Detection Of Proteins Research Articles

Protein-Labeling Reagents Selectively Activated by Copper(I).

Copper is an essential trace element that participates in many biological processes through its unique redox cycling between cuprous (Cu+) and cupric (Cu2+) oxidation states. To elucidate the biological functions of copper, chemical biology tools that enable selective visualization and detection of copper ions and proteins in copper-rich environments are required. Herein, we describe the design of Cu+-responsive reagents based on a conditional protein labeling strategy. Upon binding Cu+, the probes generated quinone methide via oxidative bond cleavage, which allowed covalent labeling of surrounding proteins with high Cu+ selectivity. Using gel- and imaging-based analyses, the best-performing probe successfully detected changes in the concentration of labile Cu+ in living cells. Moreover, conditional proteomics analysis suggested intramitochondrial Cu+ accumulation in cells undergoing cuproptosis. Our results highlight the power of Cu+-responsive protein labeling in providing insights into the molecular mechanisms of Cu+ metabolism and homeostasis.

Spatial multi-omics at subcellular resolution via high-throughput in situ pairwise sequencing.

Technology for spatial multi-omics aids the discovery of new insights into cellular functions and disease mechanisms. Here we report the development and applicability of multi-omics in situ pairwise sequencing (MiP-seq), a method for the simultaneous detection of DNAs, RNAs, proteins and biomolecules at subcellular resolution. Compared with other in situ sequencing methods, MiP-seq enhances decoding capacity and reduces sequencing and imaging costs while maintaining the efficacy of detection of gene mutations, allele-specific expression and RNA modifications. MiP-seq can be integrated with in vivo calcium imaging and Raman imaging, which enabled us to generate a spatial multi-omics atlas of mouse brain tissues and to correlate gene expression with neuronal activity and cellular biochemical fingerprints. We also report a sequential dilution strategy for resolving optically crowded signals during in situ sequencing. High-throughput in situ pairwise sequencing may facilitate the multidimensional analysis of molecular and functional maps of tissues.

Aerobic exercise training engages the canonical wnt pathway to improve pulmonary function and inflammation in COPD

BackgroundWe studied whether the exercise improves cigarette smoke (CS) induced chronic obstructive pulmonary disease (COPD) in mice through inhibition of inflammation mediated by Wnt/β-catenin-peroxisome proliferator-activated receptor (PPAR) γ signaling.MethodsFirstly, we observed the effect of exercise on pulmonary inflammation, lung function, and Wnt/β-catenin-PPARγ. A total of 30 male C57BL/6J mice were divided into the control group (CG), smoke group (SG), low-intensity exercise group (LEG), moderate-intensity exercise group (MEG), and high-intensity exercise group (HEG). All the groups, except for CG, underwent whole-body progressive exposure to CS for 25 weeks. Then, we assessed the maximal exercise capacity of mice from the LEG, MEG, and HEG, and performed an 8-week treadmill exercise intervention. Then, we used LiCl (Wnt/β-catenin agonist) and XAV939 (Wnt/β-catenin antagonist) to investigate whether Wnt/β-catenin-PPARγ pathway played a role in the improvement of COPD via exercise. Male C57BL/6J mice were randomly divided into six groups (n = 6 per group): CG, SG, LiCl group, LiCl and exercise group, XAV939 group, and XAV939 and exercise group. Mice except those in the CG were exposed to CS, and those in the exercise groups were subjected to moderate-intensity exercise training. All the mice were subjected to lung function test, lung histological assessment, and analysis of inflammatory markers in the bronchoalveolar lavage fluid, as well as detection of Wnt1, β-catenin and PPARγ proteins in the lung tissue.ResultsExercise of various intensities alleviated lung structural changes, pulmonary function and inflammation in COPD, with moderate-intensity exercise exhibiting significant and comprehensive effects on the alleviation of pulmonary inflammation and improvement of lung function. Low-, moderate-, and high-intensity exercise decreased β-catenin levels and increased those of PPARγ significantly, and only moderate-intensity exercise reduced the level of Wnt1 protein. Moderate-intensity exercise relieved the inflammation aggravated by Wnt agonist. Wnt antagonist combined with moderate-intensity exercise increased the levels of PPARγ, which may explain the highest improvement of pulmonary function observed in this group.ConclusionsExercise effectively decreases COPD pulmonary inflammation and improves pulmonary function. The beneficial role of exercise may be exerted through Wnt/β-catenin-PPARγ pathway.

