Genetic characterization of VP1 of coxsackieviruses A2, A4, and A10 associated with hand, foot, and mouth disease in Vietnam in 2012-2017: endemic circulation and emergence of new HFMD-causing lineages.

While conducting sentinel surveillance of hand, foot, and mouth disease (HFMD) in Vietnam, we found a sudden increase in the prevalence of coxsackievirus A10 (CV-A10) in 2016 and CV-A2 and CV-A4 in 2017, the emergence of which has been reported recently to be associated with various clinical manifestations in other countries. However, there have been only a limited number of molecular studies on those serotypes, with none being conducted in Vietnam. Therefore, we sequenced the entire VP1 genes of CV-A10, CV-A4, and CV-A2 strains associated with HFMD in Vietnam between 2012 and 2017. Phylogenetic analysis revealed a trend of endemic circulation of Vietnamese CV-A10, CV-A4, and CV-A2 strains and the emergence of thus-far undescribed HFMD-causing lineages of CV-A4 and CV-A2. The Vietnamese CV-A10 strains belonged to a genotype comprising isolates from patients with HFMD from several other countries; however, most of the Vietnamese strains were grouped into a local lineage. Recently, emerging CV-A4 strains in Vietnam were grouped into a unique lineage within a genotype comprising strains isolated from patients with acute flaccid paralysis from various countries. New substitutions were detected in the putative BC and HI loops in the Vietnamese CV-A4 strains. Except for one strain, Vietnamese CV-A2 isolates were grouped into a unique lineage of a genotype that includes strains from various countries that are associated with other clinical manifestations. Enhanced surveillance is required to monitor their spread and to specify their roles as etiological agents of HFMD or "HFMD-like" diseases, especially for CV-A4 and CV-A2. Further studies including whole-genome sequencing should be conducted to fully understand the evolutionary changes occurring in these newly emerging strains.

Aging-Affected MSC Functions and Severity of Periodontal Tissue Destruction in a Ligature-Induced Mouse Periodontitis Model.

Mesenchymal stem cells (MSCs) are known to play important roles in the repair of lost or damaged tissues and immunotolerance. On the other hand, aging is known to impair MSC function. However, little is currently known about how aged MSCs affect the host response to the local inflammatory condition and tissue deterioration in periodontitis, which is a progressive destructive disease of the periodontal tissue potentially leading to multiple tooth loss. In this study, we examined the relationship between aging-induced impairment of MSC function and the severity of periodontal tissue destruction associated with the decrease in host immunomodulatory response using a ligature-induced periodontitis model in young and aged mice. The results of micro computerized tomography (micro-CT) and histological analysis revealed a more severe bone loss associated with increased osteoclast activity in aged (50-week-old) mice compared to young (5-week-old) mice. Immunostaining analysis revealed that, in aged mice, the accumulation of inflammatory T and B cells was higher, whereas the percentage of platelet-derived growth factor receptor α (PDGFRα)+ MSCs, which are known to modulate the apoptosis of T cells, was significantly lower than in young mice. In vitro analysis of MSC function showed that the expression of surface antigen markers for MSCs (Sca-1, CD90, CD146), colony formation, migration, and osteogenic differentiation of aged MSCs were significantly declined compared to those of young MSCs. Moreover, a significantly higher proportion of aged MSCs were positive for the senescence-associated ß galactosidase activity. Importantly, aged MSCs presented a decreased expression of FAS-L, which was associated with a lower immunomodulatory property of aged MSCs to induce T cell apoptosis in co-cultures compared with young MSCs. In summary, this is the first study showing that aging-induced impairment of MSC function, including immunomodulatory response, is potentially correlated with progressive periodontal tissue deterioration.

BMP-2/ß-TCP Local Delivery for Bone Regeneration in MRONJ-Like Mouse Model.

