Identification of Candidate Synovial Fluid Biomarkers for the Prediction of Patient Outcome After Microfracture or Osteotomy

Background: Biomarkers are needed to predict clinical outcomes for microfracture and osteotomy surgeries to ensure patients can be better stratified to receive the most appropriate treatment. Purpose: To identify novel biomarker candidates and to investigate the potential of a panel of protein biomarkers for the prediction of clinical outcome after treatment with microfracture or osteotomy. Study Design: Descriptive laboratory study. Methods: To identify novel candidate biomarker proteins, we used label-free quantitation after liquid chromatography–tandem mass spectrometry of dynamic range-compressed synovial fluids (SFs) from individuals who responded excellently or poorly (based on change in Lysholm score) to microfracture (n = 6) or osteotomy (n = 7). Biomarkers that were identified in this proteomic analysis or that relate to osteoarthritis (OA) severity or have predictive value in another early OA therapy (autologous cell implantation) were measured in the SF of 19 and 13 patients before microfracture or osteotomy, respectively, using commercial immunoassays, and were normalized to urea. These were aggrecanase-1 (ADAMTS-4), cartilage oligomeric matrix protein (COMP), hyaluronan (HA), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), matrix metalloproteinase 1 and 3, soluble CD14, S100 calcium binding protein A13, and 14-3-3 protein theta (YWHAQ). Levels of COMP and HA were also measured in the plasma of these patients. To find predictors of postoperative function, multivariable regression analyses were performed. Results: Proteomic analyses highlighted YWHAQ and LYVE-1 as being differentially abundant between the clinical responders/improvers and nonresponders after microfracture. A linear regression model after backward variable selection could relate preoperative concentrations of SF proteins (HA, YWHAQ, LYVE-1), activity of ADAMTS-4, and patient demographic characteristics (smoker status and sex) with Lysholm score 12 months after microfracture. Further, a generalized linear model with elastic net penalization indicated that lower preoperative activity of ADAMTS-4 in SF, being a nonsmoker, and being younger at the time of operation were indicative of a higher postoperative Lysholm score (improved joint function) after osteotomy surgery. Conclusion: We have identified biomarkers and generated regression models with the potential to predict clinical outcome in patients treated with microfracture or osteotomy of the knee. Clinical Relevance: Candidate protein biomarkers identified in this study have the potential to help determine which patients will be best suited to treatment with microfracture or osteotomy.

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Multitarget, quantitative nanoplasmonic electrical field-enhanced resonating device (NE2RD) for diagnostics

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1510824112 ◽

2015 ◽

Vol 112 (32) ◽

pp. E4354-E4363 ◽

Cited By ~ 44

Author(s):

Fatih Inci ◽

Chiara Filippini ◽

Murat Baday ◽

Mehmet Ozgun Ozen ◽

Semih Calamak ◽

...

Keyword(s):

Dynamic Range ◽

Point Of Care ◽

Medical Diagnostics ◽

Clinical Samples ◽

Label Free ◽

Protein Biomarkers ◽

Electrical Field ◽

Color Changes ◽

Sensing Platform ◽

Broad Dynamic Range

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients’ homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE2RD), which addresses all these impediments on a single platform. The NE2RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE2RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE2RD’s broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients’ homes.

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MosaicNeedles: A tool for multi-omic liquid biopsy sensitivity and specificity enhancement.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e15019 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e15019-e15019

Author(s):

Qimin Quan ◽

Joe Wilkinson ◽

Joshua Ritchey ◽

Alaina Kaiser ◽

John Geanacopoulos ◽

...

Keyword(s):