Serum monoclonal immunoglobulin light-chain detection differs between immunofixation electrophoresis methods in patients with AL amyloidosis.

Serum immunofixation electrophoresis (IFE) is often performed for screening monoclonal proteins (M proteins) in immunoglobulin light-chain amyloidosis (AL amyloidosis). However, the performance of serum IFE for detecting M protein in AL amyloidosis patients is often insufficient. In this study, we examined the detection rate of serum M protein in newly diagnosed AL amyloidosis patients and analyzed differences in M protein detection between IFE methods. Among 60 patients newly diagnosed with AL amyloidosis, 22 had undetectable serum M protein by IFE with the Epalyzer2 system. Samples with undetectable M protein had significantly lower involved serum-free light-chain (iFLC) and a smaller difference between involved and uninvolved serum-free light-chain (dFLC) values than samples with IFE-detectable monoclonal light chains. When samples that tested negative for M protein by the Epalyzer2 system were retested by IFE with the HYDRASYS 2 system, 50% had IFE-detectable monoclonal light chains. The IFE system and reagents used may affect serum monoclonal immunoglobulin light-chain detection in AL amyloidosis patients, especially those with low iFLC or low dFLC samples. More attention should be paid to the performance of IFE systems, since it may affect the diagnostic and therapeutic evaluation of AL amyloidosis patients.

Label-free tracking of cytochrome C oxidation state in live cells by resonance Raman imaging.

Free interconversion of cytochrome C (CytC) between native ferrous (Cyt-FeII) and oxidized ferric (CytC-FeIII) states is necessary to maintain the respiratory function of mitochondria. Disturbances in CytC-FeIII to total CytC ratio may indicate mitochondrial dysfunction and apoptosis. Thus, tracking CytC oxidation state delivers important information about cellular physiology. In this work, we propose a novel methodology based on resonance Raman (rR) imaging optimized uniquely to track and qualitatively analyze the transition of Cyt-FeII to CytC-FeIII within live cells without affecting their morphology. None of the commonly used excitation lines allows such clear-cut differentiation, contrary to the 405 nm applied in this work. The presented methodology provides a novel pathway in the label-free detection of ferrous and ferric heme proteins.

Dielectrophoresis: Measurement technologies and auxiliary sensing applications.

Dielectrophoresis (DEP), which arises from the interaction between dielectric particles and an aqueous solution in a nonuniform electric field, contributes to the manipulation of nano and microparticles in many fields, including colloid physics, analytical chemistry, molecular biology, clinical medicine, and pharmaceutics. The measurement of the DEP force could provide a more complete solution for verifying current classical DEP theories. This review reports various imaging, fluidic, optical, and mechanical approaches for measuring the DEP forces at different amplitudes and frequencies. The integration of DEP technology into sensors enables fast response, high sensitivity, precise discrimination, and label-free detection of proteins, bacteria, colloidal particles, and cells. Therefore, this review provides an in-depth overview of DEP-based fabrication and measurements. Depending on the measurement requirements, DEP manipulation can be classified into assistance and integration approaches to improve sensor performance. To this end, an overview is dedicated to developing the concept of trapping-on-sensing, improving its structure and performance, and realizing fully DEP-assisted lab-on-a-chip systems.

Hypoxia differently regulates the proportion of ALDHhi cells in lung squamous carcinoma H520 and adenocarcinoma A549 cells via the Wnt/β-catenin pathway.

