membrane transporters or proteins associated with RNA metabolism. In conclusion, MCDM automated the decision making using well known algorithms while conformal prediction intervals varied significantly across the 8000+ proteins of T. gondii. Highly ranked proteins (e.g. the top 100) typically generated low prediction intervals, providing high levels of confidence in their ranks.

Toxoplasma↗

PubMed2000

Sub-microliter DNA sequencing for capillary array electrophoresis.

DNA sequencing from sub-microliter samples was demonstrated for capillary array electrophoresis by optimizing the analysis of 500 nl reaction aliquots of full-volume reactions and by preparing 500 nl reactions within fused-silica capillaries. Sub-microliter aliquots were removed from the pooled reaction products of 10 microl dye-primer cycle-sequencing reactions and analyzed without modifying either the reagent concentrations or instrument workflow. The impact of precipitation methods, resuspension buffers, and injection times on electrokinetic injection efficiency for 500 nl aliquots were determined by peak heights, signal-to-noise ratios, and changes in base-called readlengths. For 500 nl aliquots diluted to 5 microl in 60% formamide-1 mM EDTA and directly injected, a five-fold increase in signal-to-noise ratios was obtained by increasing injection times from 10 to 80 s without a corresponding increase in peak widths or reduction in readlengths. For 500 nl aliquots precipitated in alcohol, 80 +/- 5% template recovery and a two-fold decrease in conductivity was obtained, resulting in a two-fold increase in peak heights and 50 to 100 bases increase in readlengths. In a comparison of aliquot volumes and precipitation methods, equivalent readlengths were obtained for 500 nl, 4 microl, and 8 microl aliquots by simply adjusting the electrokinetic injection conditions. To ascertain the robustness of this methodology for genomic sequencing, 96 Arabidopsis thaliana subclones were sequenced, with a yield of 38 624 bases obtained from 500 nl aliquots versus 30 764 bases from standard scale reactions. To demonstrate 500 nl sample preparation, reactions were performed in fused-silica capillary reaction chambers using air-based thermal cycling. A readlength of 690 bases was obtained for the polymerase chain reaction product of an Arabidopsis subclone without modifying the reagent concentrations, post-reaction processing or electrokinetic injection workflow. These results demonstrated the fundamental feasibility of small-volume DNA sequencing for high-throughput capillary electrophoresis.

Arabidopsis↗

PubMed2003

Emergency department crowding: consensus development of potential measures.

STUDY OBJECTIVE: We identify measures of emergency department (ED) and hospital workflow that would be of value in understanding, monitoring, and managing crowding. METHODS: A national group of 74 experts developed 113 potential measures using a conceptual model of ED crowding that segmented the measures into input, throughput, and output categories. Ten investigators then used group consensus methods to revise and consolidate them into a refined set of 30 measures that were rated by all 74 experts, who used a magnitude estimation technique on a Web site. Each measure was compared with a standard to obtain numeric ratings for feasibility, affordability, early warning potential, long-term planning potential, a summary rating of operational usefulness, and research potential. After review of the comprehensiveness of the resulting measures, 8 additional measures were developed and also rated by all reviewers. RESULTS: The original set of 113 measures (46 input, 35 throughput, and 32 output) was reduced to 38 through the iterative revision and rating process (15 input, 9 throughput, and 14 output). Summary scores in each rating category showed significant variation in ratings among the various potential measures. For measures that address similar concepts, the priority ranking depended on the rating category chosen. CONCLUSION: The final 38 measures of ED and hospital workflow provide a useful pool from which EDs and policymakers can draw to improve their ability to understand and address the issue of ED crowding. These measures require rigorous testing for feasibility, reliability, and value.

Consensus↗

PubMed2003

From PACS to integrated EMR.

