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In a disease-state-dependent manner, the histamine-resistant itch in dry skin-based skin diseases such as atopic dermatitis (AD) and xerosis is mainly due to hyperinnervation in the epidermis. Semaphorin 3A (Sema3A) is a nerve repulsion factor expressed in keratinocytes and it suppresses nerve fiber elongation in the epidermis. Our previous studies have shown that Sema3A ointment inhibits epidermal hyperinnervation and scratching behavior and improves dermatitis scores in AD model mice. Therefore, we consider Sema3A as a key therapeutic target for improving histamine-resistant itch in AD and xerosis. This study was designed to screen a library of herbal plant extracts to discover compounds with potential to induce Sema3A in normal human epidermal keratinocytes (NHEKs) using a reporter gene assay, so that positive samples were found. Among the positive samples, only the extract of S. baicalensis was found to consistently increase Sema3A levels in cultured NHEKs in assays using quantitative real-time PCR and ELISA. In evaluation of reconstituted human epidermis models, the level of Sema3A protein in culture supernatants significantly increased by application of the extract of S. baicalensis. In addition, we investigated which components in the extract of S. baicalensis contributed to Sema3A induction and found that baicalin and baicalein markedly increased the relative luciferase activity, and that baicalein had higher induction activity than baicalin. Thus, these findings suggest that S. baicalensis extract and its compounds, baicalin and baicalein, may be promising candidates for improving histamine-resistant itch via the induction of Sema3A expression in epidermal keratinocytes., Competing Interests: Three of the authors (YY, IY, NM) are employees of Kobayashi Pharmaceutical Co., Ltd. We have lodged an application for a domestic patent. This application has not been approved, and is currently pending. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Purpose: Although dose prediction for intensity modulated radiation therapy (IMRT) has been accomplished by a deep learning approach, delineation of some structures is needed for the prediction. We sought to develop a fully automated dose-generation framework for IMRT of prostate cancer by entering the patient CT datasets without the contour information into a generative adversarial network (GAN) and to compare its prediction performance to a conventional prediction model trained from patient contours., Methods: We propose a synthetic approach to translate patient CT datasets into a dose distribution for IMRT. The framework requires only paired-images, i.e., patient CT images and corresponding RT-doses. The model was trained from 81 IMRT plans of prostate cancer patients, and then produced the dose distribution for 9 test cases. To compare its prediction performance to that of another trained model, we created a model trained from structure images. Dosimetric parameters for the planning target volume (PTV) and organs at risk (OARs) were calculated from the generated and original dose distributions, and mean differences of dosimetric parameters were compared between the CT-based model and the structure-based model., Results: The mean differences of all dosimetric parameters except for D98% and D95% for PTV were within approximately 2% and 3% of the prescription dose for OARs in the CT-based model, while the differences in the structure-based model were within approximately 1% for PTV and approximately 2% for OARs, with a mean prediction time of 5 seconds per patient., Conclusions: Accurate and rapid dose prediction was achieved by the learning of patient CT datasets by a GAN-based framework. The CT-based dose prediction could reduce the time required for both the iterative optimization process and the structure contouring, allowing physicians and dosimetrists to focus their expertise on more challenging cases., Competing Interests: The authors have declared that no competing interests exist.

Extracellular vesicles (EVs) in the tumor microenvironment facilitate intercellular communication. Cancer cell-derived EVs act as an immunosuppressor by transporting cargos and presenting transmembrane proteins. By contrast, CD8+ cytotoxic T-lymphocytes (CTLs) exert anti-cancer cytotoxicity via the pore-forming protein perforin. Here, we hypothesize that although EVs are destroyed by perforin, cancer cell-derived EVs might possess mechanisms that enable them to avoid this destruction. We used a breast cancer cell line, MDA-MB-231-luc-D3H2LN (D3H2LN), to generate EVs. Destruction of the EVs by perforin was demonstrated visually using atomic force microscopy. To investigate immunosuppressive metabolites within cancer cell-derived EVs, we performed metabolomic profiling of EVs from D3H2LN cells cultured for 48 h with or without IFN-γ, which induces metabolic changes in the cells. We found that both types of EV from IFN-γ treated D3H2LN cells and non-treated D3H2LN cells contained adenosine, which has immunosuppressive effects. When we exposed cancer cell-derived EVs to CTLs, perforin secretion by CTLs fell significantly. In addition, the decreases in perforin secretion were ameliorated by treatment with adenosine deaminase, which degrades extracellular adenosine. Taken together, these results suggest that after perforin secreted by CTLs disrupts the membrane of EVs, adenosine released from the EVs acts as an immunosuppressive metabolite by binding to the adenosine receptor on the CTL membrane. This mechanism provides a novel survival strategy using cancer cell-derived EVs., Competing Interests: The authors have declared that no competing interests exist.