Cancer stem cells (CSCs) are a specific subpopulation of cancer cells with the ability of self-renewal, infinite proliferation, multidifferentiation and tumorigenicity, and play critical roles in cancer progression and treatment resistance. CSCs are tightly regulated by the tumor microenvironment, such as hypoxia; however, how hypoxia regulates CSCs in non-small cell lung cancer (NSCLC) remains unclear. The proportion of ALDHhi cells was examined using the Aldefluor assay. Tankyrase inhibitor XAV939 and siRNA were used to inhibit β-catenin while pcDNA3-β-catenin (S33Y) plasmid enhanced the expression of β-catenin. Western blot was administered for protein detection. The mRNA expression was measured by quantitative real-time PCR. We found that hypoxia led to an increase in the proportion of ALDHhi cells in lung squamous carcinoma (LUSC) H520 cells, while causing a decrease in the ALDHhi cell proportion in lung adenocarcinoma (LUAD) A549 cells. Similarly, β-catenin expression was upregulated in H520 cells but downregulated in A549 cells upon exposure to hypoxia. Mechanically, the proportion of ALDHhi cells in both cell lines was decreased by β-catenin inhibitor or siRNA knockdown, whereas increased after β-catenin overexpression. Furthermore, hypoxia treatment suppressed E-cadherin expression in H520 cells and enhanced N-cadherin and β-catenin expression, while this effect was completely opposite in A549 cells. The hypoxia-EMT-β-catenin axis functions as an important regulator for the proportion of CSCs in NSCLC and could potentially be explored as therapeutic targets in the future.

Label-free and label-based electrochemical detection of disease biomarker proteins

Introduction: Biosensors, analytical devices integrating biological sensing elements with physicochemical transducers, have gained prominence as rapid and convenient tools for monitoring human health status using biochemical analytes. Due to its cost-effectiveness, simplicity, portability, and user-friendliness, electrochemical detection has emerged as a widely adopted method in biosensor applications. Crucially, biosensors enable early disease diagnosis by detecting protein biomarkers associated with various conditions. These biomarkers offer an objective indication of medical conditions that can be accurately observed from outside the patient. Method: This review comprehensively documents both label-free and labelled detection methods in electrochemical biosensor techniques. Label-free detection mechanisms elicit response signals upon analyte molecule binding to the sensor surface, while labelled detection employs molecular labels such as enzymes, nanoparticles, and fluorescent tags. Conclusion: The selection between label-free and labelled detection methods depends on various factors, including the biomolecular compound used, analyte type and biological binding site, biosensor design, sample volume, operational costs, analysis time, and desired detection limit. Focusing on the past six years, this review highlights the application of label-free and labelled electrochemical biosensors for detecting protein biomarkers of diseases.

Dengue: A review of laboratory diagnostics in the vaccine age

Background. Dengue is an important arboviral infection of considerable public health significance. It occurs in a wide global belt within a variety of tropical regions. The timely laboratory diagnosis of Dengue infection is critical to inform both clinical management and an appropriate public health response. Vaccination against Dengue virus is being introduced in some areas. Discussion. Appropriate diagnostic strategies will vary between laboratories depending on the available resources and skills. Diagnostic methods available include viral culture, the serological detection of Dengue-specific antibodies in using enzyme immunoassays (EIAs), microsphere immunoassays, haemagglutination inhibition or in lateral flow point of care tests. The results of antibody tests may be influenced by prior vaccination and exposure to other flaviviruses. The detection of non-structural protein 1 in serum (NS1) has improved the early diagnosis of Dengue and is available in point-of-care assays in addition to EIAs. Direct detection of viral RNA from blood by PCR is more sensitive than NS1 antigen detection but requires molecular skills and resources. An increasing variety of isothermal nucleic acid detection methods are in development. Timing of specimen collection and choice of test is critical to optimize diagnostic accuracy. Metagenomics and the direct detection by sequencing of viral RNA from blood offers the ability to rapidly type isolates for epidemiologic purposes. Conclusion. The impact of vaccination on immune response must be recognized as it will impact test interpretation and diagnostic algorithms.

Constructing a novel prognostic model for triple-negative breast cancer based on genes associated with vasculogenic mimicry.

