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.

Predicting the holistic force-displacement relation of the periodontal ligament: in-vitro experiments and finite element analysis.

BACKGROUND: The biomechanical property of the periodontal ligament (PDL) is important in orthodontics and prosthodontics. The objective of this study was to evaluate the feasibility of measuring the biomechanical behavior of the periodontal ligament using micro-computed tomography (micro-CT). METHODS: A custom-made apparatus measured the force and displacement of a porcine PDL specimen within the micro-CT environment. Synchronized computed tomography (CT) images were used to obtain the deformation and displacement of the entire specimen and to reconstruct the three-dimensional mesh model. To match the experimental results, finite element analysis was then applied to simulate the biomechanical response of the PDL. The mechanical model of the PDL was assumed as the hyperelastic material in this study. RESULTS: The volume variations of the tooth and the alveolar bone were less than 1%, which implies that tooth displacement was caused mostly by displacement of the PDL. Only translational displacement was observed with each load step because the transformation matrix acquired from the CT image registration was identical. The force-displacement curve revealed the nonlinear behavior of the PDL. There was a high correlation between the experimental displacement results and the simulation displacement results. The numerical results (based on the assumption that the PDL is the hyperelastic material) showed good agreement with the experimental results. CONCLUSIONS: Nondestructive measurements by micro-CT obtained the biomechanical behavior of the PDL. Using the hyperelastic characteristic as the constitutive model can properly predict the force-displacement relation of the PDL after loading. This study provided a feasible approach for measuring the biomechanical behavior of the PDL for further dental application.

Biomechanical Analysis of Titanium Dental Implants in the All-on-4 Treatment with Different Implant-Abutment Connections: A Three-Dimensional Finite Element Study.

The type of implant-abutment connection is one of the factors influencing the distribution of occlusal forces. This study aims to investigate the biomechanical performance of the mandibular all-on-4 treatment with different implant-abutment connections. Two connection types with 30° abutments and 18-mm implant fixtures were chosen for the posterior implants of the all-on-4 assembly. For the external hexagon connection (EHC) group, the implants with 4 mm in diameter were used. For the internal hexagon connection (IHC) group, we selected implants with 4.3 mm in diameter. A vertical force of 190 N was applied to the cantilever region. The FEA results indicated that the most stressed region in the two groups was prosthetic screws, followed by multi-unit abutments (MUAs). The lowest values of von Mises stress were both observed on the bone. The peak stress value of the implant screw and implant fixture in the EHC group were 37.75% and 33.03% lower than the IHC group, respectively. For stress distribution patterns, the load force tended to be concentrated at locations where components were interconnected. The EHC and IHC are clinically durable under the tested loading conditions, but the prosthetic screws and MUAs can be the weak point on the posterior implant within the mandibular all-on-four assembly. The peak stress values of implant screw and implant fixture in the EHC groups were lower than the IHC group.

Double Asymptotic Structures of Topologically Interlocked Molecules.

The mean square size of topologically interlocked molecules (TIMs) is presented as a linear combination of contributions from the backbone and subcomponents. Using scaling analyses and extensive molecular dynamics simulations of polycatenanes as a typical example of TIMs, we show that the effective exponent ν(m) for the size dependence of the backbone on the monomer number of subcomponent m is asymptotic to a value ν (∼0.588 in good solvents) with a correction of m-0.47, which is the same as for the covalently linked polymer. However, the effective exponent for the size dependence of subcomponents on m is asymptotic to the same value ν but with a new correction of m-1.0. The different corrections to the scaling on the backbone and subcomponent structure induce a surprising double asymptotic behavior for the architecture of the TIMs. The scaling model that takes into account the double asymptotic behavior is in good quantitative agreement with the simulation result that the effective exponent for the size dependence of TIMs on m increases with the subcomponent number n. The full scaling functional form of the size dependence on m and n for polycatenanes in a good solvent is well described by a simple sum of two limiting behaviors with different corrections.

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.

Biocompatibility of nano-hydroxyapatite/Mg-Zn-Ca alloy composite scaffolds to human umbilical cord mesenchymal stem cells from Wharton's jelly in vitro.

Seeding cells and scaffolds play pivotal roles in bone tissue engineering and regenerative medicine. Wharton's jelly-derived mesenchymal stem cells (WJCs) from human umbilical cord represent attractive and promising seeding cells in tissue regeneration and engineering for treatment applications. This study was carried out to explore the biocompatibility of scaffolds to seeding cells in vitro. Rod-like nano-hydroxyapatite (RN-HA) and flake-like micro-hydroxyapatite (FM-HA) coatings were prepared on Mg-Zn-Ca alloy substrates using micro-arc oxidation and electrochemical deposition. WJCs were utilized to investigate the cellular biocompatibility of Mg-Zn-Ca alloys after different surface modifications by observing the cell adhesion, morphology, proliferation, and osteoblastic differentiation. The in vitro results indicated that the RN-HA coating group was more suitable for cell proliferation and cell osteoblastic differentiation than the FM-HA group, demonstrating better biocompatibility. Our results suggested that the RN-HA coating on Mg-Zn-Ca alloy substrates might be of great potential in bone tissue engineering.