Medication-related osteonecrosis of the jaw (MRONJ) is a severe pathological condition associated mainly with the long-term administration of bone resorption inhibitors, which are known to induce suppression of osteoclast activity and bone remodeling. Bone Morphogenetic Protein (BMP)-2 is known to be a strong inducer of bone remodeling, by directly regulating osteoblast differentiation and osteoclast activity. This study aimed to evaluate the effects of BMP-2 adsorbed onto beta-tricalcium phosphate (ß-TCP), which is an osteoinductive bioceramic material and allows space retention, on the prevention and treatment of MRONJ in mice. Tooth extraction was performed after 3 weeks of zoledronate (ZA) and cyclophosphamide (CY) administration. For prevention studies, BMP-2/ß-TCP was transplanted immediately after tooth extraction, and the mice were administered ZA and CY for an additional 4 weeks. The results showed that while the tooth extraction socket was mainly filled with a sparse tissue in the control group, bone formation was observed at the apex of the tooth extraction socket and was filled with a dense connective tissue rich in cellular components in the BMP-2/ß-TCP transplanted group. For treatment studies, BMP-2/ß-TCP was transplanted 2 weeks after tooth extraction, and bone formation was followed up for the subsequent 4 weeks under ZA and CY suspension. The results showed that although the tooth extraction socket was mainly filled with soft tissue in the control group, transplantation of BMP-2/ß-TCP could significantly accelerate bone formation, as shown by immunohistochemical analysis for osteopontin, and reduce the bone necrosis in tooth extraction sockets. These data suggest that the combination of BMP-2/ß-TCP could become a suitable therapy for the management of MRONJ.

Exploring the Regulators of Keratinization: Role of BMP-2 in Oral Mucosa.

The oral mucosa functions as a physico-chemical and immune barrier to external stimuli, and an adequate width of the keratinized mucosa around the teeth or implants is crucial to maintaining them in a healthy and stable condition. In this study, for the first time, bulk RNA-seq analysis was performed to explore the gene expression of laser microdissected epithelium and lamina propria from mice, aiming to investigate the differences between keratinized and non-keratinized oral mucosa. Based on the differentially expressed genes (DEGs) and Gene Ontology (GO) Enrichment Analysis, bone morphogenetic protein 2 (BMP-2) was identified to be a potential regulator of oral mucosal keratinization. Monoculture and epithelial-mesenchymal cell co-culture models in the air-liquid interface (ALI) indicated that BMP-2 has direct and positive effects on epithelial keratinization and proliferation. We further performed bulk RNA-seq of the ALI monoculture stimulated with BMP-2 in an attempt to identify the downstream factors promoting epithelial keratinization and proliferation. Analysis of the DEGs identified, among others, IGF2, ID1, LTBP1, LOX, SERPINE1, IL24, and MMP1 as key factors. In summary, these results revealed the involvement of a well-known growth factor responsible for bone development, BMP-2, in the mechanism of oral mucosal keratinization and proliferation, and pointed out the possible downstream genes involved in this mechanism.

Beneficial Effects of Liposomal Formulations of Lichen Substances: A Review.

Lichens are commonly used as essential traditional medicines to treat various conditions, including skin disorders, wounds, digestive, respiratory, obstetric, and gynecological problems in many cultures in Africa, Asia, Europe, Haitian, Oceania, and North and South America. Lichens have been deeply investigated for their phytochemical properties and, to date, numerous compounds (also known as substances) have been successfully isolated from the extracts. However, the low solubility and bioavailability of pure lichen substances have been widely recognized as significant issues hindering their biological applications. Recently, several groups have investigated the properties and the potential applications of lichen metabolites-based liposomal formulations and revealed a substantial improvement in their solubility, bioactivity, and toxicity in the animal. Thus, in this topical review, we aimed to provide an overview of liposomal structures, the efficacy of liposomal formulations, as well as their beneficial effects as compared to the free compounds themselves.