Research has shown a connection between vasculogenic mimicry (VM) and cancer progression. However, the functions of genes related to VM in the emergence and progression of TNBC have not been completely elucidated. A survival risk model was constructed by screening biomarkers using DESeq2 and WGCNA based on public TNBC transcriptome data. Furthermore, gene set enrichment analysis was performed, and tumor microenvironment and drug sensitivity were analyzed. The selected biomarkers were validated via quantitative PCR detection, immunohistochemical staining, and protein detection in breast cancer cell lines. Biomarkers related to the proliferation and migration of TNBC cells were validated via in vitro experiments. The findings revealed that 235 target genes were connected to the complement and coagulation cascade pathways. The risk score was constructed using KCND2, NRP1, and VSTM4. The prognosis model using the risk score and pathological T stage yielded good validation results. The clinical risk of TNBC was associated with the angiogenesis signaling pathway, and the low-risk group exhibited better sensitivity to immunotherapy. Quantitative PCR and immunohistochemistry indicated that the expression levels of KCND2 in TNBC tissues were higher than those in adjacent nontumor tissues. In the TNBC cell line, the protein expression of KCND2 was increased. Knockdown of KCND2 and VSTM4 inhibited the proliferation and migration of TNBC cells in vitro. In this study, three VM-related biomarkers were identified, including KCND2, NRP1, and VSTM4. These findings are likely to aid in deepening our understanding of the regulatory mechanism of VM in TNBC.

Multi-epitope protein production and its application in the diagnosis of opisthorchiasis

BackgroundOpisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis.MethodsThe Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique.ResultsThe recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively.ConclusionsThe high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces.Graphical

Nicotinamide riboside alleviates brain dysfunction induced by chronic cerebral hypoperfusion via protecting mitochondria

Chronic cerebral hypoperfusion (CCH) is an enduring inadequate blood flow to the brain, resulting in vascular dementia (VaD). However, the effective treatment strategies are lacking. Supplementing with nicotinamide adenine dinucleotide (NAD+) has shown neuroprotective benefits in other neurodegenerative disorders. Nicotinamide riboside (NR), as a precursor of NAD+, is believed to hold promise in improving mitochondrial health, autophagy, and cognitive function. Meanwhile, NR has unique oral bioavailability, good tolerability, and minimal side effects, and it is the most promising for clinical translation. However, the effectiveness of NR in treating CCH-related VaD is still uncertain. The present study examined the neuroprotective effects of NR supplementation and its underlying mechanisms in a CCH rat model. The rats with CCH were given NR at a daily dosage of 400 mg/kg for 3 months. NR supplementation increased blood and brain NAD+ levels and improved brain function in CCH rats, including cognitive function and oxygenation capacity. It also reduced hippocampal neuronal loss and abnormalities and mitigated the decrease in dendritic spine density. The analysis of RNA sequencing in hippocampal tissue supports these findings. Electron microscopy and protein detection results suggest that NR may maintain mitochondrial structural integrity and exert a protective role by attenuating mitochondrial fission and impaired autophagy flux caused by CCH. In conclusion, these findings offer evidence for the neuroprotective potential of NR supplementation in ameliorating cognitive impairment induced by CCH.

Epithelium-derived 6-nitrodopamine modulates noradrenaline-induced contractions in human seminal vesicles

AimsTo evaluate the basal release of 6-nitrodopamine (6-ND) from human isolated seminal vesicles (HISV) and to characterize its action and origin. Main methodsLeft HISV obtained from patients undergoing prostatectomy surgery was suspended in a 3-mL organ bath containing warmed (37 °C) and gassed (95%O2:5%CO2) Krebs-Henseleit's solution (KHS) with ascorbic acid. An aliquot of 2 mL of the supernatant was used to quantify catecholamines by LC-MS/MS. For functional studies, concentration-responses curves to catecholamines were obtained, and pEC50 and Emax values were calculated. Detection of tyrosine hydroxylase and S100 protein were also carried out by both immunohistochemistry and fluorescence in-situ hybridization assays (FISH). Key findingsBasal release of 6-ND was higher than the other catecholamines (14.76 ± 14.54, 4.99 ± 6.92, 3.72 ± 4.35 and 5.13 ± 5.76 nM for 6-ND, noradrenaline, adrenaline, and dopamine, respectively). In contrast to the other catecholamines, the basal release of 6-ND was not affected by the sodium current (Nav) channel inhibitor tetrodotoxin (1 μM; 10.4 ± 8.9 and 10.4 ± 7.9 nM, before and after tetrodotoxin, respectively). All the catecholamines produced concentration-dependent HISV contractions (pEC50 4.1 ± 0.2, 4.9 ± 0.3, 5.0 ± 0.3, and 3.9 ± 0.8 for 6-ND, noradrenaline, adrenaline, and dopamine, respectively), but 6-ND was 10-times less potent than noradrenaline and adrenaline. However, preincubation with very low concentration of 6-ND (10−8 M, 30 min) produced significant leftward shifts of the concentration-response curves to noradrenaline. Immunohistochemical and FISH assays identified tyrosine hydroxylase in tissue epithelium of HISV strips. SignificanceEpithelium-derived 6-ND is the major catecholamine released from human isolated seminal vesicles and that modulates smooth muscle contractility by potentiating noradrenaline-induced contractions.