The aim of this study was to select a candidate deep-sea amphipod species suitable for connectivity analyses in areas around cobalt-rich ferromanganese crusts (CRCs). We applied DNA barcoding based on the mitochondrial protein-coding gene, cytochrome c oxidase subunit I (COI), to specimens collected from the Xufu Guyot (the JA06 Seamount) off southeastern Minami-Torishima Island in the North Pacific, where CRCs are distributed. We used baited traps to collect 37 specimens. Comparison of COI sequences with public reference databases (GenBank, BOLD) showed that almost all of the specimens belonged to the superfamily Lysianassoidea, which is known to be ubiquitous in deep-sea areas. In a molecular taxonomic analysis of these sequences, we detected 11 clades. One of these clades (group 9) composed of 18 sequences and was identified by DNA barcoding as a putative species belonging to Abyssorchomene, which has been reported from the New Hebrides Trench in the South Pacific. We considered this species to be a candidate for connectivity analysis and analyzed its genome by restriction site-associated DNA sequencing. The results showed that the genetic variation in this species is adequate for analyzing connectivity patterns in CRC areas in the future., Competing Interests: Kaiyo Engineering provided support in the form of salaries for authors MN and RM, and Japan Oil, Gas and Metals National Corporation (JOGMEC) provided support in the form of salaries for authors SK, TM, YI, and NO. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Rapeseed (Brassica napus L.) is one of the most economically important oilseed crops worldwide. In Japan, it has been cultivated for more than a century and has formed many feral populations. The aim of this study was to elucidate the genetic diversity of feral rapeseeds by genotyping 537 individuals (among which 130 were determined to be genetically modified) sampled from various regions in Japan. Analysis of 30 microsatellite markers amplified 334 alleles and indicated moderate genetic diversity and high inbreeding (expected heterozygosity, 0.50; observed heterozygosity, 0.16; inbreeding coefficient within individuals, 0.68) within the feral populations. The Mantel test showed only an insignificant weak positive correlation between geographic distance and genetic distance. Analysis of molecular variance showed a greater genetic diversity among individuals than between populations. These results are in accordance with population structure assessed by using principal coordinate analysis and the program STRUCTURE, which showed that the 537 individuals could be assigned to 8 genetic clusters with very large genetic differences among individuals within the same geographic population, and that among feral individuals, many are closely related to rapeseed accessions in the NARO Genebank but some have unknown origins. These unique feral rapeseeds are likely to be affected by strong selection pressure. The results for genetically modified individuals also suggest that they have two different sources and have a considerable degree of diversity, which might be explained by hybridization with nearby individuals and separation of hybrid cultivars. The information obtained in this study could help improve the management of feral rapeseed plants in Japan., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To identify blood cell markers that predict the long-term outcomes of patients with colorectal cancer., Methods: Data from 892 stage II and III colorectal cancer patients who underwent R0 resection were included. We analyzed the correlations of the preoperative blood data, previously reported prognostic indices, and clinicopathologic factors with the long-term outcomes, such as relapse-free survival and overall survival, using univariate and multivariate analyses., Results: Multivariate analysis showed that tumor location, stage, mean corpuscular volume, neutrophil-to-lymphocyte ratio, and lymphocyte-to- monocyte ratio were significantly correlated with relapse-free survival. A mean corpuscular volume ≥80.5 fL, neutrophil-to-lymphocyte ratio ≥5.5, and lymphocyte-to- monocyte ratio <3.4 had hazard ratios for disease relapse between 1.39 and 1.93. The cumulative scores of these three factors were aggregated into a laboratory prognostic score, with a maximum score at 6. The relapse-free survival and overall survival were well stratified by a laboratory prognostic score between 0-3 and 4-6, respectively, independent of the stage., Conclusion: The mean corpuscular volume, neutrophil-to-lymphocyte ratio, and lymphocyte-to- monocyte ratio can serve as blood cell markers to predict the long-term outcomes of patients who underwent R0 resection for stage II/III colorectal cancer., Competing Interests: The authors have declared that no competing interests exist.

Stereotactic body radiation therapy with CyberKnife for prostate cancer has long treatment times compared with conventional radiotherapy. This arises the need for designing treatment plans with short execution times. We propose an objective function for plan quality evaluation, which was used to determine an optimal combination between small and large collimators based on short treatment times and clinically acceptable dose distributions. Data from 11 prostate cancer patients were used. For each patient, 20 plans were created based on all combinations between one small (⌀ 10-25 mm) and one large (⌀ 35-60 mm) Iris collimator size. The objective function was assigned to each combination as a penalty, such that plans with low penalties were considered superior. This function considered the achievement of dosimetric planning goals, tumor control probability, normal tissue complication probability, relative seriality parameter, and treatment time. Two methods were used to determine the optimal combination. First, we constructed heat maps representing the mean penalty values and standard deviations of the plans created for each collimator combination. The combination giving a plan with the smallest mean penalty and standard deviation was considered optimal. Second, we created two groups of superior plans: group A plans were selected by histogram analysis and group B plans were selected by choosing the plan with the lowest penalty from each patient. In both groups, the most used small and large collimators were assumed to represent the optimal combination. The optimal combinations obtained from the heat maps included the 25 mm as a small collimator, giving small/large collimator sizes of 25/35, 25/40, 25/50, and 25/60 mm. The superior-group analysis indicated that 25/50 mm was the optimal combination. The optimal Iris combination for prostate cancer treatment using CyberKnife was determined to be a collimator size between 25 mm (small) and 50 mm (large)., Competing Interests: The authors have declared that no competing interests exist.