The integration of medical images as part of the patient record has always been a critical component of documentation and information supporting clinical decisions. In the past two decades the increased number of imaging procedures that allows a more accurate and more specific diagnosis has significantly increased and their role in patient management has grown rapidly. With the evolution toward digital modalities and management of medical images in a fully digital environment with the deployment of enterprise wide Picture Archiving Communication Systems (PACS) a wider and more rapid access to the images by referring physicians and clinicians has become possible. The parallel evolution of electronic medical records (EMR) supporting all other documents and clinical data in electronic format led to the necessity of integrating medial image data with the rest of the patient record. Although the marriage of medical images and patient record data in electronic format seems a very natural and necessary combination it has often been very slow in development due to the lack of standardization and clear understanding of clinical workflows and clinical requirements. Several early implementations demonstrated the added value of combining medical images with the patient record and have shown that the availability of data and images facilitates and improves the accuracy and efficiency of patient management. Recent efforts in industry and the academic community to harmonize and improve the integration of medical images with patient record, with the promotion of new standards and better definitions of clinical workflows and standard mechanism of integration of different types of data into unified data models, has facilitated the deployment of modern EMR. Also, a shift in paradigm due to recent technological revolutions such as the development of the World Wide Web and the concepts of portal servers for accessing data for multiple sources has significantly boosted the trends into open systems which allows easier and more functional integration of medical data from different sources. Furthermore, the emergence of a new strategy for software development, based on open source components, allows software programs to be shared and exchanged between different institutions leading to more rapid deployment of standardized electronic patient record.

Diffusion of Innovation↗

PubMed2000

Guideline-based careflow systems.

This paper describes a methodology for achieving an efficient implementation of clinical practice guidelines. Three main steps are illustrated: knowledge representation, model simulation and implementation within a health care organisation. The resulting system can be classified as a 'guideline-based careflow management system'. It is based on computational formalisms representing both medical and health care organisational knowledge. This aggregation allows the implementation of a guideline, not only as a simple reminder, but also as an 'organiser' that facilitates health care processes. As a matter of fact, the system not only suggests the tasks to be performed, but also the resource allocation. The methodology initially comprehends a graphical editor, that allows an unambiguous representation of the guideline. Then the guideline is translated into a high-level Petri net. The resources, both human and technological necessary for performing guideline-based activities, are also represented by means of an organisational model. This allows the running of the Petri net for simulating the implementation of the guideline in the clinical setting. The purpose of the simulation is to validate the careflow model and to suggest the optimal resource allocation before the careflow system is installed. The final step is the careflow implementation. In this phase, we show that the 'workflow management' technology, widely used in business process automation, may be transferred to the health care setting. This requires augmenting the typical workflow management systems with the flexibility and the uncertainty management, typical of the health care processes. For illustrating the proposed methodology, we consider a guideline for the management of patients with acute ischemic stroke.

Artificial Intelligence↗

PubMed2001

Flexible guideline-based patient careflow systems.

Workflow Management Systems integrate domain and organisational knowledge to support business processes. When applied to the medical environment, they can be termed "Careflow Management Systems", and may be used to manage care delivery by enhancing co-operation among healthcare professionals. This paper focuses on care delivery based on clinical practice guidelines. Healthcare organisations are very different from industrial or commercial companies: their main goal is not profit, but maintaining and improving the health of the public. Therefore, outcomes are difficult to measure. Firstly, physicians, while playing a variety of roles, are quite independent decision-makers; secondly, the object of the process, i.e. the patient, may be involved in choosing treatment options, and may be treated by different institutions. For these reasons, the standard functionality of typical Workflow Management Systems must be strongly enhanced in order to cope with healthcare delivery needs. A major issue is accounting for exceptions. In most non-clinical settings this is not a problem because processes are very well defined and can often be easily controlled by some higher authority. As explained above, this does not happen in healthcare organisations. Responsibilities are widely shared, and health care professionals may be non-compliant with guidelines for a variety of reasons. The paper presents a classification of possible exceptions, and shows how the sequence of tasks described by a guideline may be altered, at the implementation level, in order to meet actual user needs, while maintaining guideline intentions as much as possible. A terminology server is also exploited towards this end. This work illustrates a prototype of a Careflow Management System based on an international guideline for ischemic stroke treatment, developed by the American Heart Association.

