Salivary microbiome in children with Down syndrome: a case-control study.

BACKGROUND: Down syndrome (DS), a most frequently occurring genetic disorder, is associated with oral morphological abnormalities and higher incidence rates of oral diseases. Recent studies have analyzed the oral microbiome to elucidate their relationships with oral diseases and general health; however, reports on the oral microbiome in individuals with DS are scarce. This study aimed to characterize the oral microbiome in children with DS. METHODS: A total of 54 children aged 1-13 years were enrolled in this case-control study. Of these children, 27 had DS (Case: DS group) and 27 were age-matched healthy children (Control: ND group). Saliva in the oral cavity was collected with a swab, cultured, and tested for cariogenic and periodontopathic bacteria by quantitative polymerase chain reaction (qPCR) detection, and the salivary microbiome was analyzed using next-generation sequencing. The student's t-test, Fisher's exact test, Mann-Whitney U test, and permutational multivariate analysis of variance were used for statistical analysis. RESULTS: Results of culture and qPCR detection tests for cariogenic and periodontopathic bacteria showed no significant differences in the detected bacteria between the DS and ND groups, with the exception of a significantly higher detection rate of Candida albicans in children with DS with mixed dentition. A comparison of the salivary microbiomes by 16S sequencing showed no significant difference in α diversity; however, it showed a significant difference in ß diversity. Children with DS had a higher relative abundance of Corynebacterium and Cardiobacterium, and lower relative abundance of TM7. CONCLUSIONS: This study provided basic data on the salivary microbiome of children with DS and showed the microbiological markers peculiar to children with DS. However, further research to identify the relationship with oral diseases is warranted.

[Concept analysis of "Gojo" (mutual help)].

Objective　The purpose of this study is to redefine "Gojo" (mutual help) among residents and explore measures to develop it Gojo in the integrated community care system. In 2000, long-term care insurance accelerated Japan's movement toward an aging society, thus increasing the importance of Gojo. This study aims to clarify the conceptual definition it has come to acquire in recent years and explore ways it can be developed in comprehensive community systems.Methodology　Rodger's evolutionary concept analysis was utilized in the study. A keyword research was done using the term "Gojo" in the Japan Medical Abstracts Society (Ichushi Web) and CiNii Articles databases. The search period was from 2000 to 2016 (search date: August 30, 2016) and the database search formula was set to "Gojo/TA". Thirty documents were selected through random sampling and a total of 32 documents were selected for analysis, including landmark documents. A qualitative analysis was conducted on three frameworks: attributes (i.e., characteristics of Gojo), antecedents (i.e., factors affecting Gojo), and consequences (i.e., expected results for Gojo). These three frameworks represent how Gojo is used, the events occurring before it, and the events resulting from it, respectively.Results　The characteristics of Gojo were found to include "experiences of empathy toward residents' daily need for support," "awareness about the residents' efforts to support each other," and "interaction among residents who support other's daily needs." The factors affecting Gojo were identified to be "the need for daily life support beyond what self-help, social solidarity care, and governmental care can provide"; "interchange among residents"; "sharing needs for daily life support among residents"; and "a resident-led public system that promotes mutual support." Gojo's expected results were "a solution for residents' needs of daily support"; "promotion of self-help awareness"; "providing them with a role to enhance their purpose in life"; and "encouraging exchanges and relations between them."Conclusion　Gojo was defined as "the interaction between local residents who support their daily needs, have an empathic experience of the need for daily life support, and have the desire to support each other." In addition, the following points were suggested as measures necessary to expand Gojo. The residents must have sympathy for other residents who need daily life support, understand the benefits of supporting each other, and have a public support system, even if Gojo is led by the residents.

[Characteristics and challenges of health promotion volunteer organizations in Japan: Findings from a national municipality survey].

