Third-generation sequencing-selected Scardovia wiggsiae promotes periodontitis progression in mice.

BACKGROUNDS: Periodontitis is an oral-bacteria-directed disease that occurs worldwide. Currently, periodontal pathogens are mostly determined using traditional culture techniques, next-generation sequencing, and microbiological screening system. In addition to the well-known and cultivatable periodontal bacteria, we aimed to discover a novel periodontal pathogen by using DNA sequencing and investigate its role in the progression of periodontitis. OBJECTIVE: This study identified pathogens from subgingival dental plaque in patients with periodontitis by using the Oxford Nanopore Technology (ONT) third-generation sequencing system and validated the impact of selected pathogen in periodontitis progression by ligature-implanted mice. METHODS: Twenty-five patients with periodontitis and 25 healthy controls were recruited in this study. Subgingival plaque samples were collected for metagenomic analysis. The ONT third-generation sequencing system was used to confirm the dominant bacteria. A mouse model with ligature implantation and bacterial injection verified the pathogenesis of periodontitis. Neutrophil infiltration and osteoclast activity were evaluated using immunohistochemistry and tartrate-resistant acid phosphatase assays in periodontal tissue. Gingival inflammation was evaluated using pro-inflammatory cytokines in gingival crevicular fluids. Alveolar bone destruction in the mice was evaluated using micro-computed tomography and hematoxylin and eosin staining. RESULTS: Scardovia wiggsiae (S. wiggsiae) was dominant in the subgingival plaque of the patients with periodontitis. S. wiggsiae significantly deteriorated ligature-induced neutrophil infiltration, osteoclast activation, alveolar bone destruction, and the secretion of interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α in the mouse model. CONCLUSION: Our metagenome results suggested that S. wiggsiae is a dominant flora in patients with periodontitis. In mice, the induction of neutrophil infiltration, proinflammatory cytokine secretion, osteoclast activation, and alveolar bone destruction further verified the pathogenic role of S. wiggsiae in the progress of periodontitis. Future studies investigating the metabolic interactions between S. wiggsiae and other periodontopathic bacteria are warranted.

TIF1ß is phosphorylated at serine 473 in colorectal tumor cells through p38 mitogen-activated protein kinase as an oxidative defense mechanism.

TIF1ß is a pleiotropic regulator of a diverse range of cellular processes such as DNA repair or gene repression in stem cells. This functional switch depends on phosphorylation at serine residue 473 and multiple pathways exist to accomplish this. However, the effects of exogenous reactive oxygen species (ROS) generated by bacterial flora and dietary metabolites in the colonic lumen or chemotherapy on TIF1ß have not been determined. We report here that exposure of colorectal cancer (CRC) cell lines DLD-1 and HCT116 to hydrogen peroxide specifically induces TIF1ß Ser473 phosphorylation. Hydrogen peroxide also induces primarily p38 MAPK and some p42/44 MAPK phosphorylation. Chemical inhibition of p38 MAPK and p42/44 MAPK reduced phosphorylation of TIF1ß serine 473 and increased CRC cell death upon peroxide exposure. Taken together, this suggests that it is primarily peroxide-induced p38 MAPK that mediates Ser473 phosphorylation and activation of TIF1ß to enable more efficient DNA repair to assist in tumor cell survival against exogenous ROS.

Chitosan-assisted differentiation of porcine adipose tissue-derived stem cells into glucose-responsive insulin-secreting clusters.

The unique advantage of easy access and abundance make the adipose-derived stem cells (ADSCs) a promising system of multipotent cells for transplantation and regenerative medicine. Among the available sources, porcine ADSCs (pADSCs) deserve especial attention due to the close resemblance of human and porcine physiology, as well as for the upcoming availability of humanized porcine models. Here, we report on the isolation and conversion of pADSCs into glucose-responsive insulin-secreting cells. We used the stromal-vascular fraction of the dorsal subcutaneous adipose from 9-day-old male piglets to isolate pADSCs, and subjected the cells to an induction scheme for differentiation on chitosan-coated plates. This one-step procedure promoted differentiation of pADSCs into pancreatic islet-like clusters (PILC) that are characterized by the expression of a repertoire of pancreatic proteins, including pancreatic and duodenal homeobox (Pdx-1), insulin gene enhancer protein (ISL-1) and insulin. Upon glucose challenge, these PILC secreted high amounts of insulin in a dose-dependent manner. Our data also suggest that chitosan plays roles not only to enhance the differentiation potential of pADSCs, but also to increase the glucose responsiveness of PILCs. Our novel approach is, therefore, of great potential for transplantation-based amelioration of type 1 diabetes.

Topoisomerase II-mediated DNA cleavage and mutagenesis activated by nitric oxide underlie the inflammation-associated tumorigenesis.

