Clinical use of duplicate complete dentures: A narrative review.

Most reports on duplicate dentures are introduction to fabrication methods or clinical case reports. Only a few studies have verified their clinical effectiveness; hence, evidence to construct useful clinical guidelines for duplicate denture use is lacking. This review aimed to comprehensively investigate reports on duplicate dentures to accumulate evidences that will contribute to the formulation of clinical practice guidelines. Duplicate dentures are effectively used for impression making and bite registration when fabricating new dentures, thereby reducing the number of clinic visits and treatment time. Duplicate denture can also be used as temporary or new dentures. Older people in whom various adaptive abilities have declined, may find it difficult to adjust to new dentures and experience stress, even if the shape is appropriate. Duplicate dentures, which reproduces the shape of old dentures that they are used to, have the advantage of being more familiar to older people and less stressful. When manufacturing duplicate dentures, digital methods such as milling and three-dimensional printing are superior to conventional methods regarding working time and cost. A notable advantage of the digital method is that the denture shape can be saved as digital data, and the denture can be easily duplicated if lost.

iPS cell generation-associated point mutations include many C > T substitutions via different cytosine modification mechanisms.

Genomic aberrations are a critical impediment for the safe medical use of iPSCs and their origin and developmental mechanisms remain unknown. Here we find through WGS analysis of human and mouse iPSC lines that genomic mutations are de novo events and that, in addition to unmodified cytosine base prone to deamination, the DNA methylation sequence CpG represents a significant mutation-prone site. CGI and TSS regions show increased mutations in iPSCs and elevated mutations are observed in retrotransposons, especially in the AluY subfamily. Furthermore, increased cytosine to thymine mutations are observed in differentially methylated regions. These results indicate that in addition to deamination of cytosine, demethylation of methylated cytosine, which plays a central role in genome reprogramming, may act mutagenically during iPSC generation.

Insertion/deletion and microsatellite alteration profiles in induced pluripotent stem cells.

We here demonstrate that microsatellite (MS) alterations are elevated in both mouse and human induced pluripotent stem cells (iPSCs), but importantly we have now identified a type of human iPSC in which these alterations are considerably reduced. We aimed in our present analyses to profile the InDels in iPSC/ntESC genomes, especially in MS regions. To detect somatic de novo mutations in particular, we generated 13 independent reprogramed stem cell lines (11 iPSC and 2 ntESC lines) from an identical parent somatic cell fraction of a C57BL/6 mouse. By using this cell set with an identical genetic background, we could comprehensively detect clone-specific alterations and, importantly, experimentally validate them. The effectiveness of employing sister clones for detecting somatic de novo mutations was thereby demonstrated. We then successfully applied this approach to human iPSCs. Our results require further careful genomic analysis but make an important inroad into solving the issue of genome abnormalities in iPSCs.

Evaluating the long-term effect of space radiation on the reproductive normality of mammalian sperm preserved on the International Space Station.

Space radiation may cause DNA damage to cells and concern for the inheritance of mutations in offspring after deep space exploration. However, there is no way to study the long-term effects of space radiation using biological materials. Here, we developed a method to evaluate the biological effect of space radiation and examined the reproductive potential of mouse freeze-dried spermatozoa stored on the International Space Station (ISS) for the longest period in biological research. The space radiation did not affect sperm DNA or fertility after preservation on ISS, and many genetically normal offspring were obtained without reducing the success rate compared to the ground-preserved control. The results of ground x-ray experiments showed that sperm can be stored for more than 200 years in space. These results suggest that the effect of deep space radiation on mammalian reproduction can be evaluated using spermatozoa, even without being monitored by astronauts in Gateway.

In situ delivery of iPSC-derived dendritic cells with local radiotherapy generates systemic antitumor immunity and potentiates PD-L1 blockade in preclinical poorly immunogenic tumor models.