Objective　Various types of health promotion organizations exist in Japan, including volunteer organizations that promote healthy diets, health improvement, and maternal and child health. Health promotion volunteers, trained and recruited by municipalities, provide counseling and education on various health concerns to community members. A previous study on the effect of these activities and the organizational challenges demonstrated a decrease in the number of volunteer members. The present study aimed to identify the organizational characteristics and challenges of health promotion volunteer work in Japan to gain insight into volunteer recruitment and organizational management.Methods　A questionnaire was sent to 1,873 Japanese municipalities via e-mail or postal mail (excluding Tokyo's 23 wards). For cities of cabinet order, a questionnaire was sent to each borough in the municipality. Data were collected from February to March 2017. The survey was used to collect data on four types of health promotion volunteer organizations: healthy diet (organizations of volunteers who were shokuseikatsu kaizen suishinin), health improvement (organizations of volunteers who were kenkozukuri suishinin), and two types promoting maternal and child health (organizations of volunteers who were boshihoken suishinin and aiikuhan). We asked about the presence or absence of the organization type, year of establishment, number of members, the largest age group, recruitment methods, etc. Twelve organizational challenges were measured with a six-item Likert scale (from 1="strongly agree" to 6="strongly disagree"). Proportions of active members in the volunteer organization ranged from 0% to 10%.Results　Eight hundred eight municipalities responded to the survey (valid responses: 805, valid response rate: 43.1%). The presence or absence of the four types of volunteer organizations differed among the municipalities. The most common type was organizations of volunteers who were shokuseikatsu kaizen suishinin (84.7% municipalities), followed by kenkozukuri suishinin (64.3%), boshihoken suishinin (26.4%), and aiikuhan (10.1%). The total ratio of responses of "strongly agree," "agree," and "somewhat agree" about the organizational challenges, including "The organization cannot find new members easily" and "The participants are always the same people," was commonly more than 50% for the four types of volunteer organizations. The challenges, including "Many do not enjoy what they do," "For many volunteers, participation is limited due to work and family obligations," and "The goal of the activities is not widely shared among volunteers," were significantly correlated to the proportion of active members in the volunteer organization for all four types of organizations.Conclusion　This study revealed that organizational characteristics differed among the four types of health promotion volunteer organizations; however, many of the organizational challenges were found to be common throughout Japanese municipalities.

Poly(ethylene glycol) Crowding as Critical Factor To Determine pDNA Packaging Scheme into Polyplex Micelles for Enhanced Gene Expression.

A critical role of polyethylene glycol (PEG) crowding in the packaging of plasmid DNA (pDNA) into polyplex micelles (PMs) was investigated using a series of PEG-b-poly(l-lysine) (PEG-PLys) block copolymers with varying molecular weights of both PEG and PLys segments. Rod-shaped PMs preferentially formed when the tethered PEG chains covering pDNA in a precondensed state were dense enough to overlap one another (reduced tethering density (RTD) > 1), whereas globular PMs were obtained when they were not overlapped (RTD < 1). These results submitted a scheme that steric repulsive effect of PEG regulated packaging pathways of pDNA either through folding into rod-shape or collapsing into globular depending on whether the PEG chains are overlapped or not. The rod-shaped PMs gave significantly higher gene expression efficacies in a cell-free system compared to the globular PMs, demonstrating the practical relevance of regulating packaging structure of pDNA for developing efficient gene delivery systems.

Rod-to-Globule Transition of pDNA/PEG-Poly(l-Lysine) Polyplex Micelles Induced by a Collapsed Balance Between DNA Rigidity and PEG Crowdedness.

The role of poly(ethylene-glycol) (PEG) in rod-shaped polyplex micelle structures, having a characteristic core of folded plasmid DNA (pDNA) and a shell of tethered PEG chains, is investigated using PEG-detachable polyplex micelles. Rod shapes undergo change to compacted globule shapes by removal of PEG from polyplex micelles prepared from block copolymer with acid-labile linkage between PEG and poly(l-lysine) (PLys) through exposure to acidic milieu. This structural change supports the previous investigation on the rod shapes that PEG shell prevents the DNA structure from being globule shaped as the most favored structure in minimizing surface area. Noteworthy, despite the PEG is continuously depleted, the structural change does not occur in gradual shortening manner but the rod shapes keep their length unchanged and abruptly transform into globule shapes. Analysis of PEG density reveals the transition occurred when tethered PEG of rod shapes has decreased to a critical crowdedness, i.e., discontacted with neighboring PEG, which eventually illuminates another contribution, rigidity of DNA packaged as bundle in the rod shapes, in addition to the steric repulsion of PEG, in sustaining rod shapes. This investigation affirms significant role of PEG and also DNA rigidity as bundle in the formation of rod-shaped structures enduring the quest of compaction of charge-neutralized DNA in the polyplex micelles.

