Induction of somatic cell haploidy by premature cell division.

Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of premature cell division, where nonreplicated, G0/G1-stage somatic cell nuclei are transplanted to the metaphase cytoplasm of mouse oocytes. Subsequent cell division generates daughter cells with reduced ploidy. Unexpectedly, genome sequencing analysis revealed proper segregation of homologous chromosomes, resulting in complete haploid genomes. We observed a high occurrence of somatic genome haploidization in nuclei from inbred genetic backgrounds but not in hybrids, emphasizing the importance of sequence homology between homologs. These findings suggest that premature cell division relies on mechanisms similar to meiosis I, where genome haploidization is facilitated by homologous chromosome interactions, recognition, and pairing. Unlike meiosis, no evidence of recombination between somatic cell homologs was detected. Our study offers an alternative in vitro gametogenesis approach by directly reprogramming diploid somatic cells into haploid oocytes.

Distinct longevity mechanisms across and within species and their association with aging.

Lifespan varies within and across species, but the general principles of its control remain unclear. Here, we conducted multi-tissue RNA-seq analyses across 41 mammalian species, identifying longevity signatures and examining their relationship with transcriptomic biomarkers of aging and established lifespan-extending interventions. An integrative analysis uncovered shared longevity mechanisms within and across species, including downregulated Igf1 and upregulated mitochondrial translation genes, and unique features, such as distinct regulation of the innate immune response and cellular respiration. Signatures of long-lived species were positively correlated with age-related changes and enriched for evolutionarily ancient essential genes, involved in proteolysis and PI3K-Akt signaling. Conversely, lifespan-extending interventions counteracted aging patterns and affected younger, mutable genes enriched for energy metabolism. The identified biomarkers revealed longevity interventions, including KU0063794, which extended mouse lifespan and healthspan. Overall, this study uncovers universal and distinct strategies of lifespan regulation within and across species and provides tools for discovering longevity interventions.

Self-aligned single-electrode actuation of tangential and wineglass modes using PMN-PT.

Considering the evolution of rotation sensing and timing applications realized in micro-electro-mechanical systems (MEMS), flexural mode resonant shapes are outperformed by bulk acoustic wave (BAW) counterparts by achieving higher frequencies with both electrostatic and piezoelectric transduction. Within the 1-30 MHz range, which hosts BAW gyroscopes and timing references, piezoelectric and electrostatic MEMS have similar transduction efficiency. Although, when designed intelligently, electrostatic transduction allows self-alignment between electrodes and the resonator for various BAW modes, misalignment is inevitable regarding piezoelectric transduction of BAW modes that require electrode patterning. In this paper transverse piezoelectric actuation of [011] oriented single crystal lead magnesium niobate-lead titanate (PMN-PT) thin film disks are shown to excite the tangential mode and family of elliptical compound resonant modes, utilizing a self-aligned and unpatterned electrode that spans the entire disk surface. The resonant mode coupling is achieved by employing a unique property of [011] PMN-PT, where the in-plane piezoelectric coefficients have opposite signs. Fabricating 1-port disk transducers, RF reflection measurements are performed that demonstrate the compound mode family shapes in the 1-30 MHz range. Independent verification of mode transduction is achieved using in-plane displacement measurements with Polytec's laser Doppler vibrometer (LDV). While the tangential mode achieves a 40o/s dithering rate at 335 kHz resonant frequency, the n = 2 wine-glass mode achieves 11.46 nm tip displacement at 8.42 MHz resonant frequency on a radius of 60 µm disk resonator in air. A single electrode resonator that can excite both tangential and wine-glass modes with such metrics lays the foundation for a BAW MEMS gyroscope with a built-in primary calibration stage.

Theoretical and Experimental Studies on Sensitivity and Bandwidth of Thickness-Mode Driving Hydrophone Utilizing A 2-2 Piezoelectric Single Crystal Composite.