Artificial Intelligence↗

PubMed2001

The socio-organizational age of artificial intelligence in medicine.

The increasing pressure on Health Care Organizations (HCOs) to ensure efficiency and cost-effectiveness, balancing quality of care and cost containment, will drive them towards a more effective management of medical knowledge derived from research findings. The relation between science and health services has until recently been too casual. The primary job of medical research has been to understand the mechanisms of disease and produce new treatments, not to worry about the effectiveness of the new treatments or their implementation. As a result many new treatments have taken years to become part of routine practice, ineffective treatments have been widely used, and medicine has been opinion rather than evidence based. This results in suboptimal care for patients. Knowledge management technology may provide effective approaches in speeding up the diffusion of innovative medical procedures whose clinical effectiveness have been proved: the most interesting one is represented by computer-based utilization of evidence-based clinical guidelines. As researchers in Artificial Intelligence in Medicine (AIM), we are committed to foster the strategic transition from opinion to evidence-based decision making. Reviews of the effectiveness of various methods of guideline dissemination show that the most predictable impact is achieved when the guideline is made accessible through computer-based and patient specific reminders that are integrated into the clinician's workflow. However, the traditional single doctor-patient relationship is being replaced by one in which the patient is managed by a team of health care professionals, each specializing in one aspect of care. Such shared care depends critically on the ability to share patient-specific information and medical knowledge easily among them. Strategically there is a need to take a more clinical process view of health care delivery and to identify the appropriate organizational and information infrastructures to support this process. Thus, the great challenge for AIM researchers is to exploit the astonishing capabilities of new technologies to disseminate their tools to benefit HCOs by assuring the conditions of knowledge management and organizational learning at the fullest extent possible. To achieve such a strategic goal, a guideline can be viewed as a model of the care process. It must be combined with an organization model of the specific HCO to build patient careflow management systems. Artificial intelligence can be extensively used to design innovative tools to support all the development stages of those systems. However, exploiting the knowledge represented in a guideline to build them requires to extend today's workflow technology by solving some challenging problems.

Artificial Intelligence↗

PubMed2001

IMRT: high-definition radiation therapy in a community hospital.

A multileaf collimator (MLC)-based intensity-modulated radiation therapy (IMRT) program was implemented successfully at Monmouth Medical Center, a community hospital at Long Branch, New Jersey. Our clinical experience gained in the treatment of over 80 patients using IMRT for prostate, head and neck, and brain is reviewed, and some of the clinical issues are also, discussed. Implementation of the IMRT requires a treatment planning system, computer-controlled beam-shaping aperture, electronic record and verify system, and a good physics quality assurance program. These components, by grouping them efficiently, have created a seamless workflow for our complete radiotherapy process of IMRT. Each of these radiotherapy processes are discussed for clarity and the clinical importance is also evaluated. Of particular interest is inverse treatment planning that will impact treatment delivery such as beam orientation, treatment ports, and organ motion of IMRT. A checklist for physics and departmental quality assurance is suggested, with the intention of providing systematic workflow, making IMRT feasible at a community medical center setting. This is especially important because most of our cancer patients received radiation therapy locally. Lastly, the reimbursement issue affecting the implementation of IMRT at our medical center is also discussed to justify this new treatment protocol for future clinical outcomes.

Head and Neck Neoplasms↗

PubMed2002

Creating a culture of medication administration safety: laying the foundation for computerized provider order entry.

