Exosomes from dental pulp cells attenuate bone loss in mouse experimental periodontitis.

BACKGROUND AND OBJECTIVE: Exosomes are small vesicles secreted from many cell types. Their biological effects largely depend on their cellular origin and the physiological state of the originating cells. Exosomes secreted by mesenchymal stem cells exert therapeutic effects against multiple diseases and may serve as potential alternatives to stem cell therapies. We previously established and characterized human leukocyte antigen (HLA) haplotype homo (HHH) dental pulp cell (DPC) lines from human wisdom teeth. In this study, we aimed to investigate the effect of local administration of HHH-DPC exosomes in a mouse model of periodontitis. METHODS: Exosomes purified from HHH-DPCs were subjected to particle size analysis, and expression of exosome markers was confirmed by western blotting. We also confirmed the effect of exosomes on the migration of both HHH-DPCs and mouse osteoblastic MC3T3-E1 cells. A mouse experimental periodontitis model was used to evaluate the effect of exosomes in vivo. The morphology of alveolar bone was assessed by micro-computed tomography (µCT) and histological analysis. The effect of exosomes on osteoclastogenesis was evaluated using a co-culture system. RESULTS: The exosomes purified from HHH-DPCs were homogeneous and had a spherical membrane structure. HHH-DPC exosomes promoted the migration of both human DPCs and mouse osteoblastic cells. The MTT assay showed a positive effect on the proliferation of human DPCs, but not on mouse osteoblastic cells. Treatment with HHH-DPC exosomes did not alter the differentiation of osteoblastic cells. Imaging with µCT revealed that the exosomes suppressed alveolar bone resorption in the mouse model of periodontitis. Although no change was apparent in the dominance of TRAP-positive osteoclast-like cells in decalcified tissue sections upon exosome treatment, HHH-DPC exosomes significantly suppressed osteoclast formation in vitro. CONCLUSIONS: HHH-DPC exosomes stimulated the migration of human DPCs and mouse osteoblastic cells and effectively attenuated bone loss due to periodontitis.

Isolation, Diversity and Characterization of Ulvan-Degrading Bacteria Isolated from Marine Environments.

In this study, we aimed to isolate bacteria capable of degrading the polysaccharide ulvan from the green algae Ulva sp. (Chlorophyta, Ulvales, Ulvaceae) in marine environments. We isolated 13 ulvan-degrading bacteria and observed high diversity at the genus level. Further, the genera Paraglaciecola, Vibrio, Echinicola, and Algibacter, which can degrade ulvan, were successfully isolated for the first time from marine environments. Among the 13 isolates, only one isolate (Echinicola sp.) showed the ability not only to produce externally expressed ulvan lyase, but also to be periplasmic or on the cell surface. From the results of the full-genome analysis, lyase was presumed to be a member of the PL25 (BNR4) family of ulvan lyases, and the bacterium also contained the sequence for glycoside hydrolase (GH43, GH78 and GH88), which is characteristic of other ulvan-degrading bacteria. Notably, this bacterium has a unique ulvan lyase gene not previously reported.

Production of 4-Deoxy-L-erythro-5-Hexoseulose Uronic Acid Using Two Free and Immobilized Alginate Lyases from Falsirhodobacter sp. Alg1.

Falsirhodobacter sp. alg1 expresses two alginate lyases, AlyFRA and AlyFRB, to produce the linear monosaccharide 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from alginate, metabolizing it to pyruvate. In this study, we prepared recombinant AlyFRA and AlyFRB and their immobilized enzymes and investigated DEH production. Purified AlyFRA and AlyFRB reacted with sodium alginate and yielded approximately 96.8% DEH. Immobilized AlyFRA and AlyFRB were prepared using each crude enzyme solution and κ-carrageenan, and immobilized enzyme reuse in batch reactions and DEH yield were examined. Thus, DEH was produced in a relatively high yield of 79.6%, even after the immobilized enzyme was reused seven times. This method can produce DEH efficiently and at a low cost and can be used to mass produce the next generation of biofuels using brown algae.