Piezoelectric composites, which consist of a piezoelectric material and a polymer, have been extensively studied for the applications of underwater sonar sensors and medical diagnostic ultrasonic transducers. Acoustic sensors utilizing piezoelectric composites can have a high sensitivity and wide bandwidth because of their high piezoelectric coefficient and low acoustic impedance compared to single-phase piezoelectric materials. In this study, a thickness-mode driving hydrophone utilizing a 2-2 piezoelectric single crystal composite was examined. From the theoretical and numerical analysis, material properties that determine the bandwidth and sensitivity of the thickness-mode piezoelectric plate were derived, and the voltage sensitivity of piezoelectric plates with various configurations was compared. It was shown that the 2-2 composite with [011] poled single crystals and epoxy polymers can provide high sensitivity and wide bandwidth when used for hydrophones with a thickness resonance mode. The hydrophone element was designed and fabricated to have a thickness mode at a frequency around 220 kHz by attaching a composite plate of quarter-wavelength thickness to a hard baffle. The fabricated hydrophone demonstrated an open circuit voltage sensitivity of more than -180 dB re 1 V/µPa at the resonance frequency and a -3 dB bandwidth of more than 55 kHz. The theoretical and experimental studies show that the 2-2 single crystal composite can have a high sensitivity and wide bandwidth compared to other configurations of piezoelectric elements when they are used for thickness-mode hydrophones.

The teaching of geriatric dentistry in South Korean dental schools.

OBJECTIVE: The purpose of this study was to investigate the curriculum of geriatric dentistry for undergraduates in Korean dental schools. BACKGROUND: For development purposes, it was necessary to compare geriatric dentistry education programmes in South Korea to programmes in the United States and Europe. METHODS: The most recent curriculum and related information on geriatric dentistry at the undergraduate level in all 11 dental schools in South Korea were collected by both official letter and e-mail. A symposium for gathering expert opinions to improve geriatric dentistry education in South Korea was also held. The collected data were analysed, and the expert opinions at the symposium were summarised. RESULTS: Six of 11 schools had a didactic course as compulsory and three schools as elective. The course was usually conducted as a form of integrated lectures, and the level of standardisation of lecture content was very low. There were no topics for older people who cannot access dental clinics due to functional frailty or disability. No dental school-affiliated hospitals had an independent department for geriatric dentistry. No schools provided clinical teaching for geriatric dentistry. There were no outreach programmes for geriatric dentistry. CONCLUSIONS: The educational curriculum for geriatric dentistry in South Korea was insufficient to cope with social and demographic changes. Curriculum content should include clinical practice education and needs to be focused on frail and dependent older adult patients. An essential educational curriculum and core competency for geriatric dentistry should be prepared.

Selenoprotein TXNRD3 supports male fertility via the redox regulation of spermatogenesis.

Thioredoxin/glutathione reductase (TXNRD3) is a selenoprotein composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent thiol oxidoreductase evolved through gene duplication within the Txnrd family, is expressed in the testes, and can reduce both thioredoxin and glutathione in vitro; however, the function of this enzyme remains unknown. To characterize the function of TXNRD3 in vivo, we generated a strain of mice bearing deletion of Txnrd3 gene. We show that these Txnrd3 knockout mice are viable and without discernable gross phenotypes, and also that TXNRD3 deficiency leads to fertility impairment in male mice. We found that Txnrd3 knockout animals exhibited a lower fertilization rate in vitro, a sperm movement phenotype, and an altered thiol redox status in sperm cells. Proteomic analyses further revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of sperm quality control. Taken together, these results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.

Epigenetic aging of the demographically non-aging naked mole-rat.

The naked mole-rat (NMR) is an exceptionally long-lived rodent that shows no increase of mortality with age, defining it as a demographically non-aging mammal. Here, we perform bisulfite sequencing of the blood of > 100 NMRs, assessing > 3 million common CpG sites. Unsupervised clustering based on sites whose methylation correlates with age reveals an age-related methylome remodeling, and we also observe a methylome information loss, suggesting that NMRs age. We develop an epigenetic aging clock that accurately predicts the NMR age. We show that these animals age much slower than mice and much faster than humans, consistent with their known maximum lifespans. Interestingly, patterns of age-related changes of clock sites in Tert and Prpf19 differ between NMRs and mice, but there are also sites conserved between the two species. Together, the data indicate that NMRs, like other mammals, epigenetically age even in the absence of demographic aging of this species.

Maintenance of genome sequence integrity in long- and short-lived rodent species.