Risk factors for root caries annual incidence and progression among older people requiring nursing care: A one-year prospective cohort study.

PURPOSE: We aimed to determine root caries annual incidence (RCAI) and root caries annual progression (RCAP) and risk factors for them among older people requiring nursing care. METHODS: The target population comprised 186 dentate individuals aged ≥ 65 years who required nursing care while living in nursing homes (NHs) or their own homes (OHs) in Okayama, Japan. Survey items included presence/absence and severity of root caries, age, sex, living environment (NH or OH), the Clinical Dementia Rating, and the Barthel Index (BI). Baseline surveys were conducted from 2015 to 2017; subjects were followed up for one year. RCAI and RCAP per tooth and per person were calculated, and risk factors for them were identified using generalized estimating equations. RESULTS: In total, 104 individuals (mean age: 82.0 ± 12.4 years) completed the follow-up survey. RCAIs per tooth and per person were 14.6% (173/1188) and 59.6% (62/104), respectively. RCAP per tooth was 22.5% (51/227 teeth with root caries at baseline). Significant risk factors for RCAI were living environment (OH, odds ratio [OR]: 2.14), sex (male, OR: 1.84), clasped tooth (OR: 1.82), and older age (OR: 1.05) at baseline. Significant risk factors for RCAP were sex (male, OR: 5.20), regular dental checkup (OR: 2.74), and high BI score (OR: 1.02) at baseline. CONCLUSION: At one-year follow-up, 59.6% of the subjects developed at least one root caries. Risk factors for RCAI were living environment (OH), male, clasped tooth, and older age, whereas those for RCAP were male, regular dental checkup, and high BI score.

Six-year follow-up assessment of prosthesis survival and oral health-related quality of life in individuals with partial edentulism treated with three types of prosthodontic rehabilitation.

Purpose The purpose of the study was to compare the long-term performance of three prostheses for partial edentulism: implant-supported, fixed denture (IFD), fixed partial denture (FPD), and removable partial denture (RPD), in terms of prosthesis survival and oral health-related quality of life (OHRQoL).Methods The 138 patients in our previous study (Kimura et al., 2012) received one of the three prosthetic treatments and answered a validated OHRQoL questionnaire before and immediately after treatment. In the present study, the patients were followed up six years after treatment using medical records and OHRQoL examinations to evaluate prosthesis survival and change in OHRQoL. The cumulative survival rates were calculated using the Kaplan-Meier analysis. The Steel-Dwass test was used to compare the median OHRQoL scores at the three time points.Results For the 105 patients (66.8 ± 10.8 years, IFD/FPD/RPD: 58/27/20 patients) who successfully completed the follow-up assessments, the six-year estimated cumulative survival rates of the IFDs, FPDs, and RPDs were 94.7%, 77.4%, and 33.3%, respectively. The log-rank tests indicated that the survival curves were significantly different (IFDs vs. FPDs: p = 0.01; RPDs vs. IFDs, FPDs: p < 0.01). The median OHRQoL scores of the IFD group immediately after treatment and six years after treatment were significantly higher than those observed before treatment (p < 0.01). There was no significant difference in the median OHRQoL scores among the three time points in the RPD or FPD groups.Conclusions IFDs showed significantly longer survival rates than FPDs and RPDs in partially edentulous patients. Only in the IFD patients was the OHRQoL level six years after treatment significantly higher than that before treatment.

The transcription factor γMYB2 acts as a negative regulator of secondary cell wall thickening in anther and stem.

