Comparative lipidomic profiling of the human commensal bacterium Propionibacterium acnes and its extracellular vesicles.

Propionibacterium acnes is a lipophilic commensal bacterium mainly found on the skin and in the gastrointestinal tract. Pathophysiological effects of P. acnes have recently been reported not only in acne progression but in various diseases. As an emerging mode of bacterial communication, extracellular vesicles (EVs) have been demonstrated to conduct critical pathophysiological functions. To provide information on P. acnes lipid composition for the first time, we conducted a comparative lipidomic analysis of P. acnes and P. acnes EVs and identified 214 lipids with high confidence using triplicated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses. P. acnes EVs contained substantially more PCs, DGs, PAs, PEs, LPAs, LPCs, and MGs than P. acnes, and contained fewer PSs, SO1Ps, SA1Ps, LPGs, LPIs, and LPSs. Distinctively, P. acnes EVs possessed a markedly reduced amount of TG. These findings will provide useful clues for understanding the biological and pathophysiological mechanisms of P. acnes and for clinical applications such as vaccine development, diagnostics and therapeutics.

Construction and characterization of the Korean whole saliva proteome to determine ethnic differences in human saliva proteome.

As the first step to discover protein disease biomarkers from saliva, global analyses of the saliva proteome have been carried out since the early 2000s, and more than 3,000 proteins have been identified in human saliva. Recently, ethnic differences in the human plasma proteome have been reported, but such corresponding studies on human saliva in this aspect have not been previously reported. Thus, here, in order to determine ethnic differences in the human saliva proteome, a Korean whole saliva (WS) proteome catalogue indexing 480 proteins was built and characterized through nLC-Q-IMS-TOF analyses of WS samples collected from eleven healthy South Korean male adult volunteers for the first time. Identification of 226 distinct Korean WS proteins, not observed in the integrated human saliva protein dataset, and significant gene ontology distribution differences in the Korean WS proteome compared to the integrated human saliva proteome strongly support ethnic differences in the human saliva proteome. Additionally, the potential value of ethnicity-specific human saliva proteins as biomarkers for diseases highly prevalent in that ethnic group was confirmed by finding 35 distinct Korean WS proteins likely to be associated with the top 10 deadliest diseases in South Korea. Finally, the present Korean WS protein list can serve as the first level reference for future proteomic studies including disease biomarker studies on Korean saliva.

UPLC-QqQ/MS-Based Lipidomics Approach To Characterize Lipid Alterations in Inflammatory Macrophages.

In this study, UPLC-QqQ/MS-based lipidomics was applied to profile various lipids from RAW264.7 macrophages treated with different concentrations of lipopolysaccharide (LPS). The degree of inflammation increased with the LPS concentration. To elucidate the altered lipid metabolism of inflammatory macrophages, we targeted to analyze 25 lipid classes from LPS-treated RAW264.7 cells. As a result, 523 lipid species were successfully profiled by using the optimal UPLC and MRM. Statistical data analyses such as PCA, PLS-DA, and HCA differentiated five RAW264.7 cells treated with different concentrations of LPS. VIP plot, heat map, and bar plot also provided lists of up- or down-regulated lipids according to the LPS concentration. From the results, 11 classes of lipids, TG, DG, ChE, PE, PS, PI, PA, LyPC, LyPE, Cer, and dCer, were increased, and three classes, cholesterol, PC, and LyPA, were decreased in an LPS concentration-dependent manner. Furthermore, the treatment of an anti-inflammatory compound recovered the levels of PC, PE, PI, PA, LyPE, LyPA, and Cer from the activated macrophages. Finally, these results demonstrate the correlation between inflammation and lipid metabolism in macrophages. The differentially regulated lipids also have the potential to be used as biomarkers for inflammation.

Proteomic analysis of extracellular vesicles derived from Propionibacterium acnes.

PURPOSE: Extracellular vesicle (EV) has been reported to conduct critical pathophysiological functions as an emerging mode of communication in bacteria. Recently, Propionibacterium acnes, an anaerobic Gram-positive human commensal found in the skin and gastrointestinal tract, has drawn increasing attention as an underestimated pathogen in a variety of diseases. EXPERIMENTAL DESIGN: For the comprehensive understanding of P. acnes, here we report the isolation of P. acnes EVs for the first time and identification of 252 vesicular proteins with high confidence using triplicate LC-MS/MS analyses. RESULT: Comprehensive proteomic profiling reveals that P. acnes EVs harbor various proteins involved in biochemical processes, antibiotic resistance, bacterial competition, cell adherence, virulence, and immunogenicity. CONCLUSION AND CLINICAL RELEVANCE: We believe that this report will provide valuable information for investigating the biological role of P. acnes EVs and effective targets for developing clinical applications against P. acnes.

UPLC-MS/MS-Based Profiling of Eicosanoids in RAW264.7 Cells Treated with Lipopolysaccharide.