AIMS: Both cancer-suppressing and cancer-promoting properties of reactive nitrogen and oxygen species (RNOS) have been suggested to play a role in tumor pathology, particularly those activities associated with chronic inflammation. Here, we address the impact of nitric oxide (NO) on the induction of DNA damage and genome instability with a specific focus on the involvement of topoisomerase II (TOP2). We also investigate the contribution of NO to the formation of skin melanoma in mice. RESULTS: Similar to the TOP2-targeting drug, etoposide (VP-16), the NO-donor, S-nitrosoglutathione (GSNO), induces skin melanomas formation in 7,12-dimethyl- benz[a]anthracene (DMBA)-initiated mice. To explore the mechanism(s) underlying this NO-induced tumorigenesis, we use a co-culture model system to demonstrate that inflamed macrophages with inducible NO synthase (iNOS) expression cause γ-H2AX activation, p53 phosphorylation, and chromosome DNA breaks in the target cells. Inhibitor experiments revealed that NO and TOP2 isozymes are responsible for the above described cellular phenotypes. Notably, NO, unlike VP-16, preferentially induces the formation of TOP2ß cleavable complexes (TOP2ßcc) in cells. Moreover, GSNO induced TOP2-dependent DNA sequence rearrangements and cytotoxicity. Furthermore, the incidences of GSNO- and VP-16-induced skin melanomas were also observed to be lower in the skin-specific top2ß-knockout mice. Our results suggest that TOP2 isozymes contribute to NO-induced mutagenesis and subsequent cancer development during chronic inflammation. INNOVATION AND CONCLUSIONS: We provide the first experimental evidence for the functional role of TOP2 in NO-caused DNA damage, mutagenesis, and carcinogenesis. Notably, these studies contribute to our molecular understanding of the cancer-promoting actions of RNOS during chronic inflammation.

Single-walled carbon nanotubes induce airway hyperreactivity and parenchymal injury in mice.

Inhalation of single-walled carbon nanotubes (SWCNTs) has raised serious concerns related to potential toxic effects in the respiratory system. This study examined possible SWCNT-induced toxic mechanisms in vivo in mice. The results indicated that a single intratracheal instillation of SWCNTs could induce airway hyperreactivity and airflow obstruction and confirmed previous findings of granulomatous changes in the lung parenchyma that persisted from 7 days to 6 months after exposure. The irreversible lung pathology and functional airway alterations in the mouse model mimicked obstructive airway disease in humans. Transcriptomic analysis showed that SWCNTs might up-regulate proteinases (cathepsin K and matrix metalloproteinase [MMP]12), chemokines C-C motif ligands (CCL2 and CCL3), and several macrophage receptors (Toll-like receptor 2, macrophage scavenger receptor 1). Pathway analyses showed that NF-κB-related inflammatory responses and downstream signals affecting tissue remodeling dominated the pathologic process. The NF-κB inhibitor pyrrolidine dithiocarbamate attenuated SWCNT-induced airway hyperreactivity, chronic airway inflammation, and MMP12 and cathepsin K expression when administered in vivo, whereas a cathepsin K inhibitor could partially reduce airway hyperreactivity and granulomatous changes in the SWCNT-treated group. The up-regulation of cathepsin K and MMP12 by SWCNTs was further confirmed via in vitro coculture of bronchoalveolar macrophages with lung epithelial/mesenchymal cells but not in macrophages without coculture, indicating that SWCNT-induced MMP12 and cathespin K were cell-type specific and cell-cell interaction dependent. In conclusion, exposure to SWCNTs may cause irreversible obstructive airway disease. Nanotoxicogenomics uncovered novel mechanisms underlying SWCNT-induced lung diseases, implicating MMP12 and cathepsin K in the pathologic injury as potential biomarkers or therapeutic targets.

Au nanorings for enhancing absorption and backscattering monitored with optical coherence tomography.

Preparation of a high-concentration Au nanoring (NR) water solution and its applications to the enhancement of image contrast in optical coherence tomography (OCT) and the generation of the photothermal effect in a bio-sample through localized surface plasmon (LSP) resonance are demonstrated. Au NRs are first fabricated on a sapphire substrate with colloidal lithography and secondary sputtering of Au, and then transferred into a water solution through a liftoff process. By controlling the NR geometry, the LSP dipole resonance wavelength in tissue can cover a spectral range of 1300 nm for OCT scanning of deep tissue penetration. The extinction cross sections of the fabricated Au NRs in water are estimated to give levels of 10(-10)-10(-9) cm(2) near their LSP resonance wavelengths. The fabricated Au NRs are then delivered into pig adipose samples for OCT scanning. It is observed that, when resonant Au NRs are delivered into such a sample, LSP resonance-induced Au NR absorption results in a photothermal effect, making the opaque pig adipose cells transparent. Also, the delivered Au NRs in the intercellular substance enhance the image contrast of OCT scanning through LSP resonance-enhanced scattering. By continuously OCT scanning a sample, both photothermal and image contrast enhancement effects are observed. However, by continually scanning a sample with a low scan frequency, only the image contrast enhancement effect is observed.