Mass Spectrometry ◽

Single Molecule ◽

Liquid Biopsy ◽

Dynamic Range ◽

Color Change ◽

Protein Detection ◽

Target Sequence ◽

Label Free ◽

Protein Biomarkers ◽

Oncology Research

e15019 Background: Liquid biopsy has evolved to be an important method complementary to tissue biopsy. It is not only non-invasive, but also has the potential to detect cancer in its earliest stages and monitor patients in remission. The integration of proteomics into liquid biopsy may transform the molecular diagnostics of cancer and accelerate basic and clinical oncology research. A recent study showed that adding just 8 protein biomarkers to a panel of circulating DNA biomarkers increased the diagnostic accuracy up to 98% sensitivity and 99% specificity. Proteomics also bridges the gaps of functional information lost due to post-transcriptional and post-translational modifications in the genomic approach. However, the proteogenomic approach normally requires the use of multiple different assay technologies and laboratory workflows, including mass spectrometry. Methods: NanoMosaic’s Tessie platform employs a densely integrated nanoneedle sensor array (thus named MosaicNeedles) which can be used to detect both nucleic acids and proteins in a single assay process with reduced workflow complexity, without the need for mass spectrometry. Results: The NanoMosaic platform is a label-free, digital, single molecule counting technology using nanoneedles. It achieves sub-pg/ml (̃fM) level sensitivity with 7 logs of dynamic range. An array of nanoneedles is densely integrated and manufactured with CMOS-compatible nanofabrication processes. Each nanoneedle is a single molecule biosensor that is functionalized with capture probes. The capture probe can be either an antibody for protein detection or an oligonucleotide with a specific target sequence to a DNA fragment, mRNA, or miRNA of interest. The scattering spectrum of each nanoneedle changes when an analyte binds to its surface. At low abundance, analytes that are captured can be quantitated by counting the presence or absence of a color change on each individual nanoneedle in a binary fashion. As an analyte concentration increases the binding events increase accordingly and achieve saturation. In this range, an analog analysis on the spectrum shift will be performed, thus providing a wider dynamic range, up to 7 logs. Ultrahigh level multiplex can be achieved by parallelizing each analyte specific sensing area without loss of sensitivity or dynamic range. A 10,000-plex study can be achieved with a total of 2.5 billion nanoneedles on a 50mm by 50mm consumable. In this consumable, a 2,000-plex proteome and 8,000 cell-free DNA fragments can be detected. Conclusions: In conclusion, a full proteogenomic quantification can be performed on the NanoMosaic platform in one reaction, with higher sensitivity, lower cost and higher throughput than is currently possible by traditional methods. In addition, the high-plexibility of the NanoMosaic platform allows the discovery of new biomarkers across the whole proteome without the need for mass spectrometry.

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Label-Free Mass Spectrometry-Based Plasma Proteomics Identified LY6D, DSC3, CDSN, SERPINB12, and SLURP1,as Novel Protein Biomarkers For Pulmonary Tuberculosis

Current Proteomics ◽

10.2174/1570164617666191210105122 ◽

2019 ◽

Vol 17 ◽

Author(s):

Xiaoli Yu ◽

Lu Zhang ◽

Na Li ◽

Peng Hu ◽

Zhaoqin Zhu ◽

...

Keyword(s):

Pulmonary Tuberculosis ◽

Search Algorithm ◽

Differentially Expressed ◽

P Value ◽

Plasma Samples ◽

Label Free ◽

Protein Biomarkers ◽

Protein Database ◽

Leave One Out ◽

Potential Biomarkers

Aim: We aimed to identify new plasma biomarkers for the diagnosis of Pulmonary tuberculosis. Background: Tuberculosis is an ancient infectious disease that remains one of the major global health problems. Until now, effective, convenient, and affordable methods for diagnosis of Pulmonary tuberculosis were still lacked. Objective: This study focused on construct a label-free LC-MS/MS based comparative proteomics between six tuberculosis patients and six healthy controls to identify differentially expressed proteins (DEPs) in plasma. Method: To reduce the influences of high-abundant proteins, albumin and globulin were removed from plasma samples using affinity gels. Then DEPs from the plasma samples were identified using a label-free Quadrupole-Orbitrap LC-MS/MS system. The results were analyzed by the protein database search algorithm SEQUEST-HT to identify mass spectra to peptides. The predictive abilities of combinations of host markers were investigated by general discriminant analysis (GDA), with leave-one-out cross-validation. Results: A total of 572 proteins were identified and 549 proteins were quantified. The threshold for differentially expressed protein was set as adjusted p-value < 0.05 and fold change ≥1.5 or ≤0.6667, 32 DEPs were found. ClusterVis, TBtools, and STRING were used to find new potential biomarkers of PTB. Six proteins, LY6D, DSC3, CDSN, FABP5, SERPINB12, and SLURP1, which performed well in the LOOCV method validation, were termed as potential biomarkers. The percentage of cross-validated grouped cases correctly classified and original grouped cases correctly classified is greater than or equal to 91.7%. Conclusion: We successfully identified five candidate biomarkers for immunodiagnosis of PTB in plasma, LY6D, DSC3, CDSN, SERPINB12, and SLURP1. Our work supported this group of proteins as potential biomarkers for pulmonary tuberculosis, and be worthy of further validation.

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FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus

International Journal of Molecular Sciences ◽

10.3390/ijms22010111 ◽

2020 ◽

Vol 22 (1) ◽

pp. 111

Author(s):

Oksana M. Subach ◽

Natalia V. Barykina ◽

Elizaveta S. Chefanova ◽

Anna V. Vlaskina ◽

Vladimir P. Sotskov ◽

...