DNA mutations in somatic cells have been implicated in the causation of aging, with longer-lived species having a higher capacity to maintain genome sequence integrity than shorter-lived species. In an attempt to directly test this hypothesis, we used single-cell whole-genome sequencing to analyze spontaneous and bleomycin-induced somatic mutations in lung fibroblasts of four rodent species with distinct maximum life spans, including mouse, guinea pig, blind mole-rat, and naked mole-rat, as well as humans. As predicted, the mutagen-induced mutation frequencies inversely correlated with species-specific maximum life span, with the greatest difference observed between the mouse and all other species. These results suggest that long-lived species are capable of processing DNA damage in a more accurate way than short-lived species.

Epigenetic clocks reveal a rejuvenation event during embryogenesis followed by aging.

The notion that the germ line does not age goes back to the 19th-century ideas of August Weismann. However, being metabolically active, the germ line accumulates damage and other changes over time, i.e., it ages. For new life to begin in the same young state, the germ line must be rejuvenated in the offspring. Here, we developed a multi-tissue epigenetic clock and applied it, together with other aging clocks, to track changes in biological age during mouse and human prenatal development. This analysis revealed a significant decrease in biological age, i.e., rejuvenation, during early stages of embryogenesis, followed by an increase in later stages. We further found that pluripotent stem cells do not age even after extensive passaging and that the examined epigenetic age dynamics is conserved across species. Overall, this study uncovers a natural rejuvenation event during embryogenesis and suggests that the minimal biological age (ground zero) marks the beginning of organismal aging.

Perfluoropolyether-benzophenone as a highly durable, broadband anti-reflection, and anti-contamination coating.

Anti-reflection and anti-contamination coatings prepared from fluorinated polymers have widespread and important applications, ranging from protective films for corrosion resistance to high-tech microelectronics and medical devices due to their transparency, low refractive index, stain resistance, and antifouling properties. However, the application of existing coatings is hindered by low surface adhesion to the target substrate and weakness when exposed to mechanical stress or damage, resulting in significant limitations to their practical applications. Herein, we incorporate perfluoropolyether (PFPE) with benzophenone (BP) to develop an efficient coating material (PFPE-BP) possessing broadband anti-reflectivity, anti-contamination properties, excellent abrasion resistance, and stability under elevated temperatures and relative humidity. The presence of BP allows the coating materials to be homogeneously mixed with a commercial hard coating solution to uniformly coat the target substrate. Furthermore, UV light irradiation on the coating surface results in excellent adhesion between BP groups of PFPE-BP and the hard coating matrix.

A naked mole rat iPSC line expressing drug-inducible mouse pluripotency factors developed from embryonic fibroblasts.

Naked mole rats (NMRs, Heterocephalus glaber) are long-lived, cancer-resistant rodents. Here, we report the development of an induced pluripotent stem cell (iPSC) line generated from immortalized NMR embryonic fibroblasts transduced with a doxycycline-inducible mouse OSKM polycistronic vector. This iPSC line was shown to express pluripotency-associated markers, form embryoid bodies, differentiate in vitro to the derivatives of three germ layers, and exhibit normal karyotype. The ability of iPSCs to differentiate in vivo was supported by the contribution to interspecific chimera upon injection into mouse blastocysts. This NMR iPSC line may be a useful tool in cancer and aging research.

Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins.

Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDP-glucose:glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins.

Naked Mole Rat Induced Pluripotent Stem Cells and Their Contribution to Interspecific Chimera.

Naked mole rats (NMRs) are exceptionally long-lived, cancer-resistant rodents. Identifying the defining characteristics of these traits may shed light on aging and cancer mechanisms. Here, we report the generation of induced pluripotent stem cells (iPSCs) from NMR fibroblasts and their contribution to mouse-NMR chimeric embryos. Efficient reprogramming could be observed under N2B27+2i conditions. The iPSCs displayed a characteristic morphology, expressed pluripotent markers, formed embryoid bodies, and showed typical differentiation patterns. Interestingly, NMR embryonic fibroblasts and the derived iPSCs had propensity for a tetraploid karyotype and were resistant to forming teratomas, but within mouse blastocysts they contributed to both interspecific placenta and fetus. Gene expression patterns of NMR iPSCs were more similar to those of human than mouse iPSCs. Overall, we uncovered unique features of NMR iPSCs and report a mouse-NMR chimeric model. The iPSCs and associated cell culture systems can be used for a variety of biological and biomedical applications.

