Association between severity of dental caries and the risk of stroke.

OBJECTIVE: The relationship between dental caries and stroke remains inconclusive. The aim of this study is to investigate whether different severities of dental caries affect the risk of stroke. METHODS: This retrospective cohort study was conducted using the 2000-2013 Taiwan National Health Insurance Database. We selected 23,662 patients with advanced/severe dental caries and 23,662 patients with incipient/moderate dental caries between 2000 and 2006. These patients were followed to the occurrence of stroke or to the end of the study in 2013. Hazard ratios (HRs) and 95% confidence intervals (CIs) derived from the Cox proportional hazards models were calculated to assess the association between severity of dental caries and the risk of stroke. RESULTS: The advanced/severe dental caries group had a significantly higher risk of stroke compared with incipient/moderate dental caries group (adjusted HR, 1.16; 95% CI, 1.03-1.31). Stratified analyses showed that advanced/severe dental caries was positively associated with the risk of ischemic stroke in patients aged ≥40 years and with the risk of hemorrhagic stroke in patients aged <40 years. CONCLUSION: There is a severity-dependent association between dental caries and stroke in an Asian population.

Autotransplantation or replantation of cryopreserved teeth: a case series and literature review.

BACKGROUND: The aim of this report was to evaluate the outcome of autotransplantation or replantation of cryopreserved teeth clinically and radiographically. Donor teeth were slowly frozen in a controlled-rate freezer using 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) as protectants. Seven cryopreserved teeth, with duration of storage ranging from 4 to 36 months, were autotransplanted or replanted at Niigata University Medical and Dental Hospital. Endodontic treatment involving root canal debridement followed by interim root canal filling with calcium hydroxide was started 3 weeks after the operation and continued with replacement of the calcium hydroxide filling at 2-week to 3-month intervals. Three transplants showed periodontal regeneration clinically and radiographically, whereas replacement root resorption was observed in the remaining transplants. From the results, it can be concluded that cryopreserved tooth autotransplantation has potential for clinical use; however, the risk of replacement root resorption remains.

Keratinocytes of tissue-engineered human oral mucosa promote re-epithelialization after intraoral grafting in athymic mice.

PURPOSE: The objective of this study was to investigate the role of grafted oral keratinocytes in a transplanted ex vivo-produced oral mucosa equivalent (EVPOME) in the regeneration and/or healing process of the oral mucosa at the recipient site. MATERIALS AND METHODS: The EVPOME was developed in a serum-free defined culture system without a feeder layer. EVPOME is composed of a stratified layer of human oral keratinocytes that are seeded onto a human cadaveric dermis, AlloDerm (LifeCell, Branchburg, NJ). Intraorally grafted EVPOMEs in athymic mice (BALB/c) were excised, contiguous with the surrounding oral mucosa, on days 5, 7, 14, and 21 after grafting. Serial sections were stained with hematoxylin-eosin and immunohistochemically analyzed for cytokeratin 17 (CK17) expression to distinguish the human-cultured EVPOME epithelial keratinocytes from murine oral keratinocytes. RESULTS: All EVPOME epithelial cells showed intense immunoreactivity for CK17, whereas mouse buccal mucosal epithelial cells did not show CK17 immunoreactivity. The grafted EVPOME maintained a stratified epithelial layer for up to 5 days after grafting. By day 7 after grafting, a portion of the EVPOME epithelial layer peeled away from the AlloDerm, and a thin, CK17-immunonegative epithelial layer extended from the adjacent thick epithelial layer of the mouse and contacted the CK17-immunopositive EVPOME epithelium. From days 14 to 21 after grafting, the stratification of the CK17-immunonegative continuous mouse epithelium increased compared with earlier time points and showed a similar appearance to the epithelium of the adjacent mouse mucosa. In contrast, no epithelial coverage of the AlloDerm that was grafted without keratinocytes was observed for up to 21 days after grafting. The grafted AlloDerm without cells resulted in tissue necrosis that was accompanied by a dramatic infiltration of inflammatory cells by day 14. CONCLUSIONS: These findings suggest that grafting of EVPOME with viable oral keratinocytes onto an intraoral mucosal wound plays an active role in promotion of re-epithelialization of the oral wound during the subsequent healing process.