[Research progress on plate mixed culture of lignocellulolytic microorganisms].

Mixed culture of microorganisms has been widely used for the research of lignocellulose transformation and degradation, but the results of the mixed culture are largely affected by the interactions of different lignocellulolytic microorganisms. At present, the researches on these interactions are mainly based on plate mixed culture assay. For this assay, two types of plate were used, namely, basic medium plate and improved medium plate. The basic medium plate is mainly used for the study of colony morphology, mycelia color, exocellular volatile organic compounds, and exocellular enzyme activity, whereas the improved medium plate is used for comparative study, with the carbon sources replaced by natural lignocelloses. This paper summarized the present research status and advancement about the plate mixed culture of lignocellulolytic microorganisms, and put forward a prospect about the focuses of future research in this field.

[Finite element analysis of mechanical characteristics during retracting mandibular incisors through sliding mechanics].

PURPOSE: To analyze the mechanical characteristic changes of teeth and arch under different loading direction during retracting mandibular incisors through implant, simulating clinical loading system. METHODS: Three- dimensional finite element model, including brackets, archwire, crampable hooks and implants, was reconstructed. The force direction was determined by connecting the points in crampable hook and the center point of implant, and the force point and force direction were changed with the adjustment of the height of crampable hook and the height of implant. Then three-dimensional movement trend of teeth, stress distribution in periodontal membrane and the largest displacement of archwire nodes in each group were calculated and analyzed. SPSS13.0 software package was used for statistical analysis. RESULTS: It was found that the height of implant and the height of crampable hook were correlated with the movement of teeth and stress distribution in periodontal membrane (P<0.01). The movement trend of teeth in the condition of different height of implant and different height of crampable hook was illustrated as follows:(1)with the height increase of crampable hook, the movement trend of the central and lateral incisors varied from mesial lingual tipping to mesial labial tipping. However, canines tipped distally and lingually; the second premolars tipped mesially and lingually, and the first molar roots tipped distally and buccally with decreasing tipping angle. (2) The largest stress distribution in the whole arch was located in the labial apical one-third area of the lateral incisors, while that of canines and the first molars was located in the alveolar ridges and root bifurcations. CONCLUSIONS: These findings indicate that the different movement trend during retracting anterior teeth can be achieved through the adjustment of the height of crampable hook, and implant, anchorage can effectively control anterior movement of the posterior teeth. Supported by Research Fund of Bureau of Science and Technology of Nantong City (Grant No. S40023).

[Microbial community changes during the degradation of straw using phospholipid fatty acid analysis].

Solid-state fermentation system of rice straw was established with the inoculation of soil microorganism and Phanerochaete chrysosporium. Microbial biomass and community structure were investigated using the phospholipid fatty acid (PLFA) technique, and the changes of lignocellulose's degradation rate were also detected during the process. The experimental results show that lignocellulose degradation rate reaches 44% after fermentation. When the total amount of phospholipid fatty acid is separated into indicator phospholipid fatty acids for different groups of microorganisms, these groups show different patterns during the process. Contents of PLFA in gram-positive bacteria, gram-negative and fungi reach their peak value on the sixth day. Gram-positive bacteria have lower contents. The PLFA content ratio of fungi and bacteria is 0.2 - 0.5, so fungi are the main community decomposing lignocellulose. Principal component analysis of the PLFA data show that 18 carbon unsaturated fatty acids are the major fatty acids at the end of the process, which is consistent with the results of indicator phospholipid fatty acids and lignocellulose's degradation rates, so PLFA technique is able to fairly well detect the changes of microbial biomass and community structure in solid-state fermentation system of rice straw.

[Lignin degradation by Penicillium simplicissimum].

A strain of fungi was isolated from soil, which was identified as Penicillium simplicissimum. This strain was capable of utilizing several lignin model compounds, making aromatic dyes decoloration and degrading natural lignin. All these results proved that Penicillium simplicissimum has ligninolytic ability. Three kinds of enzymes were believed to be the most important catalyzes in the biodegrading process. They are lignin peroxidase (LiP), laccase (Lac) and hemicellulase. And they always work synergistically. After 25 days' incubation, the amount of rice straw lignin decreased 0.23g, and the degrading rate was 14.94%. Different from the degrading mechanism of the white-rot fungi, the lignin degradation by P. simplicissimum mainly happened during the primary metabolism and it was greatly influenced by the pH of media, the concentration of Cu2+ and Mn2+.