Cell-Penetrating Peptide-Peptide Nucleic Acid Conjugates as a Tool for Protein Functional Elucidation in the Native Bacterium.

Approximately 30% or more of the total proteins annotated from sequenced bacteria genomes are annotated as hypothetical or uncharacterized proteins. However, elucidation on the function of these proteins is hindered by the lack of simple and rapid screening methods, particularly with novel or hard-to-transform bacteria. In this report, we employed cell-penetrating peptide (CPP) -peptide nucleotide acid (PNA) conjugates to elucidate the function of such uncharacterized proteins in vivo within the native bacterium. Paenibacillus, a hard-to-transform bacterial genus, was used as a model. Two hypothetical genes showing amino acid sequence similarity to ι-carrageenases, termed cgiA and cgiB, were identified from the draft genome of Paenibacillus sp. strain YYML68, and CPP-PNA probes targeting the mRNA of the acyl carrier protein gene, acpP, and the two ι-carrageenase candidate genes were synthesized. Upon direct incubation of CPP-PNA targeting the mRNA of the acpP gene, we successfully observed growth inhibition of strain YYML68 in a concentration-dependent manner. Similarly, both the function of the candidate ι-carrageenases were also inhibited using our CPP-PNA probes allowing for the confirmation and characterization of these hypothetical proteins. In summary, we believe that CPP-PNA conjugates can serve as a simple and efficient alternative approach to characterize proteins in the native bacterium.

Specific Deletion of p16INK4a with Retention of p19ARF Enhances the Development of Invasive Oral Squamous Cell Carcinoma.

The cyclin-dependent kinase inhibitor 2A (CDKN2A)/alternate reading frame (ARF) locus consists of two overlapping tumor suppressor genes, p16INK4a and p14ARF (p19ARF in mice), encoding two unrelated proteins in alternative reading frames. Previous reports suggest that p16INK4a and p14ARF alterations independently exhibit differential roles, and p16INK4a is more closely associated with a poor prognosis in oral cancer. However, the role of p16INK4a-specific loss in oral squamous cell carcinogenesis remains unclear. The authors assessed chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced multistep oral squamous cell carcinogenesis in mice carrying p16INK4a-specific loss with retention of the p19ARF gene (p16INK4a-/-). 4NQO-treated p16-/- mice exhibited a higher incidence and multiplicity of oral squamous cell carcinoma (OSCC) development relative to 4NQO-treated wild-type mice. 4NQO-treated p16INK4a-/- OSCC cells exhibited higher proliferation and up-regulation of Arf, transcription factor E2f1, tumor protein p63 (tp63), and oncogenic ΔNp63, an isoform p63, compared with observations in 4NQO-treated wild-type OSCC cells. Furthermore, the overexpression of oncogenic ΔNp63 was associated with human OSCC. In conclusion, these results in mice indicate the biological significance of p16INK4a-specific loss with retention of p19ARF in oral squamous cell carcinogenesis, and ΔNp63 may be a potential target for OSCC.

Role of the DEK oncogene in the development of squamous cell carcinoma.

DEK is a highly conserved nuclear factor that plays an important role in the regulation of multiple cellular processes. DEK was discovered to be an oncogene as a fusion with NUP214 gene, which results in producing DEK-NUP214 proteins, in a subset of patients with acute myeloid leukemia. Subsequently, DEK overexpression was reported in many cancers, thus DEK itself is considered to be an oncoprotein. DEK has been reported to play important roles in the progression of early and late stage squamous cell carcinoma (SCC) and is useful for early diagnosis of the disease. These findings have made DEK an attractive therapeutic target, especially for human papillomavirus (HPV)-associated SCC. However, the mechanism of DEK in SCC remains unclear. In this review, we discuss human DEK oncogene-related SCC.

FGF2-responsive genes in human dental pulp cells assessed using a rat spinal cord injury model.