Blimp1 is a critical determinant of the germ cell lineage in mice.

Germ cell fate in mice is induced in pluripotent epiblast cells in response to signals from extraembryonic tissues. The specification of approximately 40 founder primordial germ cells and their segregation from somatic neighbours are important events in early development. We have proposed that a critical event during this specification includes repression of a somatic programme that is adopted by neighbouring cells. Here we show that Blimp1 (also known as Prdm1), a known transcriptional repressor, has a critical role in the foundation of the mouse germ cell lineage, as its disruption causes a block early in the process of primordial germ cell formation. Blimp1-deficient mutant embryos form a tight cluster of about 20 primordial germ cell-like cells, which fail to show the characteristic migration, proliferation and consistent repression of homeobox genes that normally accompany specification of primordial germ cells. Furthermore, our genetic lineage-tracing experiments indicate that the Blimp1-positive cells originating from the proximal posterior epiblast cells are indeed the lineage-restricted primordial germ cell precursors.

Birth of parthenogenetic mice that can develop to adulthood.

Only mammals have relinquished parthenogenesis, a means of producing descendants solely from maternal germ cells. Mouse parthenogenetic embryos die by day 10 of gestation. Bi-parental reproduction is necessary because of parent-specific epigenetic modification of the genome during gametogenesis. This leads to unequal expression of imprinted genes from the maternal and paternal alleles. However, there is no direct evidence that genomic imprinting is the only barrier to parthenogenetic development. Here we show the development of a viable parthenogenetic mouse individual from a reconstructed oocyte containing two haploid sets of maternal genome, derived from non-growing and fully grown oocytes. This development was made possible by the appropriate expression of the Igf2 and H19 genes with other imprinted genes, using mutant mice with a 13-kilobase deletion in the H19 gene as non-growing oocytes donors. This full-term development is associated with a marked reduction in aberrantly expressed genes. The parthenote developed to adulthood with the ability to reproduce offspring. These results suggest that paternal imprinting prevents parthenogenesis, ensuring that the paternal contribution is obligatory for the descendant.

An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.

A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles.

Pesticide multiresidue analysis of 303 compounds using supercritical fluid extraction.

Compared to generally used solvent extraction methods, supercritical fluid extraction (SFE) with carbon dioxide has the advantages of automation and simple operation in preparing samples for pesticide residue analysis. This report is the outcome of our evaluation of the practicality of SFE for the preparation of samples for pesticide residue analysis. We studied the recovery of 303 compounds with several crops by a simultaneous analytical method of SFE, cartridge column purification, followed by GC/MS determination. We achieved 70 to 120% recovery for more than 80% of the examined compounds.

Epidermal growth factor receptor gene amplification is correlated with laminin-5 gamma2 chain expression in oral squamous cell carcinoma cell lines.

Both epidermal growth factor receptor (EGFR) gene amplification and laminin (Ln)-5 gamma2 chain overexpression have been reported to be poor prognostic factors in patients with squamous cell carcinoma (SCC) of the head and neck. Here we report our investigation of the relationship between EGFR gene amplification and Ln-5 gamma2 chain expression in seven SCC cell lines, since both epidermal growth factor (EGF) signaling and Ln-5 gamma2 have been reported to be involved in cell motility. The degree of correlation between EGFR gene amplification and Ln-5 gamma2 chain expression was evaluated by Southern and Western blot analyses. EGFR gene amplification was detected in all SCC cell lines at levels 5-50 times those in DNA from normal liver tissue. EGFR gene amplification increased with Ln-5 gamma2 chain protein expression in seven cell lines, showing close correlation between EGFR gene amplification and Ln-5 gamma2 chain protein expression. In order to show the causal relationship, we analyzed the effects of transforming growth factor-alpha (TGF-alpha), tyrosine kinase inhibitor of EGFR, and neutralizing antibody against EGFR, on the expression of Ln-5 gamma2 in these cell lines. In two cell lines in which EGFR gene amplification was low, expression of both protein and mRNA of the Ln-5 gamma2 chain increased in the presence of TGF-alpha, and Ln-5 gamma2 chain expression was inhibited by neutralizing antibody against EGFR. In all cell lines, Ln-5 gamma2 chain expression was inhibited by tyrosine kinase inhibitor which acts selectively on the EGFR signal transduction pathway under the stimulus of TGF-alpha. These results suggest that EGFR gene amplification and the EGFR signaling pathway can act as positive regulators on the induction of the Ln-5 gamma2 chain secreted by tumor cells.