Circ_WASF2 regulates ferroptosis by miR-634/ GPX4 signaling in pancreatic cancer

PurposePancreatic cancer (PC) is one of the most lethal malignant gastrointestinal tumors (GI) characterized by a poor prognosis. Ferroptosis is an emerging programmed cell death that plays an essential role in the progression of various cancers. Ferroptosis is driven by iron-dependent phospholipid peroxidation and is regulated by mitochondrial activity, lipid peroxidation, and reactive oxygen species (ROS). The function and mechanism of ferroptosis in PC need more research.MethodsThe levels of circRNAs, miRNAs, and mRNAs were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was used for protein detection. CCK8 assays were used to detect cell proliferation. Cell death, lipid peroxidation, ROS, and Fe2+ were detected by indicted kits. Dual-luciferase reporter and RNA pull-down assays were conducted to confirm the interaction between circRNAs, miRNAs, and mRNAs.ResultsIn this research, we found that circular RNA hsa_circ_0000003(circ_WASF2) was upregulated in pancreatic cancer cells. The silence of circ_WASF2 inhibited cancer proliferation and increased cell death by increasing ferroptosis accompanied by up-regulation of lipid peroxidation, ROS, and Fe2+. Further studies showed that circ_WASF2 could attenuate ferroptosis by targeting miR-634 and the downstream glutathione peroxidase 4 (GPX4). GPX4 has been well-reported as a central factor in ferroptosis. Our research revealed a new pathway for regulating ferroptosis in PC.ConclusionIn summary, we have determined that circ_WASF2/miR-634/GPX4 contributed to ferroptosis-induced cell death, and provided a possible therapeutic target in PC.

Exposure to low-concentration fipronil impairs survival, behavior, midgut morphology and physiology of Aedes aegypti larvae

The Aedes aegypti mosquito is a vector for various arboviruses, including dengue and yellow fever. Insecticides, such as pyrethroids and organophosphates, are widely used to manage and control these insects. However, mosquitoes have developed resistance to these chemicals. Therefore, this study aimed to investigate the effects of the commercial formulation of fipronil (Tuit® Florestal; 80% purity) on the survival, behavior, morphology, and proteins related to signaling pathways of the midgut in A. aegypti larvae under controlled laboratory conditions. Significant reductions in immature survival were observed in all concentrations of fipronil tested. Low insecticide concentration (0.5 ppb) led to decreased locomotor activity in the larvae and caused disorganization of the epithelial tissue in the midgut. Moreover, exposure to the insecticide decreased the activity of detoxifying enzymes such as catalase, superoxide dismutase, and glutathione-S-transferase. On the other hand, the insecticide increased protein oxidation and nitric oxide levels. The detection of LC3, caspase-3, and JNK proteins, related to autophagy and apoptosis, increased after exposure. However, there was a decrease in the positive cells for ERK 1/2. Furthermore, the treatment with fipronil decreased the number of positive cells for the proteins FMRF, Prospero, PH3, Wg, Armadillo, Notch, and Delta, which are related to cell proliferation and differentiation. These findings demonstrate that even at low concentrations, fipronil exerts larvicidal effects on A. aegypti by affecting behavior and enzymatic detoxification, inducing protein oxidation, free radical generation, midgut damage and cell death, and inhibiting cell proliferation and differentiation. Thus, this insecticide may represent a viable alternative for controlling the spread of this vector.