BACKGROUND: Computerized provider order entry (CPOE) systems are recognized as an effective tool for reducing preventable adverse drug events; however, implementation is a complex process that involves much more than installing new software. The literature addresses the use of these systems in large tertiary care hospitals and university settings; yet there is little information on their implementation and use in smaller hospitals. Beaver Dam Community Hospital, a small, rural hospital, set about laying the foundation for implementing CPOE. Actions were taken in terms of context (the culture and attitude, acceptance, and importance regarding the change), process (roles, workflow, and policies relating to the change), and content (how-to, such as procedural steps and rules). USE OF THE RAPID-CYCLE IMPROVEMENT PROCESS: The team elected to use the rapid-cycle improvement process for implementation to allow it to move ahead quickly, adjusting changes as necessary for maximum success. Each change was considered an individual Plan-Do-Check-Act cycle, with its own action plan and measurement for successful implementation. PLANNING ACTUAL IMPLEMENTATION: The Patient Safety Committee has begun the planning of actual implementation--Phase II. Issues addressed include how to phase in the system--in which units to bring up first, how to structure the transitional period, how to redesign workflow, and how to plan role changes. SUMMARY: The changes already implemented contribute to medication safety and are important from that perspective alone, without the use of CPOE. The addition of an electronic system will enhance the organization's ability to provide safe, accurate medication administration.

Clinical Pharmacy Information Systems↗

PubMedDate not supplied

Time and cost analysis of repacking medications in unit-of-use containers.

OBJECTIVES: To evaluate the effects of repacking drugs in unit-of-use containers in community pharmacies. The purpose of this study was to examine whether unit-of-use repacking reduces routine mechanical "counting and pouring" to allow more time for pharmaceutical care. DESIGN: Simulation pilot study to evaluate the differences between the existing and proposed systems. Based on the literature, four variables--optimum pack size, time savings, packaging costs, and shelving requirements--were selected for evaluation. Historical prescription data from a chain were used in determining the sample drugs and their optimum pack sizes. Workflow analysis and time study were used to estimate the time savings. Manufacturer bottles, repack bottles, and shelves were measured to determine the impact of using unit-of-use containers on storage requirements. SETTING: Three community pharmacies in a major drugstore chain in Cincinnati, Ohio. RESULTS: The 25 fastest-moving solid oral dosage forms, representing 21.6% of all drugs dispensed by the chain, were selected as the sample drugs for the study. The workflow analysis and time study revealed that 0.79 minutes could be saved per prescription if repacked drugs were used. There was an increased cost of approximately $0.05 for every repack bottle used in place of a prescription vial. It was calculated that repacking in unit-of-use containers would increase storage requirements in the pharmacy by 2.5 times if current inventory levels were maintained. CONCLUSION: Repacking drugs in unit-of-use containers is potentially an inexpensive method to increase usable time in the pharmacy that does not require an increase in personnel or the purchase of additional technology at the store level.

Costs and Cost Analysis↗

PubMed2002

XML-based application interface services--a method to enhance integrability of disease specific systems.

Disease specific systems usually offer excellent functionality for the management of the covered diseases. But the restriction to a certain disease often hampers their wide spread use since they are not optimised for clinical workflow. The Giessener Tumordokumentationssystem (GTDS) is a disease specific system that is not only designed for the use in tumour registries but also to support clinical care. In order to integrate it into hospital information systems, we implemented standard communication interfaces. However, interfaces are not satisfactory since they do not consider aspects of the normal workflow of a clinical user. Therefore, we developed a strategy that should ease the access to the system in the environment of existing systems. From the technical point of view, XML with its capabilities to represent even complex data in a rather simple way helped to implement this strategy. We use XML to communicate with API-like services and created a WWW environment to demonstrate the access to these services. Since HTML based access itself is a means to integrate systems, we intend to expand this environment to an appropriate region based means to improve the communication with registries. Another application using the services is the transfer of data between two registries with common patients.

Algorithms↗

PubMed2003

Guideline validation in multiple trauma care through business process modeling.