Targeted decationized polyplexes for siRNA delivery.

The applicability of small interfering RNA (siRNA) in future therapies depends on the availability of safe and efficient carrier systems. Ideally, siRNA delivery requires a system that is stable in the circulation but upon specific uptake into target cells can rapidly release its cargo into the cytoplasm. Previously, we evaluated a novel generation of carrier systems ("decationized" polyplexes) for DNA delivery, and it was shown that folate targeted decationized polyplexes had an excellent safety profile and showed intracellular triggered release upon cell specific uptake. Targeted decationized polyplexes consist of a core of disulfide cross-linked poly(hydroxypropyl methacrylamide) (pHPMA) stably entrapping nucleic acids and a shell of poly(ethylene glycol) (PEG) decorated with folate molecules. In the present study, the applicability of folate targeted decationized polyplexes for siRNA delivery was investigated. This required optimization of the carrier system particularly regarding the cross-linking density of the core of the polyplexes. Stable and nanosized siRNA decationized polyplexes were successfully prepared by optimizing the cross-link density of their core. Upon incubation in human plasma, a significant portion of siRNA remained entrapped in the decationized polyplexes as determined by fluorescence correlation spectroscopy (FCS). When tested in a folate receptor overexpressing cell line stably expressing luciferase, Skov3-luc, sequence specific gene silencing was observed. As expected, neither interference on the intrinsic luciferase expression nor on the cell metabolic activity (determined by XTT) was induced by the free-polymer or the siRNA polyplexes. In conclusion, targeted decationized polyplexes are safe and stable carriers that interact with the targeted cells and rapidly disassemble upon cell entry making them promising siRNA delivery systems.

Toroidal Packaging of pDNA into Block Ionomer Micelles Exerting Promoted in Vivo Gene Expression.

Selectively spooling single plasmid DNA (pDNA), as a giant polyelectrolyte, into a nanosized toroidal structure or folding it into a rod-like structure has been accomplished by polyion complexation with block catiomers to form polymeric micelles in varying NaCl concentrations. The interactive potency between the pDNA and block catiomers was determined to play a critical role in defining the ultimate structure of the pDNA; the formation of toroidal or rod-like structures was achieved by complexation in 600 or 0 mM NaCl solutions, respectively. Compared with the rod-like structure, the toroidal structure possessed superior biological functions capable not only of elevating in vitro transcription but also of elevating in vivo gene transduction efficiency. This demonstrated the great utility of the toroidal pDNA packaging as a distinct structured gene carrier. Furthermore, the fact that the NaCl concentration at which the toroidal structure was specifically formed corresponds to seawater stimulates interest in this ordered nanostructure as a possible inherent structure for DNA.

A tadpole-shaped gene carrier with distinct phase segregation in a ternary polymeric micelle.

A distinct tadpole-shaped nanostructure characterized by a spherical head and an extended shaft was identified in a single plasmid DNA (pDNA)-based polymeric micelle. The tadpole-shaped structure was constructed by adding anionic chondroitin sulfate (CS) to the rod-shaped polyplex micelle containing a single pDNA molecule packaged by the PEG-polycation block copolymer through their electrostatic self-assembly. The complex consequently developed a novel structure composed of segregated domains of the CS-rich inflated head and CS-poor folded DNA tail. Hence, this tadpole structure can be regarded as evidence that distinct phase segregation occurred in a single polymeric micelle containing pDNA.

Comparison of oral microbiome profiles in 18-month-old infants and their parents.

