Alzheimer’s disease (AD) is a neurodegenerative disease that usually affects older individuals. Owing to the higher incidence of root caries and missing teeth in elderly individuals, the bacteria involved in these dental concerns might potentially deteriorate their cognitive function. Altered microbiota in the oral cavity may induce neuroinflammation through migration from the oral cavity to the brain. However, the correlation between the composition of the oral microbiota and neurodegenerative disease remains unclear. In this study, we evaluated sequence to determine the relative abundance and diversity of bacterial taxa in the dental plaque of elderly patients with AD and controls. Oral samples; the DMFT index; and other clinical examination data were collected from 17 patients with AD and 18 normal elderly individuals as the control group. Patients with AD had significantly more missing teeth and higher dental plaque weight but lower microbial diversity than controls. Significantly increased numbers of Lactobacillales, Streptococcaceae, and Firmicutes/Bacteroidetes and a significantly decreased number of Fusobacterium were observed in patients with AD. In conclusion, using the PacBio single-molecule real-time (SMRT) sequencing platform to survey the microbiota dysbiosis biomarkers in the oral cavity of elderly individuals could serve as a tool to identify patients with AD.

Current therapeutics for hepatitis B virus (HBV) patients such as nucleoside analogs (NAs) are effective; however, new antiviral drugs against HBV are still desired. Since the interaction between the epsilon (ε) sequence of HBV pregenomic RNA and viral polymerase (Pol) is a key step in the HBV replication cycle, we aimed to identify small compounds for its inhibition, and established a pull-down assay system for the detection of ε-RNA-binding-Pol. Screening showed that 5 out of 3,965 compounds inhibited ε-Pol binding, and we identified rosmarinic acid, which exhibited specificity, as a potential antiviral agent. In order to examine the anti-HBV effects of rosmarinic acid, HBV-infected primary human hepatocytes from a humanized mouse liver were treated with rosmarinic acid. The rosmarinic acid treatment decreased HBV components including the amounts of extracellular HBV DNA with negligible cytotoxicity. We also investigated the combined effects of rosmarinic acid and the NA, lamivudine. rosmarinic acid slightly enhanced the anti-HBV activity of lamivudine, suggesting that the HBV replication step targeted by rosmarinic acid is distinct from that of NA. We analyzed an additional 25 rosmarinic acid derivatives, and found that 5 also inhibited ε-Pol. Structural comparisons between these derivatives implied that the “two phenolic hydroxyl groups at both ends” and the “caffeic acid-like structure” of rosmarinic acid are critical for the inhibition of ε-Pol binding. Collectively, our results demonstrate that rosmarinic acid inhibits HBV replication in HBV-infected cells by specifically targeting ε-Pol binding.

Porphyromonas gingivalis infection is one of the causes of implant failures, which can lead to peri-implantitis. Implant surface roughness is reportedly related strongly to P. gingivalis adhesion, which can lead to peri-implantitis and, later, cell adhesion. Our aim was to evaluate the effects of Er,Cr:YSGG laser on titanium (Ti) disc surfaces and its interaction with bacterial adhesion and fibroblast viability. Ti discs underwent two treatments: autoclaving (control) and erbium, chromium-doped yttrium scandium gallium garnet (Er,Cr:YSGG) laser treatment (test). Ti disc surfaces were examined with scanning electronic microscope (SEM), Energy-dispersive spectrometry (EDX), X-ray photoelectron spectroscopy (XPS). The surface roughness same as wettability were also investigated. Fibroblast viability was assessed with the water-soluble tetrazolium 1 (WST-1) test, and osteoblast differentiation was assessed with the alkaline phosphatase (ALP) assay. Bacterial structure and colony formation were detected with scanning electron microscopy and Gram stain. In comparison to control discs, the test discs showed smoother surfaces, with 0.25-µm decrease in surface roughness (p < 0.05); lower P. gingivalis adhesion (p < 0.01); less P. gingivalis colonization (p < 0.05); and increased fibroblast viability and osteoblast differentiation (p < 0.05). Er,Cr:YSGG laser treatment improved disc surfaces by making them slightly smoother, which reduced P. gingivalis adhesion and increased fibroblast viability and osteoblast differentiation. Er,Cr:YSGG laser treatment can be considered a good option for managing peri-implantitis. Further investigations of laser-assisted therapy are necessary for better guidelines in the treatment of peri-implantitis.

