Increased virulence of the oral microbiome in oral squamous cell carcinoma revealed by metatranscriptome analyses

Oral squamous cell carcinoma(OSCC) is the most prevalent and most commonly studied oral cancer. However, there is a void regarding the role that the oral microbiome may play in OSCC. Although the relationship between microbial community composition and OSCC has been thoroughly investigated, microbial profiles of the human microbiome in cancer are understudied. Here we performed a small pilot study of community-wide metatranscriptome analysis to profile mRNA expression in the entire oral microbiome in OSCC to reveal molecular functions associated with this disease. Fusobacteria showed a statistically significantly higher number of transcripts at tumour sites and tumour-adjacent sites of cancer patients compared to the healthy controls analysed. Regardless of the community composition, specific metabolic signatures were consistently found in disease. Activities such as iron ion transport, tryptophanase activity, peptidase activities and superoxide dismutase were over-represented in tumour and tumour-adjacent samples when compared to the healthy controls. The expression of putative virulence factors in the oral communities associated with OSCC showed that activities related to capsule biosynthesis, flagellum synthesis and assembly, chemotaxis, iron transport, haemolysins and adhesins were upregulated at tumour sites. Moreover, activities associated with protection against reactive nitrogen intermediates, chemotaxis, flagellar and capsule biosynthesis were also upregulated in non-tumour sites of cancer patients. Although they are preliminary, our results further suggest that Fusobacteria may be the leading phylogenetic group responsible for the increase in expression of virulence factors in the oral microbiome of OSCC patients.

Distribution and relative activity of matrix metalloproteinase-2 in human coronal dentin

The presence of matrix metalloproteinase-2 （MMP-2） in dentin has been reported, but its distribution and activity level in mature human coronal dentin are not well understood. The purpose of this study was to determine the MMP-2 distribution and relative activity in demineralized dentin. Crowns of twenty eight human molars were sectioned into inner （ID）, middle （MD）, and outer dentin （OD） regions and demineralized. MMP-2 was extracted with 0.33 mol·L-1 EDTA/2 mol·L-1 guanidine-HCl, pH 7.4, and MMP-2 concentration was estimated with enzyme-linked immunoabsorbant assay （ELISA）. Further characterization was accomplished by Western blotting analysis and gelatin zymography. The mean concentrations of MMP-2 per mg dentin protein in the dentin regions were significantly different （P=0.043）： 0.9 ng （ID）, 0.4 ng （MD）, and 2.2 ng （OD）, respectively. The pattern of MMP-2 concentration was OD〉ID〉MD. Western blotting analysis detected -66 and -72 kDa immunopositive proteins corresponding to pro- and mature MMP-2, respectively, in the ID and MD, and a -66 kDa protein in the OD. Gelatinolytic activity consistent with MMP-2 was detected in all regions. Interestingly, the pattern of levels of Western blot immunodetection and gelatinolytic activity was MD〉ID〉OD. The eoneentration of MMP-2 in human coronal dentin was highest in the region of dentin that contains the dentinoenamel junction and least in the middle region of dentin. However, levels of Western blot immunodetection and gelatinolytic activity did not correlate with the estimated regional concentrations of MMP-2, potentially indicating region specific protein interactions.