Development of bovine oocytes reconstructed with a nucleus from growing stage oocytes after fertilization in vitro.

The developmental capacity of reconstructed bovine oocytes that contained nuclei from growing stage oocytes, 70-119 microm in diameter, was assessed after fertilization in vitro. Nuclei from growing stage oocytes of adult ovaries were transferred to enucleated, fully grown germinal vesicle (GV) stage oocytes. After culture in vitro, the reconstructed oocytes matured, forming the first polar body and MII plate. To supply the ability to form pronuclei, the resultant MII plate was transferred to enucleated MII oocytes, which were obtained by in vitro culture of cumulus-oocyte complexes. After fertilization in vitro, 11-15% of the reconstructed oocytes developed to morulae and blastocysts. To assess the ability to develop to term, a total of 27 late morulae and blastocysts were transferred to 19 recipient cows. Of the three cows that subsequently became pregnant, one recipient, who received two embryos derived from reconstructed oocytes with a nucleus from oocytes 100 to 109 microm in diameter, continued the pregnancy to Day 278 of gestation. This pregnancy, however, was unexpectedly a triplet pregnancy that included a set of identical twins and resulted in the premature birth of the calves, followed by death from lack of post-parturient treatment. These results show that bovine oocyte genomes are capable of supporting term development before the oocytes grow to their full size, which suggests that growing stage oocytes can be directly used as a source of maternal genomes.

Direct production of gene-targeted mice from ES cells by nuclear transfer and gene transmission to their progeny.

In order to evaluate the usefulness of a cloning technique to produce gene-manipulated mice for the field of laboratory animal science, we produced mice cloned from gene-targeted embryonic stem (ES) cells and examined the vertical transmission of a targeted gene to their progeny. Of 1257 eggs constructed by nuclear transfer using M-phase ES donor cells targeted with an oviduct-specific glycoprotein (OGP) gene, 990 formed a pseudo-pronucleus and a polar body after activation. Of 504 cloned embryos transferred into recipients, 20 live cloned pups (2%) were recovered by Caesarean section at 19.5 days of gestation. Fourteen of these cloned mice were studied. Genotyping of the OGP locus and 20 microsatellite loci showed that they were genetically identical to the OGP gene-targeted TT2 cells. Eight cloned pups grew into adults, of which 7 were male and 1 was female (missing the Y chromosome). Mating experiments using the cloned mice were carried out. Of 89 F1 mice produced from the mating of cloned and C57BL/6J mice, 50 had the targeted OGP gene heterozygously. Thirty-seven F2 mice from 4 pairs of the OGP-/+ mice were composed of 9 OGP-/-, 18 OGP-/+, and 10 OGP+/+. Moreover, 26 offspring of one pair of the cloned mice were composed of 10 OGP-/-, 12 OGP-/+, and 4 OGP+/+. These offspring were fertile and transmitted the mutant OGP gene to the next generation. Comparison of these results with those of germline chimeric mice indicates that gene-targeted mice can be produced at least one generation earlier by nuclear transfer than by the conventional methods. In addition, the targeted OGP gene was constantly transmitted to the progeny of the gene-targeted mice. Cloning techniques are potentially a more efficient way to generate gene-manipulated mice for laboratory animal science, although such techniques include many unresolved problems, such as low production efficiency, and selection of a cell source for gene manipulation among others.

Identification of genes aberrantly expressed in mouse embryonic stem cell-cloned blastocysts.