Selenoprotein MsrB1 promotes anti-inflammatory cytokine gene expression in macrophages and controls immune response in vivo.

Post-translational redox modification of methionine residues often triggers a change in protein function. Emerging evidence points to this reversible protein modification being an important regulatory mechanism under various physiological conditions. Reduction of oxidized methionine residues is catalyzed by methionine sulfoxide reductases (Msrs). Here, we show that one of these enzymes, a selenium-containing MsrB1, is highly expressed in immune-activated macrophages and contributes to shaping cellular and organismal immune responses. In particular, lipopolysaccharide (LPS) induces expression of MsrB1, but not other Msrs. Genetic ablation of MsrB1 did not preclude LPS-induced intracellular signaling in macrophages, but resulted in attenuated induction of anti-inflammatory cytokines, such as interleukin (IL)-10 and the IL-1 receptor antagonist. This anomaly was associated with excessive pro-inflammatory cytokine production as well as an increase in acute tissue inflammation in mice. Together, our findings suggest that MsrB1 controls immune responses by promoting anti-inflammatory cytokine expression in macrophages. MsrB1-dependent reduction of oxidized methionine in proteins may be a heretofore unrecognized regulatory event underlying immunity and inflammatory disease, and a novel target for clinical applications.

A Light Illumination Enhancement Device for Photoacoustic Imaging: In Vivo Animal Study.

Photoacoustic (PA) imaging detects acoustic signals generated by thermal expansion of a light-excited tissue or contrast agents. PA signal amplitude and image quality directly depend on the light fluence at the target depth. With conventional PA imaging systems, approximately 30% energy of incident light at the near-infrared region would be lost due to reflection on the skin surface. Such light loss directly leads to a reduction of PA signal and image quality. A new light delivery scheme that collects and redistributes reflected light energy was recently suggested, which is called the light catcher. In our previous study, proof of concept using a finite-element simulation model was shown and a laboratory-built prototype of the light catcher was applied on tissue-mimicking phantoms. In this paper, we present an elaborate prototype of a high-frequency PA probe with the light catcher fabricated using 3-D printing technology, which is conformal to a subcutaneous tumor in mice. The in vivo usefulness of the developed prototype was evaluated in a mouse tumor model. Equipped with the light catcher, PA signal amplitude from the clinical photosensitizer injected into the mouse tumor was enhanced by 33.7%, which is approximately equivalent to the percent light loss due to reflection on the skin.

Using DNA Methylation Profiling to Evaluate Biological Age and Longevity Interventions.

The DNA methylation levels of certain CpG sites are thought to reflect the pace of human aging. Here, we developed a robust predictor of mouse biological age based on 90 CpG sites derived from partial blood DNA methylation profiles. The resulting clock correctly determines the age of mouse cohorts, detects the longevity effects of calorie restriction and gene knockouts, and reports rejuvenation of fibroblast-derived iPSCs. The data show that mammalian DNA methylomes are characterized by CpG sites that may represent the organism's biological age. They are scattered across the genome, they are distinct in human and mouse, and their methylation gradually changes with age. The clock derived from these sites represents a biomarker of aging and can be used to determine the biological age of organisms and evaluate interventions that alter the rate of aging.

Age-associated molecular changes are deleterious and may modulate life span through diet.

Transition through life span is accompanied by numerous molecular changes, such as dysregulated gene expression, altered metabolite levels, and accumulated molecular damage. These changes are thought to be causal factors in aging; however, because they are numerous and are also influenced by genotype, environment, and other factors in addition to age, it is difficult to characterize the cumulative effect of these molecular changes on longevity. We reasoned that age-associated changes, such as molecular damage and tissue composition, may influence life span when used in the diet of organisms that are closely related to those that serve as a dietary source. To test this possibility, we used species-specific culture media and diets that incorporated molecular extracts of young and old organisms and compared the influence of these diets on the life span of yeast, fruitflies, and mice. In each case, the "old" diet or medium shortened the life span for one or both sexes. These findings suggest that age-associated molecular changes, such as cumulative damage and altered dietary composition, are deleterious and causally linked with aging and may affect life span through diet.

