Development of a Multiplexed Assay for Oral Cancer Candidate Biomarkers Using Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry.

Oral cancer is one of the most common cancers worldwide, and there are currently no biomarkers approved for aiding its management. Although many potential oral cancer biomarkers have been discovered, very few have been verified in body fluid specimens in parallel to evaluate their clinical utility. The lack of appropriate multiplexed assays for chosen targets represents one of the bottlenecks to achieving this goal. In the present study, we develop a peptide immunoaffinity enrichment-coupled multiple reaction monitoring-mass spectrometry (SISCAPA-MRM) assay for verifying multiple reported oral cancer biomarkers in saliva. We successfully produced 363 clones of mouse anti-peptide monoclonal antibodies (mAbs) against 36 of 49 selected targets, and characterized useful mAbs against 24 targets in terms of their binding affinity for peptide antigens and immuno-capture ability. Comparative analyses revealed that an equilibrium dissociation constant (KD ) cut-off value < 2.82 × 10-9 m could identify most clones with an immuno-capture recovery rate >5%. Using these mAbs, we assembled a 24-plex SISCAPA-MRM assay and optimized assay conditions in a 25-µg saliva matrix background. This multiplexed assay showed reasonable precision (median coefficient of variation, 7.16 to 32.09%), with lower limits of quantitation (LLOQ) of <10, 10-50, and >50 ng/ml for 14, 7 and 3 targets, respectively. When applied to a model saliva sample pooled from oral cancer patients, this assay could detect 19 targets at higher salivary levels than their LLOQs. Finally, we demonstrated the utility of this assay for quantification of multiple targets in individual saliva samples (20 healthy donors and 21 oral cancer patients), showing that levels of six targets were significantly altered in cancer compared with the control group. We propose that this assay could be used in future studies to compare the clinical utility of multiple oral cancer biomarker candidates in a large cohort of saliva samples.

Golgi tethering factor golgin-97 suppresses breast cancer cell invasiveness by modulating NF-κB activity.

BACKGROUND: Golgin-97 is a tethering factor in the trans-Golgi network (TGN) and is crucial for vesicular trafficking and maintaining cell polarity. However, the significance of golgin-97 in human diseases such as cancer remains unclear. METHODS: We searched for a potential role of golgin-97 in cancers using Kaplan-Meier Plotter ( http://kmplot.com ) and Oncomine ( www.oncomine.org ) datasets. Specific functions of golgin-97 in migration and invasion were examined in golgin-97-knockdown and golgin-97-overexpressing cells. cDNA microarray, pathway analysis and qPCR were used to identify gene profiles regulated by golgin-97. The role of golgin-97 in NF-κB signaling pathway was examined by using subcellular fractionation, luciferase reporter assay, western blot analysis and immunofluorescence assay (IFA). RESULTS: We found that low expression of golgin-97 correlated with poor overall survival of cancer patients and was associated with invasiveness in breast cancer cells. Golgin-97 knockdown promoted cell migration and invasion, whereas re-expression of golgin-97 restored the above phenotypes in breast cancer cells. Microarray and pathway analyses revealed that golgin-97 knockdown induced the expression of several invasion-promoting genes that were transcriptionally regulated by NF-κB p65. Mechanistically, golgin-97 knockdown significantly reduced IκBα protein levels and activated NF-κB, whereas neither IκBα levels nor NF-κB activity was changed in TGN46- or GCC185-knockdown cells. Conversely, golgin-97 overexpression suppressed NF-κB activity and restored the levels of IκBα in golgin-97-knockdown cells. Interestingly, the results of Golgi-disturbing agent treatment revealed that the loss of Golgi integrity was not involved in the NF-κB activation induced by golgin-97 knockdown. Moreover, both TGN-bound and cytosolic golgin-97 inhibited NF-κB activation, indicating that golgin-97 functions as an NF-κB suppressor regardless of its subcellular localization. CONCLUSION: Our results collectively demonstrate a novel and suppressive role of golgin-97 in cancer invasiveness. We also provide a new avenue for exploring the relationship between the TGN, golgin-97 and NF-κB signaling in tumor progression.

Temperature effects on the magnetic properties of silicon-steel sheets using standardized toroidal frame.

This study designed a detachable and standardized toroidal test frame to measure the electromagnetic characteristic of toroidal laminated silicon steel specimens. The purpose of the design was to provide the measurements with standardized and controlled environment. The device also can withstand high temperatures (25-300°C) for short time period to allow high temperature tests. The accompanying driving circuit facilitates testing for high frequency (50-5,000 Hz) and high magnetic flux (0.2-1.8 T) conditions and produces both sinusoidal and nonsinusoidal test waveforms. The thickness of the stacked laminated silicon-steel sheets must be 30~31 mm, with an internal diameter of 72 mm and an outer diameter of 90 mm. With the standardized setup, it is possible to carry out tests for toroidal specimen in high temperature and high flux operation. The test results show that there is a tendency of increased iron loss under high temperature operation. The test results with various driving waveforms also provide references to the required consideration in engineering designs.

Stretchable Sensors: Novel Human Motion Monitoring Wearables.

