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.

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.

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.

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.

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.

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.

Validation of a phenol-sulfuric acid method in a microplate format for the quantification of soluble sugars in ruminant feeds.

Soluble sugars in feeds are important for ruminant production; however, performing numerous sugar analyses within a short period is a laborious task. Here, we developed a phenol-sulfuric acid (PSA) assay in a microplate format to quantify soluble sugars in ruminant feeds. This method is easy and quick and requires only a small quantity of harmful reagents. We found that assay measurements were not affected by the representative organic acids and sugar alcohol contained in feeds. The treatment of activated charcoal with ethanol extract prior to the PSA assay was effective in removing interfering compounds for a more accurate determination of soluble sugars in certain feeds. Furthermore, the inter-day and intra-day repeatability of the present method was acceptable. Hence, we conclude that the method developed in this study is suitable for routine analysis of soluble sugars content in ruminant feeds.

Combination of Clptm1L and TMEM207 Expression as a Robust Prognostic Marker in Oral Squamous Cell Carcinoma.

Overexpression of Cleft Lip and Palate Transmembrane 1-Like (Clptm1L) confers cancer cell survival through the endoplasmic reticulum (ER) stress survival signaling pathway, while TMEM207 impairs the tumor suppressor function of WW domain containing oxidoreductase (WWOX), which sensitizes cancer cells to ER stress-induced apoptosis. In the present study, we examined whether these two ER stress-related proteins, Clptm1L and TMEM207, could be prognostic markers in oral squamous cell carcinoma (OSCC). Immunohistochemical staining using specific antibodies to Clptm1L or TMEM207 revealed that 31 of 89 tissue specimens exhibited concomitant expression of Clptm1L and TMEM207 at the cancer invasion front. A Kaplan-Meier plot of the patient survival curve followed by a log-rank test revealed that the coexpression of Clptm1L and TMEM207 was significantly associated with poor outcome in patients with OSCC (P = 0.00252). Coexpression of Clptm1L and TMEM207 was closely related to lymph node metastasis (P=0.000574). Both univariate and multivariate analyses demonstrated that coexpression of Clptm1L and TMEM207 predicted the poor prognosis of the patients with OSCC. The present study indicated that the double positive Clptm1L and TMEM207 immunoreactivity was closely related to lymph node metastasis with prognostic value in patients with 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.

Overexpression of JAG2 is related to poor outcomes in oral squamous cell carcinoma.

OBJECTIVES: JAG2 is one of Notch ligands, which recently appear to exert various carcinogenesis. In the present study, we aimed to unveil the relation of JAG2 expression and clinicopathological features in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: We examined JAG2 expression in OSCC plus adjacent nontumorous epithelia in eight patients. Ninety-one OSCC tissue specimens were immunohistochemically stained with specific antibodies to JAG2. The immunoreactivities of JAG2 were correlated with clinicopathological factors, including the prognosis of patients. Chi-square test, Kaplan-Meier survival, and Cox proportional hazard analysis were used to determine the statistical value of JAG2 expression in OSCC. RESULTS: JAG2 mRNA expression was much expressed in OSCC tissues compared with adjacent tissue specimens in five of eight patients. JAG2 immunoreactivity was found at invasion front in 31 of 91 OSCC. JAG2 immunoreactivity was significantly associated with age, less than 50 years old of patients (P = .048). Kaplan-Meier analysis demonstrated that the patients with JAG2 immunoreactvty have a short overall survival. With the Cox proportional hazard regression mode, the independent factors predictive of poor overall survival included JAG2 immunoreactivity (P < .05). CONCLUSIONS: The present findings suggest that JAG2 overexpression, especially at the cancer invasion front, has potential prognostic value.

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.

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.

Clavicle fracture following neck dissection: imaging features and natural course.