The central nervous system in adult mammals does not heal spontaneously after spinal cord injury (SCI). However, SCI treatment has been improved recently following the development of cell transplantation therapy. We recently reported that fibroblast growth factor (FGF) 2-pretreated human dental pulp cells (hDPCs) can improve recovery in a rat model of SCI. This study aimed to investigate mechanisms underlying the curative effect of SCI enhanced via FGF2 pretreatment; we selected three hDPC lines upon screening for the presence of mesenchymal stem cell markers and of their functionality in a rat model of SCI, as assessed using the Basso, Beattie, and Bresnahan score of locomotor functional scale, electrophysiological tests, and morphological analyses. We identified FGF2-responsive genes via gene expression analyses in these lines. FGF2 treatment upregulated GABRB1, MMP1, and DRD2, which suggested to contribute to SCI or central the nervous system. In an expanded screening of additional lines, GABRB1 displayed rather unique and interesting behavior; two lines with the lowest sensitivity of GABRB1 to FGF2 treatment displayed an extremely minor effect in the SCI model. These findings provide insights into the role of FGF2-responsive genes, especially GABRB1, in recovery from SCI, using hDPCs treated with FGF2.

An adiponectin paralog protein, CTRP6 decreased the proliferation and invasion activity of oral squamous cell carcinoma cells: possible interaction with laminin receptor pathway.

C1q/tumor necrosis factor-related protein-6 (CTRP6), also known as CTRP6 is identified adiponectin paralog. Although recent studies have revealed that adiponectin has an inhibitory role in carcinogenesis, the role of CTRP6 in carcinogenesis remains unclear. In this study, we found that eukaryotic recombinant CTRP6 protein bound to the cell surface membrane of cultured oral squamous cell carcinoma cells by immunofluorescence staining. Screening of CTRP6 binding protein in expression library followed by co-immunoprecipitation assay revealed that CTRP6 bound to the precursor of laminin receptor. CTRP6 disturbed the binding of laminin to the laminin receptor. Interestingly, the eukaryotic recombinant CTRP6 protein significantly suppressed the proliferation and Matrigel invasion activity of oral squamous cell carcinoma SAS cells in a dose-dependent manner. Moreover, administration of CTRP6 significantly attenuated the growth of SAS cells in xenoplant mice model. Laminin and laminin receptor are known to be overexpressed and promote the tumor growth in OSCC. Combined together, the present findings suggest that CTRP6 could repress progression of oral squamous cell carcinoma cells, putatively through disrupting the laminin-laminin receptor axis.

TMEM207 hinders the tumour suppressor function of WWOX in oral squamous cell carcinoma.

The WW domain-containing oxidoreductase (WWOX) functions as a tumour suppressor in oral carcinogenesis. As aberrant TMEM207 expression may lead to tumour progression by hampering the tumour suppressor function of WWOX in various cancers, we explored the expression and pathobiological properties of TMEM207, focusing on the WWOX-mediated regulation of the HIF-1α pathway in oral squamous cell carcinoma (OSCC). TMEM207 immunoreactivity was detected in 40 of 90 OSCC samples but not in neighbouring non-tumorous epithelial tissues. Moreover, TMEM207 expression was significantly correlated with lymph node metastasis and poor prognosis. An in situ proximal ligation assay demonstrated the colocalization of TMEM207 and WWOX in invasive OSCC cells, especially glycogen-rich ones. Enforced expression of TMEM207 abrogated the binding of WWOX to HIF-1α, increased HIF-1α and GLUT-1 expression, even under normoxic conditions, and promoted tumour growth in a xenoplant assay using SAS tongue squamous cancer cells. In contrast, TMEM207 knockdown decreased GLUT-1 expression in two OSCC cell lines. As a whole, our findings indicate that the aberrant expression of TMEM207 contributes to tumour progression in OSCC, possibly via promoting aerobic glycolysis.

Direct bioethanol production from brown macroalgae by co-culture of two engineered Saccharomyces cerevisiae strains.