BACKGROUND: Dendritic cells (DCs) are a promising therapeutic target in cancer immunotherapy given their ability to prime antigen-specific T cells, and initiate antitumor immune response. A major obstacle for DC-based immunotherapy is the difficulty to obtain a sufficient number of functional DCs. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSCs); however, therapeutic strategies to engage iPSC-derived DCs (iPSC-DCs) into cancer immunotherapy remain to be elucidated. Accumulating evidence showing that induction of tumor-residing DCs enhances immunomodulatory effect of radiotherapy (RT) prompted us to investigate antitumor efficacy of combining intratumoral administration of iPSC-DCs with local RT. METHODS: Mouse iPSCs were differentiated to iPSC-DCs on OP9 stromal cells expressing the notch ligand delta-like 1 in the presence of granulocyte macrophage colony-stimulating factor. Phenotype and the capacities of iPSC-DCs to traffic tumor-draining lymph nodes (TdLNs) and prime antigen-specific T cells were evaluated by flow cytometry and imaging flow cytometry. Antitumor efficacy of intratumoral injection of iPSC-DCs and RT was tested in syngeneic orthotopic mouse tumor models resistant to anti-PD-1 ligand 1 (PD-L1) therapy. RESULTS: Mouse iPSC-DCs phenotypically resembled conventional type 2 DCs, and had a capacity to promote activation, proliferation and effector differentiation of antigen-specific CD8+ T cells in the presence of the cognate antigen in vitro. Combination of in situ administration of iPSC-DCs and RT facilitated the priming of tumor-specific CD8+ T cells, and synergistically delayed the growth of not only the treated tumor but also the distant non-irradiated tumors. Mechanistically, RT enhanced trafficking of intratumorally injected iPSC-DCs to the TdLN, upregulated CD40 expression, and increased the frequency of DC/CD8+ T cell aggregates. Phenotypic analysis of tumor-infiltrating CD8+ T cells and myeloid cells revealed an increase of stem-like Slamf6+ TIM3- CD8+ T cells and PD-L1 expression in tumor-associated macrophages and DCs. Consequently, combined therapy rendered poorly immunogenic tumors responsive to anti-PD-L1 therapy along with the development of tumor-specific immunological memory. CONCLUSIONS: Our findings illustrate the translational potential of iPSC-DCs, and identify the therapeutic efficacy of a combinatorial platform to engage them for overcoming resistance to anti-PD-L1 therapy in poorly immunogenic tumors.

G9a is involved in the regulation of cranial bone formation through activation of Runx2 function during development.

The methyltransferase G9a was originally isolated as a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9) to a dimethylated state (H3K9me2). Recent studies have revealed that G9a has multiple functions in various cells, including osteoblasts. Here, we investigated G9a function during cranial bone formation. Crossing Sox9-cre with G9aflox/flox (fl/fl) mice generated conditional knockout mice lacking G9a expression in Sox9-positive neural crest-derived bone cells. Sox9-Cre/G9afl/fl mice showed severe hypo-mineralization of cranial vault bones, including defects in nasal, frontal, and parietal bones with opened fontanelles. Cell proliferation was inhibited in G9a-deleted calvarial bone tissues. Expression levels of bone marker genes, i.e., alkaline phosphatase and osteocalcin, were suppressed, whereas Runx2 expression was not significantly decreased in those tissues. In vitro experiments using G9a-deleted calvarial osteoblasts showed decreased cell proliferation after G9a deletion. In G9a-deleted osteoblasts, expression levels of fibroblast growth factor receptors and several cyclins were suppressed. Moreover, the expression of bone marker genes was decreased, whereas Runx2 expression was not altered by G9a deletion in vitro. G9a enhanced the transcriptional activity of Runx2, whereas siRNA targeting G9a inhibited the transcriptional activity of Runx2 in C3H10T1/2 mesenchymal cells. We confirmed the direct association of endogenous Runx2 with G9a. Chromatin immunoprecipitation experiments showed that G9a bound to Runx2-target regions in promoters in primary osteoblasts. Furthermore, Runx2 binding to the osteocalcin promoter was abrogated in G9-deleted osteoblasts. These results suggest that G9a regulates proliferation and differentiation of cranial bone cells through binding to and activating Runx2.

Genetic aberrations in iPSCs are introduced by a transient G1/S cell cycle checkpoint deficiency.

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions.