During development, cloned embryos often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. The long-term effects resulting from embryo cloning procedures would manifest after birth as early death, obesity, various functional disorders, and so forth. Despite extensive studies, the parameters affecting the developmental features of cloned embryos remain unclear. The present study carried out extensive gene expression analysis to screen a cluster of genes aberrantly expressed in embryonic stem cell-cloned blastocysts. Differential screening of cDNA subtraction libraries revealed 224 differentially expressed genes in the cloned blastocysts: eighty-five were identified by the BLAST search as known genes performing a wide range of functions. To confirm their differential expression, quantitative gene expression analyses were performed by real-time PCR using single blastocysts. The genes Skp1a, Canx, Ctsd, Timd2, and Psmc6 were significantly up-regulated, whereas Aqp3, Ak3l1, Rhot1, Sf3b3, Nid1, mt-Rnr2, mt-Nd1, mt-Cytb, and mt-Co2 were significantly down-regulated in the majority of embryonic stem cell-cloned embryos. Our results suggest that an extraordinarily high frequency of multiple functional disorders caused by the aberrant expression of various genes in the blastocyst stage is involved in developmental arrest and various other disorders in cloned embryos.

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells.

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.

Nuclear competence for maturation and pronuclear formation in mouse oocytes.

BACKGROUND: In response to gonadotrophins, a fully grown mouse oocyte matures to the metaphase of the second meiotic division and becomes competent for the development of female and male pronuclei after fertilization. The present study was carried out to clarify when during the growth period an oocyte nucleus acquires the ability to promote pronuclei formation after fertilization. METHODS: Fully grown germinal vesicle (GV) oocytes were enucleated and fused with nuclei from growing oocytes from 1-20 day old mice by standard nuclear transfer technique. The reconstructed oocytes were matured and fertilized in vitro, and pronuclear formation was assessed. RESULTS: The oocytes whose nuclei were exchanged for those of the non-growing-stage oocytes matured to the metaphase of the second meiotic stage, but no normal female pronuclei were formed. Female pronuclei first formed in 27% of the oocytes reconstituted with the nuclei of oocytes from 8 day old pups after fertilization. Recondensed sperm chromatin was detected in 27% of the oocytes reconstructed with oocyte nuclei from 8 day old mice, and a male pronucleus was first formed in 6% of the oocytes that had been reconstructed with the nuclei of oocytes from 15 day old mice. The sizes of the female and male pronuclei increased with oocyte donor age, and reached normal size when the oocytes from 15 and 20 day old mice respectively were used. An electron microscopic study using oocytes that had received the oocyte nuclei of 8 day old mice confirmed these results. CONCLUSION: The factors required for pronuclear formation are derived from fully grown GV oocytes, and the transformation from decondensed sperm chromatin to a recondensed male pronucleus is governed by GV-derived factors.

Rapid replacement of somatic linker histones with the oocyte-specific linker histone H1foo in nuclear transfer.

The most distinctive feature of oocyte-specific linker histones is the specific timing of their expression during embryonic development. In Xenopus nuclear transfer, somatic linker histones in the donor nucleus are replaced with oocyte-specific linker histone B4, leading to the involvement of oocyte-specific linker histones in nuclear reprogramming. We recently have discovered a mouse oocyte-specific linker histone, named H1foo, and demonstrated its expression pattern in normal preimplantation embryos. The present study was undertaken to determine whether the replacement of somatic linker histones with H1foo occurs during the process of mouse nuclear transfer. H1foo was detected in the donor nucleus soon after transplantation. Thereafter, H1foo was restricted to the chromatin in up to two-cell stage embryos. After fusion of an oocyte with a cell expressing GFP (green fluorescent protein)-tagged somatic linker histone H1c, immediate release of H1c in the donor nucleus was observed. In addition, we used fluorescence recovery after photobleaching (FRAP), and found that H1foo is more mobile than H1c in living cells. The greater mobility of H1foo may contribute to its rapid replacement and decreased stability of the embryonic chromatin structure. These results suggest that rapid replacement of H1c with H1foo may play an important role in nuclear remodeling.

Abnormalities in cloned mice are not transmitted to the progeny.

Cloned animals suffer a wide range of severe fetal and placental malformations. Whether these malformations arise from insufficient epigenetic modifications or mutations has not yet been determined. To address this question, we examined siblings from both cloned XO and XY parents. These parents, which exhibited hypertrophic placentas, increased body weights, and open eyelids at birth, were created from the same ES cell sublines. The siblings from all three cloned pairs showed normal body and placenta weights and no open eyelids at birth. The results clearly showed that the phenotypic abnormalities seen in cloned mice were not transmitted to the progeny, a finding that suggests that abnormalities in cloned mice are responsible for insufficient epigenetic modifications/reprogramming.