Effects of butyrate on intestinal ischemia-reperfusion injury via the HMGB1-TLR4-MyD88 signaling pathway.

This study combined bioinformatics and experimental verification in a mouse model of intestinal ischemia-reperfusion injury (IRI) to explore the protection mechanism exerted by butyrate against IRI. GeneCards, Bioinformatics Analysis Tool for Molecular Mechanisms of Traditional Chinese Medicine and GSE190581 were used to explore the relationship between butyrate and IRI and aging. Protein-protein interaction networks involving butyrate and IRI were constructed via the STRING database, with hub gene analysis performed through Cytoscape. Functional enrichment analysis was conducted on intersection genes. A mouse model of IRI was established, followed by direct arterial injection of butyrate. The experiment comprised five groups: normal, sham, model, vehicle, low-dose butyrate, and high-dose butyrate. Intestinal tissue observation was done via transmission electron microscopy (TEM), histological examination via hematoxylin and eosin (H&E) staining, tight junction proteins detection via immunohistochemistry, and Western blot analysis of hub genes. Drug-target interactions were evaluated through molecular docking. Butyrate protected against IRI by targeting 458 genes, including HMGB1 and TLR4. Toll-like receptor pathway was implicated. Butyrate improved intestinal IRI by reducing mucosal damage, increasing tight junction proteins, and lowering levels of HMGB1, TLR4, and MyD88. Molecular docking showed strong binding energies between butyrate and HMGB1 (-3.7 kcal/mol) and TLR4 (-3.8 kcal/mol). According to bioinformatics predictions, butyrate mitigates IRI via multiple-target and multiple-channel mechanisms. The extent of IRI can be reduced by butyrate through the inhibition of the HMGB1-TLR4-MyD88 signaling pathway, which is related to senescence.

Abstract PO5-15-06: NVIGEN X® Comprehensive Liquid Biopsy for Sensitive ctDNA, Circulating Tumor Cells (CTC), and Protein Detection in Breast Cancer: Next-Generation Sequencing, Patient Case Studies, and Clinical Implications