Hotspots of De Novo Point Mutations in Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) are generated by direct reprogramming of somatic cells and hold great promise for novel therapies. However, several studies have reported genetic variations in iPSC genomes. Here, we investigated point mutations identified by whole-genome sequencing in mouse and human iPSCs in the context of epigenetic status. In contrast to disease-causing single-nucleotide polymorphisms, de novo point mutations introduced during reprogramming were underrepresented in protein-coding genes and in open chromatin regions, including transcription factor binding sites. Instead, these mutations occurred preferentially in structurally condensed lamina-associated heterochromatic domains, suggesting that chromatin organization is a factor that can bias the regional mutation rate in iPSC genomes. Mutation signature analysis implicated oxidative stress associated with reprogramming as a likely cause of point mutations. Altogether, our study provides deeper understanding of the mutational landscape of iPSC genomes, paving an important way toward the translation of iPSC-based cell therapy.

Low- and High-LET Ionizing Radiation Induces Delayed Homologous Recombination that Persists for Two Weeks before Resolving.

Genome instability is a hallmark of cancer cells and dysregulation or defects in DNA repair pathways cause genome instability and are linked to inherited cancer predisposition syndromes. Ionizing radiation can cause immediate effects such as mutation or cell death, observed within hours or a few days after irradiation. Ionizing radiation also induces delayed effects many cell generations after irradiation. Delayed effects include hypermutation, hyper-homologous recombination, chromosome instability and reduced clonogenic survival (delayed death). Delayed hyperrecombination (DHR) is mechanistically distinct from delayed chromosomal instability and delayed death. Using a green fluorescent protein (GFP) direct repeat homologous recombination system, time-lapse microscopy and colony-based assays, we demonstrate that DHR increases several-fold in response to low-LET X rays and high-LET carbon-ion radiation. Time-lapse analyses of DHR revealed two classes of recombinants not detected in colony-based assays, including cells that recombined and then senesced or died. With both low- and high-LET radiation, DHR was evident during the first two weeks postirradiation, but resolved to background levels during the third week. The results indicate that the risk of radiation-induced genome destabilization via DHR is time limited, and suggest that there is little or no additional risk of radiation-induced genome instability mediated by DHR with high-LET radiation compared to low-LET radiation.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196.

Restoration of ATM Expression in DNA-PKcs-Deficient Cells Inhibits Signal End Joining.

Unlike most DNA-dependent protein kinase, catalytic subunit (DNA-PKcs)-deficient mouse cell strains, we show in the present study that targeted deletion of DNA-PKcs in two different human cell lines abrogates VDJ signal end joining in episomal assays. Although the mechanism is not well defined, DNA-PKcs deficiency results in spontaneous reduction of ATM expression in many cultured cell lines (including those examined in this study) and in DNA-PKcs-deficient mice. We considered that varying loss of ATM expression might explain differences in signal end joining in different cell strains and animal models, and we investigated the impact of ATM and/or DNA-PKcs loss on VDJ recombination in cultured human and rodent cell strains. To our surprise, in DNA-PKcs-deficient mouse cell strains that are proficient in signal end joining, restoration of ATM expression markedly inhibits signal end joining. In contrast, in DNA-PKcs-deficient cells that are deficient in signal end joining, complete loss of ATM enhances signal (but not coding) joint formation. We propose that ATM facilitates restriction of signal ends to the classical nonhomologous end-joining pathway.

Generation of mouse pluripotent stem cell-derived proliferating myeloid cells as an unlimited source of functional antigen-presenting cells.

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow-derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2K(b)-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs.

Suppression of production of baboon endogenous virus by dominant negative mutants of cellular factors involved in multivesicular body sorting pathway.

Baboon endogenous virus (BaEV) is an infectious endogenous gammaretrovirus isolated from a baboon placenta. BaEV-related sequences have been identified in both Old World monkeys and African apes, but not in humans or Asian apes. Recently, it was reported that BaEV-like particles were produced from Vero cells derived from African green monkeys by chemical induction, and thus BaEV-like particles may contaminate biological products manufactured using Vero cells. In this study, we constructed an infectious molecular clone of BaEV strain M7. We found two putative L-domain motifs, PPPY and PSAP, in the pp15 region of Gag. To examine the function of the L-domain motifs, we conducted virus budding assay using L-domain motif mutants. We revealed that the PPPY motif, but not the PSAP motif, plays a major role as the L-domain in BaEV budding. We also demonstrated that Vps4A/B are involved in BaEV budding. These data suggest that BaEV Gag recruits the cellular endosomal sorting complex required for transport (ESCRT) machinery through the interaction of the PPPY L-domain with cellular factors. These data will be useful for controlling contamination of BaEV-like particles in biological products in the future.