RNA-binding protein insulin-like growth factor II mRNA-binding protein 3 expression promotes tumor invasion and predicts early recurrence and poor prognosis in hepatocellular carcinoma.

UNLABELLED: Insulin-like growth factor II mRNA-binding protein 3 (IMP3) is an RNA-binding protein expressed in embryonic tissues and multiple cancers. To investigate the role of IMP3 in hepatocellular carcinoma (HCC), its protein expression in the surgically resected unifocal tumors of 377 HCC patients (296 men and 81 women) with ages ranging from 7 to 88 years (mean, 55.49 years) was analyzed by immunohistochemistry. IMP3 was expressed in 255 (67.6%) of 377 resected unifocal primary HCCs. IMP3 protein was predominantly expressed in tumor border and invasive front, and it was more abundant in the satellite nodules and tumor thrombi than in the main tumors. The expression correlated with high alpha-fetoprotein (>200 ng/mL, P < 1 x 10(-7)), larger tumor size (>5 cm, P = 0.006), high tumor grade (P < 1 x 10(-7)), and high tumor stage with vascular invasion and various degrees of intrahepatic metastasis (P < 1 x 10(-7)). IMP3 expression predicted early tumor recurrence (P < 1 x 10(-7)) and was a strong indicator of poor prognosis (P < 0.0001). Depletion of IMP3 with RNA interference in HCC cell line HA22T caused a decrease in cell motility, invasion, and transendothelial migration. Microarray analysis revealed that IMP3 depletion was associated with downregulation of multiple genes involved in tumor invasion. CONCLUSION: Our results indicate that IMP3 plays an important role in tumor invasion and metastasis and is a strong prognostic factor for patients with HCC.

Role of L2DTL, cell cycle-regulated nuclear and centrosome protein, in aggressive hepatocellular carcinoma.

L2DTL is a human ortholog of Drosophila lethal (2) denticleless, l(2)dtl. This study is to elucidate its function and clinicopathological significance in hepatocellular carcinoma (HCC) progression. We used RT-PCR, immunostaining, Western blotting, and centrosome isolation to determine the L2DTL expression and protein localization, and RNAi to analyze its role in tumor cell growth. L2DTL protein located to the nucleus in interphase and centered to centrosomes, with colocalization of gamma-tubulin and Aurora-A, throughout the cell cycle, and cofractionated with gamma-tubulin. L2DTL gene expression increased during G1/S phase, and the DNA synthesis in liver regeneration. L2DTL protein decreased in mitosis via degradation by the APC/C-Cdh1 complex. L2DTL was downregulated in the induced differentiation of HepG2 and NT2 cells. L2DTL downregulation by RNAi oligos led to reduced cancer cell growth and invasion capability in vitro, in which microarray analysis disclosed dysregulation of genes involved in cell cycle regulation, chromosome segregation, and cell division. L2DTL overexpressed in 59% of 270 resected, unifocal, primary HCCs. L2DTL overexpression correlated with bigger tumor (p=0.000003), high-grade (p=0.00003), and high-stage tumors with portal vein invasion (p=1x10(-8)). L2DTL overexpression was associated with a lower 10-year survival, particularly in the p53-mutated HCCs (p=0.00006). In conclusion, L2DTL encodes a nuclear protein with centrosome targeting in mitosis, and plays important roles in DNA synthesis, cell cycle progression, cytokinesis, proliferation, and differentiation. L2DTL overexpression is associated with enhanced metastatic potential of HCC, and contributes synergistically with p53 mutation, which leads to the loss of p53-governed checkpoints, toward advanced HCC with poor prognosis.

CDK-dependent activation of poly(ADP-ribose) polymerase member 10 (PARP10).

Proteins of the poly(ADP-ribose) polymerase (PARP) family play a wide array of functions, covering virtually every aspect of DNA metabolism and function, most notably with the response to DNA damage, transcription, and the maintenance of genomic stability. Here we report the identification and characterization of a novel PARP family member, PARP10 (FLJ14464 or hypothetical protein LOC84875). Overexpression of PARP10 results in loss of cell viability, although down-expression by short hairpin RNA leads to delayed G1 progression and concomitant cell death. PARP10 exists in both cytoplasm and nucleus, but only nucleolar PARP10 acquires CDK-dependent phosphorylation through late-G1 to S phase, and from prometaphase to cytokinesis in the nucleolar organizing regions. The PARP activity of PARP10 depends on phosphorylation by CDK2-cyclin E in vitro. CDK-phosphorylated PARP10 is absent in growth-arrested cells. These results suggest that PARP10 functions in cell proliferation and may serve as a marker for proliferating cells.