Keyword(s):

Schizosaccharomyces Pombe ◽

Hela Cells ◽

Calcium Binding ◽

Dynamic Range ◽

Spectral Characteristics ◽

Binding Domain ◽

Calcium Transients ◽

Neuronal Cultures ◽

Decay Kinetics ◽

Calcium Binding Domain

Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with Kd of 441 nM and 2.4–6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3–3.5, respectively.

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Critical angle reflection imaging for quantification of molecular interactions on glass surface

Nature Communications ◽

10.1038/s41467-021-23730-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Guangzhong Ma ◽

Runli Liang ◽

Zijian Wan ◽

Shaopeng Wang

Keyword(s):

Refractive Index ◽

Small Molecule ◽

Molecular Interactions ◽

Glass Surface ◽

Critical Angle ◽

Dynamic Range ◽

Refractive Index Change ◽

Label Free ◽

Index Change ◽

Sensing Range

AbstractQuantification of molecular interactions on a surface is typically achieved via label-free techniques such as surface plasmon resonance (SPR). The sensitivity of SPR originates from the characteristic that the SPR angle is sensitive to the surface refractive index change. Analogously, in another interfacial optical phenomenon, total internal reflection, the critical angle is also refractive index dependent. Therefore, surface refractive index change can also be quantified by measuring the reflectivity near the critical angle. Based on this concept, we develop a method called critical angle reflection (CAR) imaging to quantify molecular interactions on glass surface. CAR imaging can be performed on SPR imaging setups. Through a side-by-side comparison, we show that CAR is capable of most molecular interaction measurements that SPR performs, including proteins, nucleic acids and cell-based detections. In addition, we show that CAR can detect small molecule bindings and intracellular signals beyond SPR sensing range. CAR exhibits several distinct characteristics, including tunable sensitivity and dynamic range, deeper vertical sensing range, fluorescence compatibility, broader wavelength and polarization of light selection, and glass surface chemistry. We anticipate CAR can expand SPR′s capability in small molecule detection, whole cell-based detection, simultaneous fluorescence imaging, and broader conjugation chemistry.

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Label-free electrochemical immunosensor based on gold nanoparticle/polyethyleneimine/reduced graphene oxide nanocomposite for the ultrasensitive detection of cancer biomarker matrix metalloproteinase-1

The Analyst ◽

10.1039/d1an00537e ◽

2021 ◽

Author(s):

Xinke Liu ◽

Lu-Yin Lin ◽

Fu-Yen Tseng ◽

Yu-Cheng Tan ◽

Jian Li ◽

...

Keyword(s):

Graphene Oxide ◽

Matrix Metalloproteinase ◽

Gold Nanoparticle ◽

Reduced Graphene Oxide ◽

Electrochemical Immunosensor ◽

Ultrasensitive Detection ◽

Label Free ◽

Matrix Metalloproteinase 1 ◽

Reduced Graphene ◽

P Matrix

Matrix metalloproteinase-1 (MMP-1) is associated with many types of cancers, including oral, colorectal, and brain cancers. This paper describes the fabrication of an MMP-1 immunosensor based on a gold nanoparticle/polyethyleneimine/reduced...

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Quantitative Assessment of Periodontal Bacteria Using a Cell-Based Immunoassay with Functionalized Quartz Crystal Microbalance

Chemosensors ◽

10.3390/chemosensors9070159 ◽

2021 ◽

Vol 9 (7) ◽

pp. 159

Author(s):

Satit Rodphukdeekul ◽

Miyuki Tabata ◽

Chindanai Ratanaporncharoen ◽

Yasuo Takeuchi ◽

Pakpum Somboon ◽

...

Keyword(s):

Monoclonal Antibody ◽

Quartz Crystal Microbalance ◽

Quantitative Assessment ◽

Quartz Crystal ◽

Dynamic Range ◽

Gold Surface ◽

Enzyme Linked Immunosorbent Assay ◽

Inflammatory Disorder ◽

Label Free ◽

Periodontal Bacteria

Periodontal disease is an inflammatory disorder that is triggered by bacterial plaque and causes the destruction of the tooth-supporting tissues leading to tooth loss. Several bacteria species, including Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, are considered to be associated with severe periodontal conditions. In this study, we demonstrated a quartz crystal microbalance (QCM) immunoassay for quantitative assessment of the periodontal bacteria, A. actinomycetemcomitans. An immunosensor was constructed using a self-assembled monolayer of 11-mercaptoundecanoic acid (11-MUA) on the gold surface of a QCM chip. The 11-MUA layer was evaluated using a cyclic voltammetry technique to determine its mass and packing density. Next, a monoclonal antibody was covalently linked to 11-MUA using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide to act as the biorecognition element. The specificity of the monoclonal antibody was confirmed by an enzyme-linked immunosorbent assay. A calibration curve, for the relationship between the frequency shifts and number of bacteria, was used to calculate the number of A. actinomycetemcomitans bacteria in a test sample. Based on a regression equation, the lower detection limit was 800 cells, with a dynamic range up to 2.32 × 106 cells. Thus, the QCM biosensor in this study provides a sensitive and label-free method for quantitative analysis of periodontal bacteria. The method can be used in various biosensing assays for practical application and routine detection of periodontitis pathogens.