Inhibition of budding/release of porcine endogenous retrovirus.

PERV is integrated into the genome of all pigs. PERV-A and PERV-B are polytropic and can productively infect human cell lines, whereas PERV-C is ecotropic. Recombinant PERV-A/C can infect human cells and exhibits high titer replication. Therefore, use of pigs for human xenotransplantation raises concerns about the risks of transfer of this infectious agent from donors to xenotransplantation recipients. To establish strategies to inhibit PERV production from cells, in the present study, we investigated the mechanism of PERV budding and anti-PERV activity of Tetherin/BST-2. The results showed that DN mutants of WWP-2, Tsg101, and Vps4A/B markedly reduced PERV production in human and porcine cell lines, suggesting that PERV budding uses these cellular factors and the cellular MVB sorting pathway as well as many other retroviruses. Moreover, PERV production was also reduced by human and porcine Tetherin/BST-2. These data are useful for developing strategies to inhibit PERV production and may reduce the risk of PERV infection in xenotransplantation.

Induced pluripotent stem cell generation-associated point mutations arise during the initial stages of the conversion of these cells.

A large number of point mutations have been identified in induced pluripotent stem cell (iPSC) genomes to date. Whether these mutations are associated with iPSC generation is an important and controversial issue. In this study, we approached this critical issue in different ways, including an assessment of iPSCs versus embryonic stem cells (ESCs), and an investigation of variant allele frequencies and the heterogeneity of point mutations within a single iPSC clone. Through these analyses, we obtained strong evidence that iPSC-generation-associated point mutations occur frequently in a transversion-predominant manner just after the onset of cell lineage conversion. The heterogeneity of the point mutation profiles within an iPSC clone was also revealed and reflects the history of the emergence of each mutation. Further, our results suggest a possible approach for establishing iPSCs with fewer point mutations.

Identification of cellular factors required for the budding of koala retrovirus.

Koala retrovirus (KoRV) is a unique gammaretrovirus that is currently endogenizing into its host and considered to be associated with leukemia, lymphoma and immunosuppression in koalas (Phascolactos cinereus). In this study, it was demonstrated that WWP2 or WWP2-like E3 ubiquitin ligases possessing the WW domain closely related to WWP2 and Vps4A/B are involved in KoRV budding. These data suggest that KoRV Gag recruits the cellular endosomal sorting complex required for transport machinery through interaction of the PPPY L-domain with the WW domain(s) of WWP2 and that progeny virions are released from cells by utilizing the multivesicular body sorting pathway.

Investigation of transcriptional responses of juvenile mouse bone marrow to power frequency magnetic fields.

To seek alterations in gene transcription in bone marrow cells following in vivo exposure of juvenile mice to power frequency magnetic fields, young (21-24-day old) C57BL/6 mice were exposed to a 100µT 50Hz magnetic field for 2h. Transcription was analysed by three methods, High Coverage Expression Profiling (HiCEP), Illumina microarrays and quantitative real-time polymerase chain reaction (QRT-PCR). A pilot HiCEP experiment with 6 exposed (E) and 6 non-exposed (NE) mice identified four candidate responsive transcripts (two unknown transcripts (AK152075 and F10-NED), phosphatidylinositol binding clathrin assembly protein (Picalm) and exportin 7 (Xpo7)). A larger experiment compared 19 E and 15 NE mice using two independent QRT-PCR assays and repeated microarray assays. No significant field-dependent changes were seen, although Picalm showed a trend to significance in one QRT-PCR assay (E/NE=0.91; P=0.06). However, the study was underpowered to detect an effect of this magnitude (52% power at P=0.05). These data indicate the current experimental constraints in detecting small changes in transcription that may occur in response to magnetic fields. These constraints result from technical limitations in the accuracy of assays and biological variation, which together were sufficient to account statistically for the number of differentially expressed transcripts identified in the pilot experiment.