Recombinant Lignin Peroxidase with Superior Thermal Stability and Melanin Decolorization Efficiency in a Typical Human Skin-Mimicking Environment.

Recently, the desire for a safe and effective method for skin whitening has been growing in the cosmetics industry. Commonly used tyrosinase-inhibiting chemical reagents exhibit side effects. Thus, recent studies have focused on performing melanin decolorization with enzymes as an alternative due to the low toxicity of enzymes and their ability to decolorize melanin selectively. Herein, 10 different isozymes were expressed as recombinant lignin peroxidases (LiPs) from Phanerochaete chrysosporium (PcLiPs), and PcLiP isozyme 4 (PcLiP04) was selected due to its high stability and activity at pH 5.5 and 37 °C, which is close to human skin conditions. In vitro melanin decolorization results indicated that PcLiP04 exhibited at least 2.9-fold higher efficiency than that of well-known lignin peroxidase (PcLiP01) in a typical human skin-mimicking environment. The interaction force between melanin films measured by a surface forces apparatus (SFA) revealed that the decolorization of melanin by PcLiP04 harbors a disrupted structure, possibly interrupting π-π stacking and/or hydrogen bonds. In addition, a 3D reconstructed human pigmented epidermis skin model showed a decrease in melanin area to 59.8% using PcLiP04, which suggests that PcLiP04 exhibits a strong potential for skin whitening.

Development and Optimization of a Rapid Colorimetric Membrane Immunoassay for Porphyromonas gingivalis.

Porphyromonas gingivalis (P. gingivalis) is a major bacterial pathogen that causes periodontitis, a chronic inflammatory disease of tissues around the teeth. Periodontitis is known to be related to other diseases, such as oral cancer, Alzheimer's disease, and rheumatism. Thus, a precise and sensitive test to detect P. gingivalis is necessary for the early diagnosis of periodontitis. The objective of this study was to optimize a rapid visual detection system for P. gingivalis. First, we performed a visual membrane immunoassay using 3,3',5,5'-tetramethylbenzidine (TMB; blue) and coating and detection antibodies that could bind to the host laboratory strain, ATCC 33277. Antibodies against the P. gingivalis surface adhesion molecules RgpB (arginine proteinase) and Kgp (lysine proteinase) were determined to be the most specific coating and detection antibodies, respectively. Using these two selected antibodies, the streptavidin-horseradish peroxidase (HRP) reaction was performed using a nitrocellulose membrane and visualized with a detection range of 103-105 bacterial cells/ml following incubation for 15 min. These selected conditions were applied to test other oral bacteria, and the results showed that P. gingivalis could be detected without crossreactivity to other bacteria, including Streptococcus mutans and Escherichia fergusonii. Furthermore, three clinical strains of P. gingivalis, KCOM 2880, KCOM 2803, and KCOM 3190, were also recognized using this optimized enzyme immunoassay (EIA) system. To conclude, we established optimized conditions for P. gingivalis detection with specificity, accuracy, and sensitivity. These results could be utilized to manufacture economical and rapid detection kits for P. gingivalis.

Extra disulfide and ionic salt bridge improves the thermostability of lignin peroxidase H8 under acidic condition.

The development of a lignin peroxidase (LiP) that is thermostable even under acidic pH conditions is a main issue for efficient enzymatic lignin degradation due to reduced repolymerization of free phenolic products at acidic pH (< 3). Native LiP under mild conditions (half-life (t1/2) of 8.2 days at pH 6) exhibits a marked decline in thermostability under acidic conditions (t1/2 of only 14 min at pH 2.5). Thus, improving the thermostability of LiP in acidic environments is required for effective lignin depolymerization in practical applications. Here, we show the improved thermostability of a synthetic LiPH8 variant (S49C/A67C/H239E, PDB: 6ISS) capable of strengthening the helix-loop interactions under acidic conditions. This variant retained excellent thermostability at pH 2.5 with a 10-fold increase in t1/2 (2.52 h at 25 °C) compared with that of the native enzyme. X-ray crystallography analysis showed that the recombinant LiPH8 variant is the only unique lignin peroxidase containing five disulfide bridges, and the helix-loop interactions of the synthetic disulfide bridge and ionic salt bridge in its structure are responsible for stabilizing the Ca2+-binding region and heme environment, resulting in an increase in overall structural resistance against acidic conditions. Our work will allow the design of biocatalysts for ligninolytic enzyme engineering and for efficient biocatalytic degradation of plant biomass in lignocellulose biorefineries.

Endodontic biofilms: contemporary and future treatment options.

Apical periodontitis is a biofilm-mediated infection. The biofilm protects bacteria from host defenses and increase their resistance to intracanal disinfecting protocols. Understanding the virulence of these endodontic microbiota within biofilm is essential for the development of novel therapeutic procedures for intracanal disinfection. Both the disruption of biofilms and the killing of their bacteria are necessary to effectively treat apical periodontitis. Accordingly, a review of endodontic biofilm types, antimicrobial resistance mechanisms, and current and future therapeutic procedures for endodontic biofilm is provided.

Antifungal Effects of Synthetic Human Beta-defensin-3-C15 Peptide on Candida albicans-infected Root Dentin.