Abstract Background Breast cancer (BC) represents the most prevalent cancer and is a leading cause of cancer related mortality among women worldwide. Despite notable therapeutic advancements, concern for metastatic recurrence remains a significant challenge. Liquid biopsy has emerged as a promising method for monitoring cancer treatment efficacy and disease progression. However, existing technologies predominantly focus on ctDNA, and their clinical utility is hindered by limited detection sensitivity and accuracy. To address these challenges, NVIGEN has developed NVIGEN X® comprehensive liquid biopsy, a novel minimally invasive approach that integrates ctDNA, CTC, and protein biomarkers. This comprehensive method could enable longitudinal monitoring of tumor dynamics, offering valuable insights into cancer biology, treatment responses, and emerging therapeutic targets. Here, we showcase compelling patient case studies, and the potential clinical implications of our technology for an exciting and evolving field in cancer diagnostics. Method Patients with advanced breast cancer were prospectively identified and consented. 10mL blood per timepoint was collected in Streck tubes. ctDNA, CTC and proteins were captured from a single tube of blood using NVIGEN magnetic nanoparticles according to established protocols. Following DNA extraction, ctDNA and CTC samples underwent NVIGEN’s highly sensitive sample workflow, optimized for NGS data accuracy and efficiency, employing hybridization capture with a 180 gene customizable cancer panel. Sequenced data were processed with NVIGEN’s proprietary high-sensitivity data analysis pipeline. Gene alterations identified from both ctDNA and CTC samples were analyzed and integrated to provide a comprehensive view of the tumor profile. Protein biomarkers of interest were detected using NVIGEN’s on-beads ELISA assay. Correlation analysis was performed to examine the relationship between the detected gene alterations, protein biomarkers, ctDNA/CTC data, and clinicopathologic features. Treatment response and identification of potential new treatment targets were evaluated based on the integrated analysis of ctDNA, CTC, protein biomarkers, and patient information. Result We collected 94 blood samples from 85 patients. Here we will present data from initial 36 patient samples. Of these 36 patients, 18 were tested with ctDNA/protein biomarkers (duo assays), and 18 were tested with ctDNA/CTC/protein biomarkers (trio assays). Comparison between duo and trio samples revealed that trio samples provided 2.5 times the number of detected variants compared to duo samples, with the median of variant numbers in duo assays being 52 (range 22-442), and in trio assays being 130 (range 73-703). Over 50% of the detected variants fell within the 0.1-0.5% variant allele frequency (VAF) range (number 2599) with total variant number being 5123. Several patient case studies were conducted using serial blood samples. These case studies revealed notable patterns of important gene alterations, such as ESR1, ERBB2 and PIK3CA, and demonstrated trends in the presence of these genes in ctDNA, CTC, or their coexistence. Protein biomarker information was correlated with ctDNA, CTC NGS data, and patient clinical status. Conclusions The analysis of the presented data from 36 patient samples with advanced breast cancer highlight the clinical relevance and potential significance of utilizing ctDNA, CTC and protein biomarkers in monitoring disease dynamics, treatment response, and guiding personalized treatment approaches. Our results suggest that individual protein biomarker analysis may be crucial in supporting personalized diagnostics and enabling more effective personalized medicine strategies. Citation Format: Aihua Fu, Minh Ton, Kevan Wang, Weiwei Gu, Henry Jin, Nuzhat Shaikh, Sasha Madan, Shreya Perepa, Tianhong Li, Minetta Liu, Heather Parsons, George Sledge, Fauzia Riaz. NVIGEN X® Comprehensive Liquid Biopsy for Sensitive ctDNA, Circulating Tumor Cells (CTC), and Protein Detection in Breast Cancer: Next-Generation Sequencing, Patient Case Studies, and Clinical Implications [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-15-06.

Recent Progress in the Application of Tau Protein Biosensors for Diagnosis of Neurodegenerative Diseases.

The microtubule-associated Tau protein is found in the central nervous system (CNS) in six major isoforms. Neurodegenerative diseases have been linked to post-translational changes of Tau, most notably phosphorylation. Tau protein's molecular diversity is highly helpful in the identification of neurodegenerative illnesses. Nonetheless, one major obstacle to the early detection of brain illness is the nanoscale identification of tau proteins. The standard methods for identifying tau protein include western blotting, polymerase chain reaction (PCR), and real-time PCR. Enzyme-linked immunosorbent assay (ELISA) is another approach used. The limited sensitivity and specificity of these detections, together with the need for sophisticated equipment, are some of their drawbacks. The development of innovative and complex methods for tau protein screening is necessary to address the aforementioned issues. Biosensors are a cutting-edge instrument that may help identify various neurodegenerative biomarkers as early as feasible. This paper provides an overview of the most recent developments in the detection of neurodegenerative diseases employing biosensors built on nanotechnology and methods for imaging, electrochemical, and optical detection of the Tau protein. Furthermore, we outline the present difficulties and suggest a possible course for biosensor-based detection and intervention in the future.

Dominant Circulating Cell-free Mycobacterial Proteins in in-use Machining Fluid and their Antigenicity Potential.