Due to autogenous bone limitations, some substitute bone grafts were developed. Collagenated porcine graft (CPG) is able to regenerate new bone, although the number of studies is insufficient, highlighting the need for future studies to better understand the biomaterial. In order to understand better CPG′s possible dental guided bone regeneration indications, the aim of this work was to determine CPG′s biological capacity to induce osteoblast differentiation in vitro and guided bone regeneration in vivo, whilst being compared with commercial hydroxyapatite and beta tricalcium phosphate (HA/β-TCP) and porcine graft alone. Cell cytotoxicity (WST-1), alkaline phosphatase activity (ALP), and real-time polymerase chain reaction (qPCR) were assessed in vitro. Critical size defects of New Zealand white rabbits were used for the in vivo part, with critical size defect closures and histological analyses. WST-1 and ALP indicated that CPG directly stimulated a greater proliferation and confluency of cells with osteoblastic differentiation in vitro. Gene sequencing indicated stable bone formation markers, decreased resorption makers, and bone remodeling coupling factors, making the transition from osteoclast to osteoblast expression at the end of seven days. CPG resulted in the highest new bone regeneration by osteoconduction in critical size defects of rabbit calvaria at eight weeks. Nonetheless, all biomaterials achieved nearly complete calvaria defect closure. CPG was found to be osteoconductive, like porcine graft and HA/β-TCP, but with higher new bone formation in critical size defects of rabbit calvaria at eight weeks. CPG can be used for different dental guided bone regeneration procedures; however, further studies are necessary.

Peri-implantitis is the pathological condition of connective tissue inflammation and the progressive loss of supporting bone around dental implants. One of the primary causes of peri mucositis evolving into peri-implantitis is bacterial infection, including infection from Porphyromonas gingivalis. Enhancing the surface smoothness of implants helps to prevent P. gingivalis adhesion to the implant’s surface. Interaction analyses between bacteria and the surface roughness of zirconia (Zr) discs subjected to a glow discharge plasma (GDP) treatment compared with non-plasma-treated autoclaved control Zr discs were done. Examinations of the material prosperities revealed that the GDP-treated Zr group had a smoother surface for a better wettability. The GDP-treated Zr discs improved the proliferation of the osteoblast-like cells MG-63, and the osteoblastic differentiation was assessed through alkaline phosphatase detection and marker gene bone sialoprotein (Bsp) and osteocalcin (OC) induction. Scanning electron microscopy demonstrated a relatively low P. gingivalis adhesion on GDP-treated Zr disks, as well as lower colonization of P. gingivalis compared with the control. Our findings confirmed that the GDP treatment of Zr discs resulted in a significant reduction of P. gingivalis adhesion and growth, demonstrating a positive correlation between surface roughness and bacteria adhesion. Therefore, the GDP treatment of Zr dental implants can provide a method for reducing the risk of peri-implantitis.

Currently, there is no available therapy to eradicate hepatitis B virus (HBV) in chronically infected individuals. This is due to the difficulty in eliminating viral covalently closed circular (ccc) DNA, which is central to the gene expression and replication of HBV. We developed an assay system for nuclear circular DNA using an integration-deficient lentiviral vector. This vector produced non-integrated circular DNA in nuclei of infected cells. We engineered this vector to encode firefly luciferase to monitor the lentiviral episome DNA. We screened 3,840 chemicals by this assay for luciferase-reducing activity and identified dicumarol, which is known to have anticoagulation activity. We confirmed that dicumarol reduced lentiviral episome DNA. Furthermore, dicumarol inhibited HBV replication in cell culture using NTCP-expressing HepG2 and primary human hepatocytes. Dicumarol reduced intracellular HBV RNA, DNA, supernatant HBV antigens and DNA. We also found that dicumarol reduced the cccDNA level in HBV infected cells, but did not affect HBV adsorption/entry. This is a novel assay system for screening inhibitors targeting nuclear cccDNA and is useful for finding new antiviral substances for HBV.