INTRODUCTION: The aim of this study was to assess the antifungal efficacy of a synthetic human beta-defensin-3-C15 peptide (HBD3-C15) in Candida albicans-infected human root dentin. METHODS: Standardized root dentin blocks were prepared (6-mm thick, 0.7-mm-wide canal) from single-rooted human permanent premolars and infected with C. albicans for 3 weeks. They were randomly divided into 4 groups (n = 8/group), and their canals were filled with calcium hydroxide (CH), HBD3-C15 peptide, or chlorhexidine digluconate (CHX, 2%) as disinfectants or saline as control. After 1 week of disinfection, dentinal debris were harvested at depths of 200 and 400 µm from the canal lumen, and incubated in Yeast broth for 72 hours at 37°C. Then, colony-forming units (CFU) were measured to assess the antifungal efficacy of each medicament and analyzed statistically. RESULTS: All medicaments showed significantly lower CFU than saline (P < .05), and their antifungal efficacies were similar at both 200- and 400-µm tubular depths (P > .05). HBD3-C15 had similar antifungal efficacy to that of CHX at both depths (P > .05), and both medicaments had significantly lower CFU than CH at both depths (P < .05). CONCLUSIONS: In this ex vivo model of C. albicans-infected human root dentin, the antifungal efficacy of synthetic HBD3-C15 was comparable with CHX.

Bacterial entombment by intratubular mineralization following orthograde mineral trioxide aggregate obturation: a scanning electron microscopy study.

The time domain entombment of bacteria by intratubular mineralization following orthograde canal obturation with mineral trioxide aggregate (MTA) was studied by scanning electron microscopy (SEM). Single-rooted human premolars (n=60) were instrumented to an apical size #50/0.06 using ProFile and treated as follows: Group 1 (n=10) was filled with phosphate buffered saline (PBS); Group 2 (n=10) was incubated with Enterococcus faecalis for 3 weeks, and then filled with PBS; Group 3 (n=20) was obturated orthograde with a paste of OrthoMTA (BioMTA, Seoul, Korea) and PBS; and Group 4 (n=20) was incubated with E. faecalis for 3 weeks and then obturated with OrthoMTA-PBS paste. Following their treatments, the coronal openings were sealed with PBS-soaked cotton and intermediate restorative material (IRM), and the roots were then stored in PBS for 1, 2, 4, 8 or 16 weeks. After each incubation period, the roots were split and their dentin/MTA interfaces examined in both longitudinal and horizontal directions by SEM. There appeared to be an increase in intratubular mineralization over time in the OrthoMTA-filled roots (Groups 3 and 4). Furthermore, there was a gradual entombment of bacteria within the dentinal tubules in the E. faecalis inoculated MTA-filled roots (Group 4). Therefore, the orthograde obturation of root canals with OrthoMTA mixed with PBS may create a favorable environment for bacterial entombment by intratubular mineralization.

Induction of long-term immunity against respiratory syncytial virus glycoprotein by an osmotic polymeric nanocarrier.

Respiratory syncytial virus (RSV) is one of the most common causes of viral deaths in infants worldwide, yet no effective vaccines are available. Here, we report an osmotically active polysaccharide-based polysorbitol transporter (PST) prepared from sorbitol diacrylate and low-molecular-weight polyethylenimine (PEI) showing a potent, yet safe, adjuvant activity and acting as an effective delivery tool for RSV glycoprotein (RGp) antigen. PST showed no toxicity in vitro or in vivo, unlike PEI and the well-known experimental mucosal adjuvant cholera toxin (CT). PST formed nano-sized complexes with RGp by simple mixing, without affecting antigenic stability. The complexes exhibited negative surface charges that made them highly efficient in the selective activation of phagocytic cells and enhancement of phagocytic uptake. This resulted in an improved cytokine production and in the significant augmentation of RGp-specific antibody production, which persisted for over 200 days. Interestingly, PST/RGp enhanced phagocytic uptake owing to the osmotic property of PST and its negative zeta potential, suggesting that PST could selectively stimulate phagocytic cells, thereby facilitating a long-lived antigen-specific immune response, which was presumably further enhanced by the polysaccharide properties of PST.

Heavy metal analysis of ortho MTA and ProRoot MTA.

INTRODUCTION: Recently, several kinds of mineral trioxide aggregate (MTA)-based products have been introduced in endodontics. Ortho MTA (BioMTA, Seoul, Republic of Korea) is one of those products, which was developed for retrograde filling, perforation repair, orthograde root canal obturation, and direct pulp capping. The inclusion of heavy metals in MTA-based materials is of concern because they come into direct contact with hard and soft tissues. The aim of this study was to investigate and compare the levels of arsenic (As), chromium (Cr), hexavalent chromium (Cr(6+)), and lead (Pb) in Ortho MTA and ProRoot MTA. METHODS: One gram of each MTA was digested using a mixture of hydrochloric and nitric acids and filtered. The As, Cr, and Pb in the resulting filtrates were analyzed by inductively coupled plasma-optical emission spectrometry. The level of Cr(6+) was measured by the methods suggested in the Korean Standard L 5221. The results were statistically analyzed using the Mann-Whitney U test. RESULTS: The concentration of As in ProRoot MTA was 1.16 ppm, but As was not detected in Ortho MTA. Cr(6+) and Pb were not detected in either MTA. Ortho MTA contained significantly less Cr than ProRoot MTA (P < .05). CONCLUSIONS: Ortho MTA and ProRoot MTA meet the ISO specification 9917-1 regarding the safety limits of As and Pb and are safe biomaterials when the purity of As, Cr(6+), and Pb is considered.