A co-culture platform for bioethanol production from brown macroalgae was developed, consisting of two types of engineered Saccharomyces cerevisiae strains; alginate- and mannitol-assimilating yeast (AM1), and cellulase-displaying yeast (CDY). When the 5% (w/v) brown macroalgae Ecklonia kurome was used as the sole carbon source for this system, 2.1 g/L of ethanol was produced, along with simultaneous consumption of alginate, mannitol, and glucans.

Construction of bioengineered yeast platform for direct bioethanol production from alginate and mannitol.

Brown macroalgae are a sustainable and promising source for bioethanol production because they are abundant in ocean ecosystems and contain negligible quantities of lignin. Brown macroalgae contain cellulose, hemicellulose, mannitol, laminarin, and alginate as major carbohydrates. Among these carbohydrates, brown macroalgae are characterized by high levels of alginate and mannitol. The direct bioconversion of alginate and mannitol into ethanol requires extensive bioengineering of assimilation processes in the standard industrial microbe Saccharomyces cerevisiae. Here, we constructed an alginate-assimilating S. cerevisiae recombinant strain by genome integration and overexpression of the genes encoding endo- and exo-type alginate lyases, DEH (4-deoxy-L-erythro-5-hexoseulose uronic acid) transporter, and components of the DEH metabolic pathway. Furthermore, the mannitol-metabolizing capacity of S. cerevisiae was enhanced by prolonged culture in a medium containing mannitol as the sole carbon source. When the constructed strain AM1 was anaerobically cultivated in a fermentation medium containing 6% (w/v) total sugars (approximately 1:2 ratio of alginate/mannitol), it directly produced ethanol from alginate and mannitol, giving 8.8 g/L ethanol and yields of up to 32% of the maximum theoretical yield from consumed sugars. These results indicate that all major carbohydrates of brown macroalgae can be directly converted into bioethanol by S. cerevisiae. This strain and system could provide a platform for the complete utilization of brown macroalgae.

Engineered yeast whole-cell biocatalyst for direct degradation of alginate from macroalgae and production of non-commercialized useful monosaccharide from alginate.

Alginate is a major component of brown macroalgae. In macroalgae, an endolytic alginate lyase first degrades alginate into oligosaccharides. These oligosaccharides are further broken down into monosaccharides by an exolytic alginate lyase. In this study, genes encoding various alginate lyases derived from alginate-assimilating marine bacterium Saccharophagus degradans were isolated, and their enzymes were displayed using the yeast cell surface display system. Alg7A-, Alg7D-, and Alg18J-displaying yeasts showed endolytic alginate lyase activity. On the other hand, Alg7K-displaying yeast showed exolytic alginate lyase activity. Alg7A, Alg7D, Alg7K, and Alg18J, when displayed on yeast cell surface, demonstrated both polyguluronate lyase and polymannuronate lyase activities. Additionally, polyguluronic acid could be much easily degraded by Alg7A, Alg7K, and Alg7D than polymannuronic acid. In contrast, polymannuronic acid could be much easily degraded by Alg18J than polyguluronic acid. We further constructed yeasts co-displaying endolytic and exolytic alginate lyases. Degradation efficiency by the co-displaying yeasts were significantly higher than single alginate lyase-displaying yeasts. Alg7A/Alg7K co-displaying yeast had maximum alginate degrading activity, with production of 1.98 g/L of reducing sugars in a 60-min reaction. This system developed, along with our findings, will contribute to the efficient utilization and production of useful and non-commercialized monosaccharides from alginate by Saccharomyces cerevisiae.

Complete bone regeneration in hemophilic pseudotumor of the mandible.

Hemophilic pseudotumor (HP) is rare, seen in 1-2% of patients with hemophilia, and is extremely uncommon in the mandible. A 6-year-old boy with moderate hemophilia A presented to our hospital with left mandibular swelling. Based on clinical and radiological findings, a tentative diagnosis of HP was made. After factor VIII administration, the lesion was curetted and HP was confirmed on histopathology. The patient was treated with twice-weekly factor VIII until the lesion had completely resolved and bone had regenerated at 1 year. The best treatment for HP is not established; however, appropriate initial treatment and postoperative prophylaxis are effective.