Relevance of α-defensins(HNP1-3) and defensin β-1 in diabetes

AIM: To investigate the genetic background of human defensin expression in type 1 and 2 diabetes.METHODS: Associations between DEFA1/DEFA3 gene copy number polymorphism and diabetes as well as between the promoter polymorphisms of DEFB1 and diabetes were studied. The copy number variation of the DEFA1/DEFA3 genes was determined in 257 diabetic patients(117 patients with type 1 and 140 with type 2 diabetes). The control group consisted of 221 age- and gender-matched healthy blood donors. The cumulative copy numbers of the DEFA1/DEFA3 genes were detected by using quantitative PCR analysis. To evaluate the HNP 1-3(human neutrophil peptide 1-3 or α-defensin) levels in the circulation, plasma HNP 1-3 concentrations were measured by ELISA. The expression of DEFA1/A3 in peripheral leukocytes of the diabetic patients was measured by quantitative RT PCR analysis. Three SNPs of the human DEFB1(human defensin β-1) gene: DEFB1 G-20A(rs11362), DEFB1 C-44G(rs1800972) and DEFB1 G-52A(rs1799946) were genotyped by Custom TaqMan? Real Time PCR assay.RESULTS: Significant differences were observed in HNP1-3 levels between the healthy subjects and both groups of diabetic patients. The mean ± SE was 28.78 ± 4.2 ng/mL in type 1 diabetes, and 29.82 ± 5.36 ng/mL in type 2 diabetes, vs 11.94 ± 2.96 ng/mL in controls; P < 0.01 respectively. There was no significant difference between patients with type 1 and type 2 diabetes in the high plasma concentrations of HNP1-3. The highest concentrations of α-defensin were found in diabetic patients with nephropathy(49.4 ± 4.8 ng/mL), neuropathy(38.7 ± 4.8 ng/mL) or cardiovascular complications(45.6 ± 1.45 ng/L). There was no significant difference in the cumulative copy numbers of DEFA1/DEFA3 genes between controls and patients, or between patients with the two types of diabetes. Comparisons of HNP 1-3 plasma level and DEFA1/A3 copy number of the same patient did not reveal significant relationship between defensin-α levels and the gene copy numbers(r2 = 0.01). Similarly, no positive correlation was observed between the copy numbers and the mRNA expression levels of DEFA1/A3. Regarding the C-44G polymorphism of DEFB1, the GG "protective" genotype was much less frequent(1%-2%) among both groups of patients than among controls(9%).CONCLUSION: Elevated HNP1-3 levels in diabetes are independent of DEFA1/DEFA3 copy numbers, but GG genotype of C-44G SNP in DEFB1 gene may result in decreased defensin β-1 production.

Survivin activity in normal human osteoblasts and osteosarcoma cells

PURPOSE: This study was designed to investigate and compare in vitro survivin activity in normal human osteoblast and MG-63 osteosarcoma cell cultures with and without vitamin D3. METHODS: Normal human alveolar bone explants were recovered from extraction sites of non-carious teeth of 9 healthy donors and cultured to the 2nd passage. MG-63 osteosarcoma cells were obtained from ATCC. To compare the survivin activities in these two types of cells and to determine the effect of vitamin D3 on survivin expression and associated activities of cell proliferation and differentiation, levels of survivin, osteocalcin and alkaline phosphatase were measured at 7-20 day cultures with and without vitamin D3 in both cultures of normal osteoblasts and osteosarcoma cells. Matched pair t-test and two sample independent t-test were applied for statistical analysis of the data. P values of 0.05 or less were considered statistically significant. RESULTS: A significantly higher level of survivin was detected in normal osteoblasts compared to osteosarcoma cells at 7 days without vitamin D3 treatment (P<0.01). Survivin expression in normal osteoblasts significantly decreased after vitamin D3 treatment at 7 days (P<0.05), but not at 20 days of culture (P=NS), compared to that in normal osteoblast culture without vitamin D3 treatment. Vitamin D3 had no effect on survivin expression in osteosarcoma cells at 7 or 20 days (P=NS). CONCLUSIONS: Expression of survivin in cultured normal human osteoblasts and osteosarcoma cells was positively identified. There is a positive correlation between higher expression of survivin and less differentiated osteoblasts that still retain their proliferative ability. Vitamin D3 has significant negative effect on expression of survivin in normal human osteoblasts but not on that in osteosarcoma cells.

NOTCH3 is expressed in human apical papilla and in subpopulations of stem cells isolated from the tissue

NOTCH plays a role in regulating stem cell function and fate decision.It is involved in tooth development and injury repair.Information regarding NOTCH expression in human dental root apical papilla(AP)and its residing stem cells(SCAP)is limited.Here we investigated the expression of NOTCH3,its ligand JAG1,and mesenchymal stem cell markers CD146 and STRO-1 in the AP or in the primary cultures of SCAP isolated from AP.Our in situ immunostaining showed that in the AP NOTCH3 and CD146 were co-expressed and associated with blood vessels having NOTCH3 located more peripherally.In cultured SCAP,NOTCH3 and JAG1 were co-expressed.Flow cytometry analysis showed that 7%,16%and 98%of the isolated SCAP were positive for NOTCH3,STRO-1 and CD146,respectively with a rare 1.5%subpopulation of SCAP co-expressing all three markers.The expression level of NOTCH3 reduced when SCAP underwent osteogenic differentiation.Our findings are the first step towards defining the regulatory role of NOTCH3 in SCAP fate decision.