While both the pro- and anti-inflammatory effects of several eicosanoids have been widely studied, the degree of inflammation in cells that results from various eicosanoids has yet to be comprehensively studied. The objective of this study was to assess the effect of lipopolysaccharide (LPS) treatment on eicosanoid content in RAW264.7 cells. An Ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS)-based profiling method was used to analyze the eicosanoid contents of RAW264.7 cells treated with different LPS concentrations. The profiling data were subjected to statistical analyses, such as principal component analysis (PCA) and hierarchical clustering analysis. LPS treatment increased nitric oxide production and secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6, in a concentration-dependent manner. In total, 79 eicosanoids were identified in the cells. RAW264.7 cells treated with different LPS concentrations were well differentiated in the PCA score plot. A heatmap was used to identify the eicosanoids that were up- or down-regulated according to the degree of inflammation and LPS concentration. Thirty-nine eicosanoids were upregulated and seven were down-regulated by LPS treatment in a concentration-dependent manner. Our novel UPLC-MS/MS technique can profile eicosanoids, and can evaluate the correlations between inflammation and eicosanoid metabolism.

Small polyanion recognition of a triazolium cyclodextrin click cluster in water.

In order to detect small polyanions (sPAs), which play important roles in many biological systems, a triazolium cyclodextrin click cluster (5, hexakis{6-(3-methyl-4-hydroxymethyl-1H-1,2,3-triazolium-1-yl)-6-deoxy}-α-cyclodextrin iodide) was synthesized and characterized. The competition binding to 5 occupied by 5-carboxyfluorescein of inositol-1,4,5-trisphosphate (IP3), phytic acid, adenosine triphosphate (ATP), ethylenediaminetetraacetic acid (EDTA), glucose, and glucose-6-phosphate was evaluated by UV/vis titration in HEPES (10 mM, pH 7.4) : methanol (1 : 1, v/v). We obtained the binding constants of IP3 and phytic acid to 5 (1.4 × 10(6) and 1.9 × 10(6) M(-1), respectively); however, the binding constants of ATP and EDTA were significantly lower (2.1 × 10(5) and 4.5 × 10(4) M(-1), respectively). Moreover, glucose and glucose-6-phosphate did not show any detectable binding. In addition, the sPA recognition of the triazolium cyclodextrin click cluster in water was confirmed by fluorescence titration.

A comparative study of bone formation following grafting with different ratios of particle dentin and tricalcium phosphate combinations.

This study aimed to evaluate the bone regeneration relative to tooth powder and tricalcium phosphate (TCP) mixing ratios using the rabbit cranium defect model. The tooth powder was mixed with TCP in 1:1, 3:1, and 1:3 ratios, and the different ratios were implanted in the rabbit cranium defect for 4 and 8 weeks. Powders crystal structure evaluated using scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and new bone formation (NBF) was analyzed using micro-computed tomography (CT) and histologic examination. NBF in the control group was restricted to the defect margins. More NBF was observed around the defect margins in the experimental groups compared with the control group. Specifically, active NBF was identified around the implant materials of the centrifugal part of the defect and defect margins in the 3:1 tooth powder: TCP group. Our results suggested that tooth powder and TCP may be useful in bone regeneration.

Effect of platelet-rich plasma and platelet-rich fibrin on peri-implant bone defects in dogs.

The purpose of this study was to evaluate the effect of tooth ash and platelet-rich plasma (PRP), and platelet-rich fibrin (PRF) grafts into bone defects around implants on bone formation. Six adult dogs were used as experimental subjects. Graft materials were used to create a particulate material. Forty-eight tapered-type implants, 3.7 mm in diameter, 10 mm in length, and with surface treated with hydroxyapatite (HA) coating, were used as implant fixtures. Using a trephine bur, four bone defects were formed and implants were placed in the femurs of the adult dogs. Bone grafts were not performed in the control group. Tooth ash was grafted into the defects in group 1. In group 2, a mixture of tooth ash and PRP (1:1 ratio by volume) was grafted into the defects. In group 3, a mixture of tooth ash and PRF (ratio of 1:1) was grafted in the defect area. Animals were sacrificed after 4 or 8 weeks. Based on histopathological examination, the amount and rate of new bone formation were evaluated. Histomorphometric examination revealed that the rate of new bone formation in group 3 of the 4-week group was significantly higher than that in the control group. In addition, in the 8-week group, a significant increase in new bone formation was confirmed in group 3. In this study, a bone graft method using a mixture of tooth ash and PRF was found to increase new bone formation compared to the method using PRP. In addition, it was confirmed that this effect was more prominent in the initial stage of the bone graft.

Novel insertion mutation of ABCB1 gene in an ivermectin-sensitive Border Collie.

P-glycoprotein (P-gp) is encoded by the ABCB1 gene and acts as an efflux pump for xenobiotics. In the Border Collie, a nonsense mutation caused by a 4-base pair deletion in the ABCB1 gene is associated with a premature stop to P-gp synthesis. In this study, we examined the full-length coding sequence of the ABCB1 gene in an ivermectin-sensitive Border Collie that lacked the aforementioned deletion mutation. The sequence was compared to the corresponding sequences of a wild-type Beagle and seven ivermectin-tolerant family members of the Border Collie. When compared to the wild-type Beagle sequence, that of the ivermectin-sensitive Border Collie was found to have one insertion mutation and eight single nucleotide polymorphisms (SNPs) in the coding sequence of the ABCB1 gene. While the eight SNPs were also found in the family members' sequences, the insertion mutation was found only in the ivermectin-sensitive dog. These results suggest the possibility that the SNPs are species-specific features of the ABCB1 gene in Border Collies, and that the insertion mutation may be related to ivermectin intolerance.