Secondary cell wall (SCW) thickening provides the mechanical force for anther dehiscence and plays an important role in the formation of xylem structure. We have previously reported that γMYB2, a MYB coiled-coil protein, directly binds to the P1BS cis-element of the PLA2-γ promoter and acts as a co-activator of γMYB1 in controlling the expression of PLA2-γ. In this study, we analyzed morphological phenotypes of the constitutive overexpression (γMYB2-OE) and knock-down (γMYB2-KD) lines of γMYB2. We found that γMYB2 overexpression caused the collapse of the endothecium layer, thereby suppressing anther dehiscence and forming short infertile siliques. The γMYB2-OE also showed less cellulose deposition in the xylem and had a longer primary stem than the wild-type, while γMYB2-KD had greater cellulose accumulation and a shorter primary stem than the wild-type. We demonstrated that the male sterility and the longer primary stem in γMYB2-OE were caused by reduced expression of SCW thickening-related genes. Our results suggest that γMYB2 acts as a negative regulator in controlling the SCW thickening in Arabidopsis.

Bone Marrow Cells Inhibit BMP-2-Induced Osteoblast Activity in the Marrow Environment.

Bone morphogenetic protein 2 (BMP-2) is widely known as a potent growth factor that promotes bone formation. However, an increasing number of studies have demonstrated side effects of BMP-2 therapy. A deeper understanding of the effect of BMP-2 on cells other than those involved directly in bone remodeling is of fundamental importance to promote a more effective delivery of BMP-2 to patients. In this study, we aimed to investigate the effect of BMP-2 in the marrow environment. First, BMP-2 adsorbed onto titanium implants was delivered at the tooth extraction socket (marrow-absent site) or in the mandible marrow of beagle dogs. BMP-2 could induce marked bone formation around the implant at the tooth extraction socket. Surprisingly, however, no bone formation was observed in the BMP-2-coated titanium implants inserted in the mandible marrow. In C57BL/6 mice, BMP-2 adsorbed in freeze-dried collagen pellets could induce bone formation in marrow-absent calvarial bone. However, similar to the canine model, BMP-2 could not induce bone formation in the femur marrow. Analysis of osteoblast differentiation using Col1a1(2.3)-GFP transgenic mice revealed a scarce number of osteoblasts in BMP-2-treated femurs, whereas in the control group, osteoblasts were abundant. Ablation of femur marrow recovered the BMP-2 ability to induce bone formation. In vitro experiments analyzing luciferase activity of C2C12 cells with the BMP-responsive element and alkaline phosphatase activity of MC3T3-E1 osteoblasts further revealed that bone marrow cells inhibit the BMP-2 effect on osteoblasts by direct cell-cell contact. Collectively, these results showed that the effect of BMP-2 in inducing bone formation is remarkably repressed by marrow cells via direct cell-cell contact with osteoblasts; this opens new perspectives on the clarification of the side-effects associated with BMP-2 application. © 2018 American Society for Bone and Mineral Research.

Type IV collagen α6 chain is a regulator of keratin 10 in keratinization of oral mucosal epithelium.

Keratinized mucosa is of fundamental importance to maintain healthy gingival tissue, and understanding the mechanisms of oral mucosa keratinization is crucial to successfully manage healthy gingiva. Previous studies have shown a strong involvement of the basement membrane in the proliferation and differentiation of epithelial cells. Therefore, first, to identify the keratinized mucosa-specific basement membrane components, immunohistochemical analysis for the six alpha chains of type IV collagen was performed in 8-week-old mice. No difference in the expression pattern of type IV collagen α1(IV) and α2(IV) chains was observed in the keratinized and non-keratinized mucosa. Interestingly, however, type IV collagen α5(IV) and α6(IV) chains specifically were strongly detected in the keratinized mucosa. To analyze the functional roles of the type IV collagen isoform α6(IV) in oral mucosa keratinization, we analyzed Col4a6-knockout mice. Epithelial developmental delay and low levels of KRT10 were observed in new-born Col4a6-knockout mice. Additionally, in vitro experiments with loss-of function analysis using human gingival epithelial cells confirmed the important role of α6(IV) chain in epithelial keratinization. These findings indicate that α112:α556 (IV) network, which is the only network that includes the α6(IV) chain, is one regulator of KRT10 expression in keratinization of oral mucosal epithelium.