The onset and progress of dental caries and periodontal disease is associated with the oral microbiome. Therefore, it is important to understand the factors that influence oral microbiome formation. One of the factors that influence oral microbiome formation is the transmission of oral bacteria from parents. However, it remains unclear when the transmission begins, and the difference in contributions of father and mother. Here, we focused on the oral microbiome of 18-month-old infants, at which age deciduous dentition is formed and the oral microbiome is likely to become stable, with that of their parents. We collected saliva from forty 18-month-old infants and their parents and compared the diversity and composition of the microbiome using next-generation sequencing of 16S rRNA genes. The results showed that microbial diversity in infants was significantly lower than that in parents and composition of microbiome were significantly different between infants and parents. Meanwhile, the microbiome of the infants was more similar to that of their mothers than unrelated adults. The bacteria highly shared between infants and parents included not only commensal bacteria but also disease related bacteria. These results suggested that the oral microbiome of the parents influences that of their children aged < 18 months.

Post-cardiotomy venovenous extracorporeal membrane oxygenation without heparinization.

We present the cases of eight patients (mean age 75 years; EuroSCORE II 17.0 ± 22.0) who underwent post-cardiotomy venovenous extracorporeal membrane oxygenation (ECMO) without heparinization due to serious bleeding. Three liver cirrhosis, two chronic hemodialysis, three redo sternotomy, and two urgent surgery cases were included. Respiratory ECMO Survival Prediction score was - 5.1 ± 4.2 (estimated survival rate: approximately 30%). Mean ECMO duration was 14 days with 9 circuit exchanges. Five patients were weaned from ECMO and three were discharged alive at 90 days (survival 37.5%). There was a case of pump-head thrombosis requiring urgent circuit exchange. All experienced bleeding complications without clinically apparent pulmonary thromboembolism. Disseminated Intravascular Coagulation scores (Pre 1.3 ± 0.8 vs. Post 3.8 ± 1.7; p < 0.05) significantly increased (N = 6). Post-cardiotomy ECMO without heparinization facilitated patient rescue at a reasonable survival rate. However, bleeding complications were still observed. More sophisticated management protocols are warranted.

Effect of shear stress on structure and function of polyplex micelles from poly(ethylene glycol)-poly(l-lysine) block copolymers as systemic gene delivery carrier.

Structural stability of polyplex micelles (PMs), prepared from plasmid DNA (pDNA) and poly(ethylene glycol)-b-poly(l-lysine) block catiomer (PEG-PLys), was evaluated in terms of their resistance against shear stress. When exposed to shear stress at magnitudes typically present in the blood stream, structural deterioration was observed in PMs owing to the partial removal of PEG-PLys strands. Eventually, impaired PEG coverage of the polyplex core led to accelerated degradation by nucleases, implying that structural deterioration by shear stress in blood stream may be a major cause of rapid clearance of PMs from blood circulation. To address this issue, introduction of disulfide crosslinking into the PM core was shown to be an efficient strategy, which successfully mitigated unfavorable effects of shear stress. Furthermore, improved in vivo blood retention profile and subsequently enhanced antitumor efficacy in systemic treatment of pancreatic adenocarcinoma were confirmed for the crosslinked PMs loaded with pDNA encoding an anti-angiogenic protein, suggesting that high stability under the shear stress during blood circulation may be a critical factor in systemically applicable gene delivery systems.

Polyplex micelle installing intracellular self-processing functionalities without free catiomers for safe and efficient systemic gene therapy through tumor vasculature targeting.