Clinical guidelines can improve the quality of care in multiple trauma. In our Department of Trauma Surgery a specific guideline is available paper-based as a set of flowcharts. This format is appropriate for the use by experienced physicians but insufficient for electronic support of learning, workflow and process optimization. A formal and logically consistent version represented with a standardized meta-model is necessary for automatic processing. In our project we transferred the paper-based into an electronic format and analyzed the structure with respect to formal errors. Several errors were detected in seven error categories. The errors were corrected to reach a formally and logically consistent process model. In a second step the clinical content of the guideline was revised interactively using a process-modeling tool. Our study reveals that guideline development should be assisted by process modeling tools, which check the content in comparison to a meta-model. The meta-model itself could support the domain experts in formulating their knowledge systematically. To assure sustainability of guideline development a representation independent of specific applications or specific provider is necessary. Then, clinical guidelines could be used for eLearning, process optimization and workflow management additionally.

Humans↗

PubMed2026

13C Stable Isotope Tracing-Based MFA Reveals the Contribution of Glucose to Glycolytic and TCA Fluxes and Its Application in Depression Research.

Metabolomics is widely applied to dissect metabolic pathways and their correlations with biological phenotypes. Unlike genomics and proteomics, metabolites exhibit substantial heterogeneity in chemical structure, physicochemical properties, and biological origin. Accordingly, pathway enrichment and annotation relying merely on alterations in metabolite abundance are prone to incomplete coverage, ionization bias, and ambiguous annotation, which inevitably impair the accuracy of pathway interpretation. Metabolic flux analysis (MFA) coupled with stable isotope-resolved metabolomics (SIRM) offers a powerful quantitative framework for tracing in vivo carbon flow and estimating reaction fluxes across key metabolic nodes. Glucose metabolism lies at the core of systemic energy homeostasis; however, most current investigations are confined to cell lines or in vitro systems, and a simple, easy-to-implement computational pipeline for in vivo glucose flux analysis in animal models is still lacking. Herein, we established an in vivo 13C-labeling-based MFA workflow to trace and resolve the systemic metabolic fate of glucose in rats. The pipeline covers tracer administration, sample preparation, LC-MS detection, isotopologue data acquisition and correction, construction of a glucose-metabolism-related metabolite database, MFA model establishment, and metabolic flux quantification. By infusing rats with [U-13C6]-glucose and [U-13C3]-sodium L-lactate, we precisely characterized the in vivo metabolic fates of circulating glucose and lactate and quantified their respective contributions to glycolytic flux and tricarboxylic acid (TCA) cycle flux. We further applied this workflow to profile energy metabolic reprogramming in depression. The results revealed a systemic shift toward aerobic glycolysis in rats exposed to chronic unpredictable mild stress (CUMS). Overall, the expanded application of this MFA strategy can provide mechanistic and quantitative insights into the regulation of metabolic pathways.

Animals↗

PubMed2026

An Instrumental Optimization of a Label-Free Proteomic Method for Trace Protein Input.

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics of trace-level samples, such as tens of cells or spatially resolved tissue regions, offers unique biological insights but is often constrained by the requirement for specialized, costly instrumentation. In this study, we developed a scalable workflow for the deep proteomic analysis of low- to ultralow-input samples by systematically optimizing a widely adopted Orbitrap and UHPLC platform to maximize sensitivity, precision, and throughput. This optimized workflow identified over 5600 proteins from 5 ng of peptides and 3400 proteins from 20 sorted cells, achieving a throughput of 30 analyses per day while maintaining deep proteome coverage and high quantitative reproducibility. Furthermore, by applying this method to spatially resolved proteomics, we identified over 6100 proteins from microscale regions of interest (ROIs) within a formalin-fixed, paraffin-embedded (FFPE) tissue. A data-driven normalization strategy was employed to correct for variable cellularity across tissue regions, effectively revealing intratumor heterogeneity and distinct molecular and functional signatures, including pathway activations not apparent in parallel spatial transcriptomic analysis. Ultimately, this accessible, high-performance method substantially lowers the instrumentation barrier for the deep proteomic profiling of trace-level biological samples.

