Age estimation based on blood DNA methylation levels in brown bears.

Age is an essential trait for understanding the ecology and management of wildlife. A conventional method of estimating age in wild animals is counting annuli formed in the cementum of teeth. This method has been used in bears despite some disadvantages, such as high invasiveness and the requirement for experienced observers. In this study, we established a novel age estimation method based on DNA methylation levels using blood collected from 49 brown bears of known ages living in both captivity and the wild. We performed bisulfite pyrosequencing and obtained methylation levels at 39 cytosine-phosphate-guanine (CpG) sites adjacent to 12 genes. The methylation levels of CpGs adjacent to four genes showed a significant correlation with age. The best model was based on DNA methylation levels at just four CpG sites adjacent to a single gene, SLC12A5, and it had high accuracy with a mean absolute error of 1.3 years and median absolute error of 1.0 year after leave-one-out cross-validation. This model represents the first epigenetic method of age estimation in brown bears, which provides benefits over tooth-based methods, including high accuracy, less invasiveness, and a simple procedure. Our model has the potential for application to other bear species, which will greatly improve ecological research, conservation, and management.

Synchronous intracellular delivery of EGFR-targeted antibody-drug conjugates by p38-mediated non-canonical endocytosis.

Monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), including cetuximab and panitumumab, have been used in clinic settings to treat cancer. They have also recently been applied to antibody-drug conjugates (ADCs); however, their clinical efficacy is limited by several issues, including lower internalization efficiency. The binding of cetuximab to the extracellular domain of EGFR suppresses ligand-induced events; therefore, we focus on ligand-independent non-canonical EGFR endocytosis for the delivery of ADCs into cells. Tumor necrosis factor-α (TNF-α) strongly induces the endocytosis of the cetuximab-EGFR complex within 15 min via the p38 phosphorylation of EGFR in a tyrosine kinase-independent manner. A secondary antibody conjugated with saporin, a ribosome-inactivating protein, also undergoes internalization with the complex and enhances its anti-proliferative activity. Anti-cancer agents, including cisplatin and temozolomide, also induce the p38-mediated internalization. The results of the present study demonstrate that synchronous non-canonical EGFR endocytosis may be a feasible strategy for promoting the therapeutic efficacy of EGFR-targeting ADCs in clinical settings.

Plastid translation is essential for lateral root stem cell patterning in Arabidopsis thaliana.

The plastid evolved from a symbiotic cyanobacterial ancestor and is an essential organelle for plant life, but its developmental roles in roots have been largely overlooked. Here, we show that plastid translation is connected to the stem cell patterning in lateral root primordia. The RFC3 gene encodes a plastid-localized protein that is a conserved bacterial ribosomal protein S6 of ß/γ proteobacterial origin. The rfc3 mutant developed lateral roots with disrupted stem cell patterning and associated with decreased leaf photosynthetic activity, reduced accumulation of plastid rRNAs in roots, altered root plastid gene expression, and changes in expression of several root stem cell regulators. These results suggest that deficiencies in plastid function affect lateral root stem cells. Treatment with the plastid translation inhibitor spectinomycin phenocopied the defective stem cell patterning in lateral roots and altered plastid gene expression observed in the rfc3 mutant. Additionally, when prps17 defective in a plastid ribosomal protein was treated with low concentrations of spectinomycin, it also phenocopied the lateral root phenotypes of rfc3 The spectinomycin treatment and rfc3 mutation also negatively affected symplasmic connectivity between primary root and lateral root primordia. This study highlights previously unrecognized functions of plastid translation in the stem cell patterning in lateral roots.

Validation of new bite block-type head-immobilization devices for radiotherapy in dogs.

An ideal head-immobilization method provides a high level of accuracy and reproducibility in the immobilization. Various head-immobilization methods for radiotherapy have been published and are excellent in terms of accuracy; however, these methods are complicated to use, and labor intensive. The present study describes two new bite block-type head-immobilization devices designed for higher stability and lower vertical variation. The device designed in our previous study (the bite block-type head-immobilization device; Device A) was modified by making a groove on the top the horizontal plate (Device B) for a stable ventral-dorsal position, or beneath the horizontal plate (Device C) for a stable dorsal-ventral position. The three devices were objectively compared with respect to setup time, and accuracy of the computed tomography scan images by two authors independently. Five male healthy beagles were used in this study. For each device, the setup time and the variation in the coordinates were measured five times for each dog. The mean setup times for Devices A, B, and C were 3.3, 1.5, and 2.4 min, respectively, showing the groove modifications were able to reduce the setup time (in device B, by at least 50%). Moreover, three-dimensional analysis of the computed tomography images revealed that the measurement variability of Device A (1.6 ± 1.0 mm) was significantly higher than that of Device C (0.7 ± 0.4 mm; P < 0.001). Collectively, our results show that use of a bite block-type head-immobilization device with a groove improves the setup time and head-immobilization accuracy.

Longitudinal Changes in the Government-Certified Index Stage and Requisite Costs for Long-Term Care Insurance System among the Community-Dwelling Demented Elderly in Japan.

Background. A new public long-term care (LTC) insurance was launched in 2000 in Japan. However, there have been few studies involving factors that increase LTC costs of demented subjects; no follow-up studies involving the Government-Certified Index (GCI) and requisite costs related to the causes of dementia. Method. An epidemiological survey was conducted in a rural area in Japan in 1999, and 271 subjects were diagnosed as dementia patients. Age, sex, mini-mental state examination, clinical dementia rating, activity of daily living, causes of dementia, and coexisting physical disease were confirmed. After the LTC insurance has been launched, we tracked the GCI stages and payment amounts every month for 8 years. Result. 209 subjects were certified to be eligible for LTC insurance; however, 13 did not receive any payment. Only 49 out of 209 were alive after the follow-up period. The most common cause of dementia was Alzheimer's disease (AD), followed by vascular dementia (VaD). There was no significant difference between the mortality rates of the two groups. VaD subjects required higher costs than AD subjects in the total certified period and in GCI stage 5. Conclusion. Our results indicate that causes of dementia can have an impact on the requisite costs for the LTC insurance.

Identification of high-risk dementia cohorts in a community sample of Japanese elderly.

AIM: The aim of this study was to develop a simple diagnostic procedure for subjects at high risk of developing dementia using the Clinical Dementia Rating (CDR), which is applicable to community-based activities. METHODS: This study divided 252 community-dwelling elderly with a CDR score of 0.5 into two groups based on the presence or absence of cognitive decline within the previous one year of the baseline, as assessed by a semi-structured interview. One hundred subjects were in the 'previously progressive group' (PP group) and 152 subjects were in the 'previously stable group' (PS group). After 6 years of observation, a total of 111 subjects were assessed in the follow-up investigation. RESULTS: Among the 39 subjects from the PP group (82.9 +/- 6.8 years old, 11 male, 28 female), 34 developed dementia (87%). Among the 72 subjects from the PS group (84.4 +/- 6.0 years old, 22 male, 50 female), 44 developed dementia (61%). The relative risk of developing dementia for the PP group versus the PS group was 1.43. The rate of conversion to dementia was 12.9% per 100 person-years in the PP group, and 9.8% in the PS group. In the PP group, the Mini-Mental State Examination score was significantly lower and the CDR score was significantly higher than in the PS group. CONCLUSION: Although there have been many attempts to identify subjects with high risk of dementia, this preliminary study suggests that information about temporal changes in cognitive function is useful when performing community-based surveys.