Both efficiency and safety profiles are crucial for promotion of gene delivery systems towards practical applications. A promising template system was previously developed based on block catiomer of poly(ethylene glycol) (PEG)-b-poly{N'-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide}-cholesteryl [PEG-PAsp(DET)-cholesteryl] with strategies of ligand conjugation at the α-terminus for specific affinity to the targeted cells and cholesteryl conjugation at the ω-terminus for structural stabilization to obtain systemic retention. Aiming for advocating this formulation towards practical applications, in the current study, the binding profile of this polymer to plasmid DNA (pDNA) was carefully studied to address an issue of toxicity origin. Quantification of free polymer composition confirmed that the toxicity mainly results from unbound polymer and polyplex micelle itself has negligible toxicity. This evaluation allowed for identifying an optimal condition to prepare safe polyplex micelles for systemic application that possess maximal polymer-binding but exclude free polymers. The identified polyplex micelles then faced a drawback of limited transfection efficiency due to the absence of free polymer, which is an acknowledged tendency found in various synthetic gene carriers. Thus, series of functional components was strategically compiled to improve the transfection efficiency such as attachment of cyclic (Arg-Gly-Asp) (cRGD) peptide as a ligand onto the polyplex micelles to facilitate cellular uptake, use of endosome membrane disruptive catiomer of PAsp(DET) for facilitating endosome escape along with use of the conjugated cholesteryl group to amplify the effect of PAsp(DET) on membrane disruption, so as to obtain efficient transfection. The mechanistic investigation respecting the appreciated pH dependent protonation behavior of PAsp(DET) permitted to depict an intriguing scenario how the block catiomers manage to escape from the endosome entrapment in response to the pH gradient. Subsequent systemic application to the pancreatic tumor demonstrated a capability of vascular targeting mediated by the cRGD ligand, which was directly confirmed based on in situ confocal laser scanning microscopy observation. Encouraging this result, the vascular targeting to transfect a secretable anti-angiogenic gene was attempted to treat the intractable pancreatic tumor with anticipation that the strategy could circumvent the intrinsic physiological barriers derived from hypovascular and fibrotic characters. The obtained therapeutic efficiency demonstrates promising utilities of the proposed formulation as a safe systemic gene delivery carrier in practical use.

Systemic delivery of messenger RNA for the treatment of pancreatic cancer using polyplex nanomicelles with a cholesterol moiety.

Systemic delivery of messenger RNA (mRNA) is technically challenging because mRNA is highly susceptible to enzymatic degradation in the blood circulation. In this study, we used a nanomicelle-based platform, prepared from mRNA and poly(ethylene glycol) (PEG)-polycation block copolymers. A cholesterol (Chol) moiety was attached to the ω-terminus of the block copolymer to increase the stability of the nanomicelle by hydrophobic interaction. After in vitro screening, polyaspartamide with four aminoethylene repeats in its side chain (PAsp(TEP)) was selected as the cationic segment of the block copolymer, because it contributes to enhance nuclease resistance and high protein expression from the mRNA. After intravenous injection, PEG-PAsp(TEP)-Chol nanomicelles showed significantly enhanced blood retention of mRNA in comparison to nanomicelles without Chol. We used the nanomicelles for treating intractable pancreatic cancer in a subcutaneous inoculation mouse model through the delivery of mRNA encoding an anti-angiogenic protein (sFlt-1). PEG-PAsp(TEP)-Chol nanomicelles generated efficient protein expression from the delivered mRNA in tumor tissue, resulting in remarkable inhibition of the tumor growth, whereas nanomicelles without Chol failed to show a detectable therapeutic effect. In conclusion, the stabilized nanomicelle system led to the successful systemic delivery of mRNA in therapeutic application, holding great promise for the treatment of various diseases.

Anti-tumor activity of fucoidan is mediated by nitric oxide released from macrophages.

Fucoidan, a sulfated polysaccharide, has significant cytotoxic activity against tumor cells; however, the mechanism(s) of this action remains poorly understood. The present study was designed to determine the in vitro and in vivo effects of fucoidan and their molecular mechanisms. Fucoidan from Cladosiphon okamuranus Tokida cultivated in Okinawa, Japan, delayed tumor growth in Sarcoma 180 (S-180)-bearing mice. However, it failed to inhibit S-180 cell growth in vitro. Activated macrophages are known to have anti-tumor effects. Murine RAW264.7 macrophages stimulated with fucoidan exerted cytotoxicity towards S-180 cells in vitro. This cytotoxicity was associated with nitric oxide (NO) production. Both cytocidal effect and NO production were significantly inhibited by L-NAME, an inhibitor of NO synthase (NOS). Furthermore, activation of nuclear factor-κB was a key step in the transcriptional activation of the inducible NOS gene. Taken together, our results indicate that the anti-tumor activity of fucoidan on S-180 cells is mediated through increased NO production by fucoidan-stimulated macrophages via nuclear factor-κB-dependent signaling pathway.