OBJECTIVE: This study aimed to assess the imaging features and natural course of clavicle fracture following neck dissection (ND). METHODS AND MATERIALS: Eight patients with clavicle fracture following ND were included. Because bilateral clavicle fractures occurred in one patient, a total of nine clavicle fractures following ND were assessed using CT images. Pathological fracture due to bone metastasis or local recurrence was carefully ruled out. RESULTS: The time interval from ND to clavicle fracture ranged from 2 to 8 months (median, 4 months). On CT images, all the nine fractures occurred in the proximal clavicles and displayed increased bone marrow density with extraosseous soft-tissue mass formation. Osteolysis of the clavicle was not observed. Intra articular fractures were observed in three (33%), displaced fractures in two (22%), and free bone fragments in three (33%) cases. Further follow-up using CT showed that six (67%) fractures resulted in pseudoarthrosis. For the remaining three (33%) fractures with bone union during follow-up, the time interval between clavicle fracture and bone union ranged from 4 to 16 months (median, 6 months). CONCLUSION: Proximal clavicle fractures, which are rarely observed following ND, always display extraosseous soft-tissue mass formation without osteolysis. They frequently result in pseudoarthrosis; however, occasionally, they also result in bone union within 24 weeks. ADVANCES IN KNOWLEDGE: The location of proximal clavicle, the lack of osteolysis, and the presence of free bone fragments may be the key to diagnosis of clavicle fracture following ND.

Pathobiological role of cleft palate transmembrane protein 1 family proteins in oral squamous cell carcinoma.

PURPOSE: Cleft palate transmembrane protein 1 (Clptm1) and its paralog protein, Cisplatin resistance-related protein 9 (CRR9) constitute a highly conserved protein family, from Caenorhabditis elegans to Homo sapiens. In the present study, we examined the clinicopathological and biological significance of Clptm1 and CRR9 expression in oral squamous cell carcinoma (OSCC). METHODS: Ninety-eight OSCC tissue specimens were immunohistochemically stained with specific antibodies to Clptm1 and CRR9. The immunoreactivity of Clptm1 and CRR9 was then correlated with clinicopathological factors, including the prognosis of patients. siRNA-mediated gene silencing of CRR9 followed by cell proliferation, Matrigel invasion, anoikis assay, and gelatin zymography were performed using cultured OSCC cells. Subsequently, immunohistochemical examination including double staining was performed to determine the correlation between CRR9 and Bcl-xL expression in OSCC cells. RESULTS: Non-tumorous oral squamous cells exhibited vague, weak, or little cytoplasmic staining with anti-Clptm1 and CRR9 antibodies. By contrast, robust Clptm1 and CRR9 immunoreactivity was found at the cancer invasion front in 55 and 54 of the 98 OSCC tissue specimens, respectively. Notably, CRR9 immunoreactivity was associated with more than 5 mm of depth of invasion, poor prognosis of the patients, and smoking habits (P < 0.05). siRNA-mediated gene silencing of CRR9 did not alter the cell proliferation but decreased Matrigel invasion and impaired anoikis resistance in cultured Ca9-22 and SAS cells. CRR9 and anti-apoptotic Bcl-xL expression levels were correlated in pT1 OSCC tissue specimens. CONCLUSIONS: Clptm1 and CRR9 were overexpressed in many OSCC tissues. In particular, CRR9 expression may promote tumor development and have a significant poor prognostic value in OSCC, possibly through conferring invasion ability and resistance to apoptotic stimuli possibly related to Bcl-xL expression. CRR9 could be a novel molecular target for patients with OSCC.

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.

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.

Analysis of functional components and radical scavenging activity of 21 algae species collected from the Japanese coast.

The functional chemical substances and the antioxidant activity of lipids in 21 marine algae along the Japanese coast were investigated. Principal component analysis was performed to detect any correlation between the chemical substances and algae phylum. Chlorophyta contained a high level of ß-carotene. Rhodophyta contained high amounts of cholesterol, ß-sitosterol, and saturated fatty acids. Phaeophyta were rich in fucosterol, α-tocopherol, fucoxanthin, and polyphenol. Phaeophyta algae also showed the highest antioxidant activity compared with other phylum. This suggests that Phaeophyta has the greatest potential to be used as a functional food. Consumption of the beneficial Phaeophyta species, such as Eisenia arborea Areschoug and Ecklonia cava Kjellman should be encouraged as not only as food products but also as nutraceuticals and dietary supplements. These beneficial ingredients should be encouraged to be studied in depth with the possibility to develop specific formulated products target to special consumer's population with added nutritional value.