Occupational exposure to industrial Metalworking Fluid (MWF) colonized by Mycobacterium immunogenum (MI) has been associated with immune lung disease hypersensitivity pneumonitis (HP) in machinists. This warrants regular fluid monitoring for early detection of mycobacterial proteins, especially those with antigenic potential. To detect and identify dominant MI proteins and antigens directly from the field-drawn in-use MWF using an integrated immunoproteomic and immunoinformatic approach. An MI-positive MWF selected by DNA-based screening of several field-drawn MWF samples were cultured to isolate the colonizing strain and profiled for dominant circulating cell- free (ccf) MI proteins, including antigens using an integrated immunoproteomic (1D- and 2Dgel fractionation of seroreactivity proteins combined with shotgun proteomic analysis using LC-MS/ MS) and immunoinformatic strategy. A new MI strain (MJY-27) was identified. The gel fractionated MI protein bands (1Dgel) or spots (2D-gel) seroreactive with anti-MI sera probes (Rabbit and Patient sera) yielded 86 MI proteins, 29 of which showed peptide abundance. T-cell epitope analysis revealed high (90-100%) binding frequency for HLA-I& II alleles for 13 of the 29 proteins. Their antigenicity analysis revealed the presence of 6 to 37 antigenic determinants. Interestingly, one of the identified candidates corresponded to an experimentally validated strong B- and T-cell antigen (AgD) from our laboratory culture-based studies. This first report on dominant proteins, including putative antigens of M. immunogenum prevalent in field in-use MWF, is a significant step towards the overall goal of developing fluid monitoring for exposure and disease risk assessment for HP development in machining environments.

Abstract PO4-06-12: Distinction of basal-like and triple-negative basal-like breast cancers utilizing a novel comprehensive single-cell liquid biopsy-based test

Abstract Introduction: Breast cancer is a highly heterogeneous disease and is a leading cause of death for women worldwide. Triple-negative breast cancer (TNBC), the most aggressive form of the disease, is clinically defined by the lack of expression of estrogen receptor (ER) and progesterone receptor (PR), as well as lack of over-expression of human epidermal growth factor receptor-2 (HER2). Approximately 70% of all TNBC cases exhibit basal-like cells, characterized by high basal expression of keratins as well as extensive genomic instability with copy number gains and losses across most chromosomes. In this study we show that novel single cell genomic evaluation coupled with immunofluorescence (IF) protein analysis of CTCs can detect TNBC with basal-like genomic features as well as basal-like tumors with expression of ER and HER2. Materials and Methods: Epic Sciences’ DefineMBC™ liquid biopsy test characterizes CTC proteomics and genomics as well as ctDNA for the treatment of metastatic breast cancer. Blood samples from 813 metastatic breast cancer patients, with either newly diagnosed or progressing disease, with historical tissue subtype [HR(+)HER2(-), HR(+/-)HER2(+), and TNBC] supplied by the referring physician were collected and tested. Up to 5 CTCs were selected and isolated from each sample based on morphology and IF signal. DNA was extracted from individual cells, and whole genome amplified and sequenced at low coverage. Copy number analysis was performed to determine copy number gains and losses across chromosomes, as well as large scale-state transitions (LSTs) and ERBB2 amplification. CTCs characterized with high chromosomal instability (e.g., exhibiting >30 LSTs; 1Q firestorm and 8Q stair step) and high levels of cytokeratin expression (mean fluorescence intensity signal >10K) were categorized as basal-like. Results: By previous metastatic tissue biopsy, 77% were HR(+), 8% were HER2(+), and 12% were TNBC. CTCs categorized in DefineMBC as basal-like subtype (9.8 % of all samples tested) showed to be highly present in patients called as TNBC (79% basal-like). Additionally, 54% of HR(+) and 23% of HER2(+) had basal-like CTCS. Patients characterized with TNBC by metastatic tissue biopsy and by DefineMBC exhibited basal-like cells in 88% of the cases. Conclusion: The results show a novel clinical assay which utilizes CTC protein detection as well as single-cell genomic analysis to identify tumors driven by basal-like cells. Generally, TNBC is classified with negative biomarker expression. We show that CTCs in TNBC display genomically basal-like cells. CTCs from HR(+) and HER2(+) tumors may express basal-like features with preserved ER and HER2 expression. The comprehensive nature of DefineMBC™ utilizing genomic analysis of detected CTCs has provided a new tool to diagnose both basal-like TNBC and basal-like hormone receptor positive and HER2 over-expressing tumors. Citation Format: Alessandra Cunsolo, David Bourdon, Ernest Lam, Giuseppe Di Caro, Nilesh Dharajiya, Timothy Pluard, Lee Schwartzberg. Distinction of basal-like and triple-negative basal-like breast cancers utilizing a novel comprehensive single-cell liquid biopsy-based test [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-12.