Formosa haliotis sp. nov., a brown-alga-degrading bacterium isolated from the gut of the abalone Haliotis gigantea.

Four brown-alga-degrading, Gram-stain-negative, aerobic, non-flagellated, gliding and rod-shaped bacteria, designated LMG 28520T, LMG 28521, LMG 28522 and LMG 28523, were isolated from the gut of the abalone Haliotis gigantea obtained in Japan. The four isolates had identical random amplified polymorphic DNA patterns and grew optimally at 25 °C, at pH 6.0-9.0 and in the presence of 1.0-4.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences placed the isolates in the genus Formosa with Formosa algae and Formosa arctica as closest neighbours. LMG 28520T and LMG 28522 showed 100 % DNA-DNA relatedness to each other, 16-17 % towards F. algae LMG 28216T and 17-20 % towards F. arctica LMG 28318T; they could be differentiated phenotypically from these established species. The predominant fatty acids of isolates LMG 28520T and LMG 28522 were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), iso-C15 : 1 G and iso-C15 : 0. Isolate LMG 28520T contained menaquinone-6 (MK-6) as the major respiratory quinone and phosphatidylethanolamine, two unknown aminolipids and an unknown lipid as the major polar lipids. The DNA G+C content was 34.4 mol% for LMG 28520T and 35.5 mol% for LMG 28522. On the basis of their phylogenetic and genetic distinctiveness, and differential phenotypic properties, the four isolates are considered to represent a novel species of the genus Formosa, for which the name Formosa haliotis sp. nov. is proposed. The type strain is LMG 28520T ( = NBRC 111189T).

A more efficient method to generate integration-free human iPS cells.

We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match ∼20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.

Genome sequence of Vibrio sp. strain 10N, an ulvan-degrading bacterium isolated from coastal seawater collected at Ise Bay, Japan.

Genome sequence of an ulvan-degrading bacterium, Vibrio sp. strain 10N, is presented. The genome is 5,358,550 bp with a G + C content of 46.5%. A total of 4,712 coding sequences, including two novel ulvan lyase genes encoding a BNR4 and a glycoside hydrolase (GH88) motif, are known to be involved in the degradation of green algae.

Analysis of Concentrated COVID-19 Outbreaks in Elderly Facilities in Suita City, Osaka Prefecture, Japan.

There are several types of facilities for elderly individuals in Japan. Infection control efforts, such as care provision and medical care access, differ according to the type of facility. Elderly individuals at these facilities who were infected with coronavirus disease 2019 (COVID-19) experienced severe illness and mortality. This study aimed to determine the characteristics of concentrated COVID-19 outbreaks that occurred in nursing homes and care facilities in Suita City. During this study, twenty-five elderly facilities in Suita City with a capacity of 40 or more individuals where an outbreak occurred during the sixth or seventh wave of infection were included. We investigated whether there was a difference in the COVID-19 incidence and the percentage of positive cases according to the type of facility. We also investigated the relationship between the facility capacity and positive case rate and that between the number of positive cases and outbreak duration. The incidence rate of COVID-19 was significantly different according to the facility type (p < 0.001). No association was found between the facility capacity and positive case rate. The outbreak duration increased as the number of positive cases increased (p = 0.004).

Change over Time in the Risk of Death among Japanese COVID-19 Cases Caused by the Omicron Variant Depending on Prevalence of Sublineages.