Proteomics↗

PubMed2026

Spatial Proteomics of the Human Atherosclerotic Microenvironment Reveals Heterogeneity in Intraplaque Proteomes and Extracellular Matrix Remodeling.

Plaque heterogeneity underlies the propensity of atherosclerotic lesions to rupture and trigger cardiovascular events. Most proteomic studies examine bulk changes, obscuring key spatial differences in protein abundance. We report a high-resolution spatial proteomics workflow exploring the molecular landscape of human plaques and a murine myocardium. By combining laser capture microdissection with high-sensitivity ion-mobility mass spectrometry, spatial profiling of cellular and extracellular matrix (ECM) proteomes was achieved. Over 2700 proteins were detected from 50,000 μm2 areas, revealing substantial intraplaque heterogeneity across distinct regions (lipid-rich, media, shoulder, necrotic core, intima) and distance from the artery lumen. Inverse correlations between proteases (cathepsin B) and core structural ECM proteins (perlecan, HSPG2) indicated active ECM remodeling. Analysis of media layers indicated distinct protein signatures associated with smooth muscle contraction and cell-cell communication. Blood coagulation signatures, including platelet degranulation and fibrin formation, were enriched at the intima. Inflammatory (clusters of differentiation 4/68, CD4/CD68; vascular cell adhesion molecule 1, VCAM1) and vascular damage markers (tenascin-C, TNC) were enriched in shoulder regions. The necrotic core was dominated by blood proteins, consistent with intraplaque hemorrhage. This workflow resolves proteomic changes over ∼200 μm distances, providing unprecedented insights into plaque morphology and offers a powerful tool for elucidating plaque biology.

Humans↗

PubMed2026

Low-Cost Nucleic-Acid-Based Radial Flow Assay for the Detection of GSTP1 Promoter DNA Methylation in Prostate Cancer.

DNA methylation of the glutathione S-transferase pi 1 (GSTP1) promoter is a widely studied epigenetic biomarker for prostate cancer; however, its direct detection in genomic DNA remains analytically challenging without complex chemical or amplification-based workflows. Here, we report a nucleic acid-based radial flow assay (NABRFA) that enables visual and pattern-based detection of gene-specific DNA methylation using gold nanoparticle (AuNP)-conjugated oligonucleotide probes. Thiol-modified single-stranded DNA probes targeting the GSTP1 CpG island (5'ThG) were conjugated to AuNPs to form stable probe-nanoparticle constructs that retain colloidal stability under high ionic strength conditions (0.5 M NaCl). Upon hybridization with methylation-protected GSTP1 DNA, the resulting AuNP-DNA complexes exhibit hybridization-dependent modulation of transport and retention on a porous nylon membrane, generating characteristic concentric radial patterns. These patterns arise from spatial separation between retained hybridized complexes and outwardly transported unbound probe-functionalized nanoparticles, enabling direct visual discrimination of target presence. The assay demonstrated concentration-dependent pattern evolution, with visual detection achievable down to 1 ng of target DNA and an analytically determined limit of detection of approximately 32 ng, based on image-derived gray value analysis. The human prostate cancer cell line LNCaP, known for GSTP1 promoter hypermethylation, was used as the test model for assay validation. Comparative analysis using methyl-sensitive restriction enzyme-treated native genomic DNA from the human osteosarcoma MG-63 cell line (non-prostate cancer, GSTP1 methylation-negative control) and the human lung fibroblast WI-38 cell line (non-cancerous, GSTP1 methylation-negative control) confirmed assay specificity. By coupling sequence-specific hybridization with transport-mediated nanoparticle pattern formation, NABRFA provides a label-free and conversion-free analytical strategy for detection of methylation-protected GSTP1 DNA using minimal instrumentation. This work establishes a proof-of-concept membrane-based, transport-driven sensing approach for epigenetic biomarker detection and highlights its potential for integration into simplified molecular diagnostic workflows.

Humans↗