A human body monitoring system remains a significant focus, and to address the challenges in wearable sensors, a nanotechnology-enhanced strategy is proposed for designing stretchable metal-organic polymer nanocomposites. The nanocomposite comprises reduced graphene oxide (rGO) and in-situ generated silver nanoparticles (AgNPs) within elastic electrospun polystyrene-butadiene-polystyrene (SBS) fibers. The resulting Sandwich Structure Piezoresistive Woven Nanofabric (SSPWN) is a tactile-sensitive wearable sensor with remarkable performance. It exhibits a rapid response time (less than three milliseconds) and high reproducible stability over 5500 cycles. The nanocomposite also demonstrates exceptional thermal stability due to effective connections between rGO and AgNPs, making it suitable for wearable electronic applications. Furthermore, the SSPWN is successfully applied to human motion monitoring, including various areas of the hand and RGB sensing shoes for foot motion monitoring. This nanotechnology-enhanced strategy shows promising potential for intelligent healthcare, health monitoring, gait detection, and analysis, offering exciting prospects for future wearable electronic products.

Electrodeposited NiSe on a forest of carbon nanotubes as a free-standing electrode for hybrid supercapacitors and overall water splitting.

NiSe nanoparticles are electrodeposited over a forest of carbon nanotubes (CNTs) to form an intertwined and porous network. The assynthesized composite (denoted as CNT@NiSe/SS) is used as a free-standing and multifunctional electrode for bothsupercapacitorsand overallwater splitting applications. For a supercapacitor application, CNT@NiSe/SS exhibits higher specific capacity and improved rate capability compared with individual NiSe and CNTs. A hybrid supercapacitor device consisting of battery-like CNT@NiSe/SS and EDLC-like graphene delivers a maximum energy density of 32.1 Wh kg-1 at a power density of 823 W kg-1 and has excellent stability after a floating test of 50 h. On the other hand, CNT@NiSe/SS also serves as a bifunctional electrocatalyst with high activity for overall water splitting. The CNT@NiSe/SS electrode displays excellent hydrogen and oxygen evolution reaction performance with the lowest overpotential of 174 mV at 10 mA cm-2 and 267 mV at 50 mA cm-2, respectively. The symmetrical two-electrode system requires an operating potential of 1.71 V to achieve a current density of 10 mA cm-2. Furthermore, this electrolyzer shows a negligible increment in potential after 24 hof continuouswater splitting. The outstanding performances of CNT@NiSe/SS can be attributed to the synergistic effect of NiSe and CNTs.

An immuno-MALDI mass spectrometry assay for the oral cancer biomarker, matrix metalloproteinase-1, in dried saliva spot samples.

Oral cavity cancer is a common cancer type that presents an increasingly serious global problem. Oral squamous cell carcinoma (OSCC) accounts for >90% oral cancer cases. No biomarker tests are currently available for management of this cancer type in clinical practice. Previously, we validated matrix metalloproteinase-1 (MMP1) as one of the most promising salivary biomarkers for OSCC detection. Development of a convenient, rapid and high-throughput assay should further facilitate application of salivary MMP1 measurement for early detection of OSCC. The present study aimed to develop a workflow comprising dry saliva spot (DSS) sampling and immunoenrichment-coupled MALDI-TOF MS (immuno-MALDI) analysis to quantify salivary MMP1. We generated recombinant MMP1 protein and anti-peptide antibodies against MMP1, which were used to optimize the procedures of the entire workflow, including DSS sampling, on-paper protein digestion and elution, KingFisher magnetic particle processor-assisted immuno-enrichment and MALDI-TOF MS analysis. The established workflow was applied to measure salivary MMP1 levels in DSS samples from 5 healthy donors and 9 OSCC cases. The newly developed workflow showed good precision (intra-day and inter-day variations <10%) and accuracy (80-100%) in quantification of MMP1 in DSS samples, with the limit of quantification at 3.07 ng/ml. Using this assay, we successfully detected elevated salivary MMP1 levels (ranging from 5.95 to 242.52 ng/ml) in 7 of 9 OSCC cases while MMP1 was not detectable in samples from the 5 healthy donors. In comparison, the traditional immunoassay was not effective in measuring MMP1 in DSS samples, highlighting the significant advantage of our immuno-MALDI assay. The DSS sampling format confers high flexibility and convenience of collection, storage and delivery of saliva specimens and the KingFisher-assisted immuno-MALDI analysis renders the assay as suitable for high-throughput screening. By combining the two features, the workflow developed in this study should facilitate improvement of molecular diagnostic tests for OSCC using salivary MMP1 as a biomarker.

Assessment of candidate biomarkers in paired saliva and plasma samples from oral cancer patients by targeted mass spectrometry.