To assess temporal changes to the risk of death in COVID-19 cases caused by the Omicron variant, we calculated age-standardized case fatality rates (CFR) in patients aged ≥40 years over nine diagnostic periods (3 January to 28 August 2022) in ten Japanese prefectures (14.8 million residents). Among 552,581 study subjects, we found that there were 1836 fatalities during the isolation period (up to 28 days from date of onset). The highest age-standardized CFR (0.85%, 95% confidence interval (CI):0.78-0.92) was observed in cases diagnosed in the second 4-week period (January 31 to February 27), after which it declined significantly up to the 6th 4-week period (0.23%, 95% CI: 0.13-0.33, May 23 to June 19). The CFR then increased again but remained at 0.39% in the eighth period (July 18 to August 28). The CFR in cases with the BA.2 or BA.5 sublineages in the age range 60-80 years was significantly lower than that with BA.1 infections (60 years: 0.19%, 0.02%, 0.053%, respectively; 70 years: 0.91%, 0.33%, 0.39%; ≥80 years: 3.78%, 1.96%, 1.81%, respectively). We conclude that the risk of death in Japanese COVID-19 patients infected with Omicron variants declined through February to mid-June 2022.

Three phlorotannins from Sargassum carpophyllum are effective against the secretion of allergic mediators from antigen-stimulated rat basophilic leukemia cells.

Sargassum carpophyllum (Sargassaceae) is a brown seaweed that contains phlorotannins, which are phloroglucinol polymers with reported anti-inflammatory activities. The phlorotannins 2-[2-(3,5-dihydroxyphenoxy)-3,5-dihydroxyphenoxy]-1,3,5-benzenetriol (1), 2,2'-[[2-(3,5-dihydroxyphenoxy)-5-hydroxy-1,3-phenylene]bis(oxy)]bis(1,3,5-benzenetriol) (2), and 2-[2-[4-[2-(3,5-dihydroxyphenoxy)-3,5-dihydroxyphenoxy]-3,5-dihydroxyphenoxy]-3,5-dihydroxyphenoxy]-1,3,5-benzenetriol (3) were isolated from S. carpophyllum. Here, we evaluated the anti-allergic activities of these compounds and comprehensively explored their effects on intracellular protein levels. Immunoglobulin E-sensitized rat basophilic leukemia cells pretreated with any of these three compounds exhibited reduced ß-hexosaminidase, prostaglandin D2, and tumor necrosis factor-α secretion compared with dinitrophenyl-human serum albumin (DNP-HSA)-stimulated cells. Reduction of ß-hexosaminidase release was dose-dependent but the half-maximal inhibitory concentrations of the compounds were similar (36-51 µM). Proteomics analysis revealed that the three compounds up-regulated 25 proteins and down-regulated 33 proteins compared with DNP-HSA stimulation alone, and slightly suppressed proteasome 5 expression linked to the regulation of IκB. These results demonstrate that these phlorotannins are potentially useful for preventing immediate hypersensitivity. S. carpophyllum may be a functional food.

Shorter Incubation Period among COVID-19 Cases with the BA.1 Omicron Variant.

We aimed to elucidate the range of the incubation period in patients infected with the SARS-CoV-2 Omicron variant in comparison with the Alpha variant. Contact tracing data from three Japanese public health centers (total residents, 1.06 million) collected following the guidelines of the Infectious Diseases Control Law were reviewed for 1589 PCR-confirmed COVID-19 cases diagnosed in January 2022. We identified 77 eligible symptomatic patients for whom the date and setting of transmission were known, in the absence of any other probable routes of transmission. The observed incubation period was 3.03 ± 1.35 days (mean ± SDM). In the log-normal distribution, 5th, 50th and 95th percentile values were 1.3 days (95% CI: 1.0−1.6), 2.8 days (2.5−3.1) and 5.8 days (4.8−7.5), significantly shorter than among the 51 patients with the Alpha variant diagnosed in April and May in 2021 (4.94 days ± 2.19, 2.1 days (1.5−2.7), 4.5 days (4.0−5.1) and 9.6 days (7.4−13.0), p < 0.001). As this incubation period, mainly of sublineage BA.1, is even shorter than that in the Delta variant, it is thought to partially explain the variant replacement occurring in late 2021 to early 2022 in many countries.