Aggregation of cloned embryos in empty zona pellucida improves derivation efficiency of pig ES-like cells.

The development of embryonic stem cells (ESCs) from large animal species has become an important model for therapeutic cloning using ESCs derived by somatic cell nuclear transfer (SCNT). However, poor embryo quality and blastocyst formation have been major limitations for derivation of cloned ESCs (ntESCs). In this study, we have tried to overcome these problems by treating these cells with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. First, cloned embryos were treated with Scriptaid to confirm the effect of HDACi on cloned embryo quality. The Scriptaid-treated blastocysts showed significantly higher total cell numbers (29.50 ± 2.10) than non-treated blastocysts (22.29 ± 1.50, P < 0.05). Next, cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free, reconstructed, four-cell-stage SCNT embryos were injected into the empty zona of hatched parthenogenetic (PA) blastocysts. Blastocyst formation and total cell number of cloned blastocysts increased significantly for all aggregates (76.4% and 83.18 ± 8.33) compared with non-aggregates (25.5% and 27.11 ± 1.67, P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine ES-like cell derivation. Aggregated blastocysts showed a higher primary colony formation rate than non-aggregated cloned blastocysts (17.6 ± 12.3% vs. 2.2 ± 1.35%, respectively, P < 0.05). In addition, derived ES-like cells showed typical characters of ESCs. In conclusion, the aggregation of porcine SCNT embryos at the four-cell stage could be a useful technique for improving the development rate and quality of porcine-cloned blastocysts and the derivation efficiency of porcine ntESCs.

Selenophosphate synthetase 1 is an essential protein with roles in regulation of redox homoeostasis in mammals.

Selenophosphate synthetase (SPS) was initially detected in bacteria and was shown to synthesize selenophosphate, the active selenium donor. However, mammals have two SPS paralogues, which are designated SPS1 and SPS2. Although it is known that SPS2 catalyses the synthesis of selenophosphate, the function of SPS1 remains largely unclear. To examine the role of SPS1 in mammals, we generated a Sps1-knockout mouse and found that systemic SPS1 deficiency led to embryos that were clearly underdeveloped by embryonic day (E)8.5 and virtually resorbed by E14.5. The knockout of Sps1 in the liver preserved viability, but significantly affected the expression of a large number of mRNAs involved in cancer, embryonic development and the glutathione system. Particularly notable was the extreme deficiency of glutaredoxin 1 (GLRX1) and glutathione transferase Omega 1 (GSTO1). To assess these phenotypes at the cellular level, we targeted the removal of SPS1 in F9 cells, a mouse embryonal carcinoma (EC) cell line, which affected the glutathione system proteins and accordingly led to the accumulation of hydrogen peroxide in the cell. Furthermore, we found that several malignant characteristics of SPS1-deficient F9 cells were reversed, suggesting that SPS1 played a role in supporting and/or sustaining cancer. In addition, the overexpression of mouse or human GLRX1 led to a reversal of observed increases in reactive oxygen species (ROS) in the F9 SPS1/GLRX1-deficient cells and resulted in levels that were similar to those in F9 SPS1-sufficient cells. The results suggested that SPS1 is an essential mammalian enzyme with roles in regulating redox homoeostasis and controlling cell growth.

Polymorphic sites in complete genome sequences of Asian badger, Meles leucurus amurensis (Mustelidae, Melinae) mitochondria.

The badger is a common carnivorous animal maintain ecosystem by regulating the population of their prey. However, genomic information such as sequence polymorphisms has been restricted until recently. In this study, we have sequenced and assembled complete mitogenomes of Asian badgers, Meles leucurus amurensis, and polymorphic sites were identified. A total 182 singleton polymorphic sites were identified, and the 92 sites were located in protein-coding genes. Phylogenetic analysis showed that Asian badgers are evolutionary closed to Japanese badgers rather than European badgers. This study will provide important genomic information to assign taxon of species and to identify species of mustelids.