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Electrokinetic Formation of “Microbridges” for Protein Biomarkers as Sensors

JALA Journal of the Association for Laboratory Automation ◽

10.1016/j.jala.2007.06.001 ◽

2007 ◽

Vol 12 (5) ◽

pp. 311-317 ◽

Cited By ~ 2

Author(s):

Vindhya Kunduru ◽

Shalini Prasad

Keyword(s):

Electric Fields ◽

Microelectrode Array ◽

Protein Detection ◽

High Specificity ◽

Detection Methods ◽

Label Free ◽

Protein Biomarkers ◽

Detection Scheme ◽

Polystyrene Beads ◽

Conducting Path

We demonstrate a technique to detect protein biomarkers contained in vulnerable coronary plaque using a platform-based microelectrode array (MEA). The detection scheme is based on the property of high specificity binding between antibody and antigen similar to most immunoassay techniques. Rapid clinical diagnosis can be achieved by detecting the amount of protein in blood by analyzing the protein's electrical signature. Polystyrene beads which act as transportation agents for the immobile proteins (antigen) are electrically aligned by application of homogenous electric fields. The principle of electrophoresis is used to produce calculated electrokinetic movement among the anti-C-reactive protein (CRP), or in other words antibody funtionalized polystyrene beads. The electrophoretic movement of antibody-functionalized polystyrene beads results in the formation of “Microbridges” between the two electrodes of interest which aid in the amplification of the antigen—antibody binding event. Sensitive electrical equipment is used for capturing the amplified signal from the “Microbridge” which essentially behaves as a conducting path between the two electrodes. The technique circumvents the disadvantages of conventional protein detection methods by being rapid, noninvasive, label-free, repeatable, and inexpensive. The same principle of detection can be applied for any receptor—ligand-based system because the technique is based only on the volume of the analyte of interest. Detection of the inflammatory coronary disease biomarker CRP is achieved at concentration levels spanning over the lower microgram/milliliter to higher order nanogram/milliliter ranges.

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Thrombospondin-4 knockout in hypertension protects small-artery endothelial function but induces aortic aneurysms

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00046.2016 ◽

2016 ◽

Vol 310 (11) ◽

pp. H1486-H1493 ◽

Cited By ~ 11

Author(s):

Teresa Palao ◽

Catarina Rippe ◽

Henk van Veen ◽

Ed VanBavel ◽

Karl Swärd ◽

...

Keyword(s):

Calcium Binding ◽

Matrix Protein ◽

Pressure Overload ◽

Aortic Aneurysms ◽

Endoplasmic Reticulum Stress Response ◽

Extracellular Matrix Protein ◽

Ang Ii ◽

Wild Type ◽

Resistance Arteries ◽

Thrombospondin 4

Thrombospondin-4 (TSP-4) is a multidomain calcium-binding protein that has both intracellular and extracellular functions. As an extracellular matrix protein, it is involved in remodeling processes. Previous work showed that, in the cardiovascular system, TSP-4 expression is induced in the heart in response to experimental pressure overload and infarction injury. Intracellularly, it mediates the endoplasmic reticulum stress response in the heart. In this study, we explored the role of TSP-4 in hypertension. For this purpose, wild-type and TSP-4 knockout ( Thbs4 −/−) mice were treated with angiotensin II (ANG II). Hearts from ANG II-treated Thbs4 −/− mice showed an exaggerated hypertrophic response. Interestingly, aortas from Thbs4 −/− mice treated with ANG II showed a high incidence of aneurysms. In resistance arteries, ANG II-treated wild-type mice showed impaired endothelial-dependent relaxation. This was not observed in ANG II-treated Thbs4 −/− mice or in untreated controls. No differences were found in the passive pressure-diameter curves or stress-strain relationships, although ANG II-treated Thbs4 −/− mice showed a tendency to be less stiff, associated with thicker diameters of the collagen fibers as revealed by electron microscopy. We conclude that TSP-4 plays a role in hypertension, affecting cardiac hypertrophy, aortic aneurysm formation, as well as endothelial-dependent relaxation in resistance arteries.

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