For oral cancer, numerous saliva- and plasma-derived protein biomarker candidates have been discovered and/or verified; however, it is unclear about the behavior of these candidates as saliva or plasma biomarkers. In this study, we developed two targeted assays, MRM and SISCAPA-MRM, to quantify 30 potential biomarkers in both plasma and saliva samples collected from 30 healthy controls and 30 oral cancer patients. Single point measurements were used for target quantification while response curves for assay metric determination. In comparison with MRM assay, SISCAPA-MRM effectively improved (>1.5 fold) the detection sensitivity of 11 and 21 targets in measurement of saliva and plasma samples, respectively. The integrated results revealed that the salivary levels of these 30 selected biomarkers weakly correlated (r < 0.2) to their plasma levels. Five candidate biomarkers (MMP1, PADI1, TNC, CSTA and MMP3) exhibited significant alterations and disease-discriminating powers (AUC = 0.914, 0.827, 0.813, 0.77, and 0.753) in saliva sample; nevertheless, no such targets could be found in plasma samples. Our data support the notion that saliva may be more suitable for the protein biomarker-based detection of oral cancer, and the newly developed SISCAPA-MRM assay could be applied to verify multiple oral cancer biomarker candidates in saliva samples. SIGNIFICANCE: In this work we systematically determined the abundance of 30 selected targets in the paired saliva and plasma samples to evaluate the utility of saliva and plasma samples for protein biomarker-based detection of oral cancer. Our study provides significant evidence to support the use of saliva, but not blood samples, offer more opportunity to achieve the success of protein biomarker discovery for oral cancer detection.

Variability Assessment of 90 Salivary Proteins in Intraday and Interday Samples from Healthy Donors by Multiple Reaction Monitoring-Mass Spectrometry.

PURPOSE: Saliva is an attractive sample source for the biomarker-based testing of several diseases, especially oral cancer. Here, we sought to apply multiplexed LC-MRM-MS to precisely quantify 90 disease-related proteins and assess their intra- and interindividual variability in saliva samples from healthy donors. EXPERIMENTAL DESIGN: We developed two multiplexed LC-MRM-MS assays for 122 surrogate peptides representing a set of disease-related proteins. Saliva samples were collected from 10 healthy volunteers at three different time points (Day 1 morning and afternoon, and Day 2 morning). Each sample was spiked with a constant amount of a 15 N-labeled protein and analyzed by MRM-MS in triplicate. Quantitative results from LC-MRM-MS were calculated by single-point quantification with reference to a known amount of internal standard (heavy peptide). RESULTS: The CVs for assay reproducibility and technical variation were 13 and 11%, respectively. The average concentrations of the 99 successfully quantified proteins ranged from 0.28 ± 0.58 ng mL-1 for profilin-2 (PFN2) to 8.55 ±8.96 µg mL-1 for calprotectin (S100A8). For the 90 proteins detectable in >50% of samples, the average CVs for intraday, interday, intraindividual, and interindividual samples were 38%, 43%, 45%, and 69%, respectively. The fluctuations of most target proteins in individual subjects were found to be within ± twofold. CONCLUSIONS AND CLINICAL RELEVANCE: Our study elucidated the intra- and interindividual variability of 90 disease-related proteins in saliva samples from healthy donors. The findings may facilitate the further development of salivary biomarkers for oral and systemic diseases.

Enhanced CO2 Adsorption on Activated Carbon Fibers Grafted with Nitrogen-Doped Carbon Nanotubes.

In this paper, multiscale composites formed by grafting N-doped carbon nanotubes (CNs) on the surface of polyamide (PAN)-based activated carbon fibers (ACFs) were investigated and their adsorption performance for CO2 was determined. The spaghetti-like and randomly oriented CNs were homogeneously grown onto ACFs. The pre-immersion of cobalt(II) ions for ACFs made the CNs grow above with a large pore size distribution, decreased the oxidation resistance, and exhibited different predominant N-functionalities after chemical vapor deposition processes. Specifically, the CNs grafted on ACFs with or without pre-immersion of cobalt(II) ions were characterized by the pyridine-like structures of six-member rings or pyrrolic/amine moieties, respectively. In addition, the loss of microporosity on the specific surface area and pore volume exceeded the gain from the generation of the defects from CNs. The adsorption capacity of CO2 decreased gradually with increasing temperature, implying that CO2 adsorption was exothermic. The adsorption capacities of CO2 at 25 °C and 1 atm were between 1.53 and 1.92 mmol/g and the Freundlich equation fit the adsorption data well. The isosteric enthalpy of adsorption, implying physical adsorption, indicated that the growth of CNTs on the ACFs benefit CO2 adsorption.

Whey protein coating efficiency on surfactant-modified hydrophobic surfaces.

Whey protein oxygen-barrier coatings on peanuts are not effective, due to incomplete peanut-surface coverage, as well as some cracking and flaking of the coating. Addition of sorbitan laurate (Span 20) in the whey protein coating solution up to the critical micelle concentration (cmc) of 0.05% (w/w) significantly improved coating coverage to 88% of the peanut surface. Increasing the Span 20 concentration in the coating solution to 3 times the cmc (0.15% w/w) produced a substantial increase in peanut surface energy (>70 dyn/cm), indicating adsorption of the surfactant to the peanut surface. With this level of Span 20, the whey protein coating coverage on peanuts increased to 95%. These results suggest that a concentration of surfactant above the cmc in the coating solution is required for formation of self-assembled structures of surfactant molecules on peanut surfaces, which significantly increases the hydrophilicity, and thus coatability, of peanut surfaces.