In Japan, which is thought to be a rapidly growing super-aging society, a national campaign named "the Dementia Supporter Caravan" has been deployed. The aim of this study was to assess the educational benefits of the dementia supporter training program for nurses and nursing students. We conducted dementia supporter training, and measured knowledge and attitudes regarding people with dementia as educational benefits pre- and post-training. Data sets of 134 nursing students and 63 nurses were analyzed. The results indicated that the two groups gained knowledge, understanding, and the confidence to care for people with dementia after attending the dementia supporter training program. Moreover, the two groups derived different benefits from the program. Nursing students gained substantial knowledge and learnt the importance of early detection and treatment, to levels similar to those of nurses prior to training. The training program reduced the difficulties of nurses to interact with and care for people with dementia. We can conclude that the dementia supporter training program has considerable educational benefits for nurses and nursing students., Competing Interests: The authors have declared that no competing interests exist.

Sweat includes active components and metabolites, which are needed to maintain skin homeostasis. Component changes in sweat derived from atopic dermatitis (AD) have been reported. To investigate the influence of sweat components on the pathogenesis of AD, we performed a multifaceted assessment, including nuclear magnetic resonance spectroscopy-based metabolomic analysis, and linked these features to clinical features of AD. Distinctive properties of AD sweat are the quite-variation in protein, anti-microbial peptides and glucose concentrations. pH, sodium, and other salt levels in sweat of AD were comparable to that of healthy subjects. Sweat from AD patients with acute inflammation had a more prominent increase in glucose concentration than sweat from healthy individuals or those with AD with chronic inflammation. Topical glucose application delayed recovery of transepidermal water loss in barrier-disrupted mice. Furthermore, the glucose transporter GLUT2 was highly expressed in the lumen of sweat glands from AD patients. AD patients with chronic inflammation had significantly increased GLUT2 mRNA expression and near normal sweat glucose levels. Despite the small sample size in our study, we speculate that the increased glucose levels might be affected by AD severity and phenotype. We hope that this report will bring novel insight into the impact of sweat components on the clinical manifestation of AD.

For CyberKnife-mediated prostate cancer treatment, a tumour-tracking approach is applied to correct the target location by acquiring X-ray images of implanted fiducial markers intermittently. This study investigated the dosimetric impact of intra-fraction prostate motion during CyberKnife treatment. We retrospectively analyzed 16 patients treated using the CyberKnife (35 Gy delivered in five fractions). Using log files of recorded prostate motion, the intra-fraction prostate motion was simulated. We defined the worst-case intra-fraction prostate motion as the difference between pre- and post-deviation on log files and shifted structure sets according to the corresponding offsets for each beam. The dose-volume indices were calculated and compared with the original plan in terms of clinical target volume (CTV), planning target volume (CTV plus a 2-mm margin), rectum, bladder, and urethra. Prostate motions of >3, >5, and >10 mm were observed for 31.3, 9.1, and 0.5% of the 1929 timestamps, respectively. Relative differences between the simulated and original plans were mostly less than 1%. Although significant decreases were observed in D50% and D98% of the target, absolute dose differences were <0.1 Gy compared with the planned dose. The dosimetric impact of intra-fraction prostate motion may be small even with longer treatment durations, indicating that the tumour tracking using the CyberKnife could be a robust system for examining prostate motion.

This study evaluated a method for prostate intensity-modulated radiation therapy (IMRT) based on edge-enhanced (EE) intensity in the presence of intrafraction organ deformation using the data of 37 patients treated with step-and-shoot IMRT. On the assumption that the patient setup error was already accounted for by image guidance, only organ deformation over the treatment course was considered. Once the clinical target volume (CTV), rectum, and bladder were delineated and assigned dose constraints for dose optimization, each voxel in the CTV derived from the DICOM RT-dose grid could have a stochastic dose from the different voxel location according to the probability density function as an organ deformation. The stochastic dose for the CTV was calculated as the mean dose at the location through changing the voxel location randomly 1000 times. In the EE approach, the underdose region in the CTV was delineated and optimized with higher dose constraints that resulted in an edge-enhanced intensity beam to the CTV. This was compared to a planning target volume (PTV) margin (PM) approach in which a CTV to PTV margin equivalent to the magnitude of organ deformation was added to obtain an optimized dose distribution. The total monitor units, number of segments, and conformity index were compared between the two approaches, and the dose based on the organ deformation of the CTV, rectum, and bladder was evaluated. The total monitor units, number of segments, and conformity index were significantly lower with the EE approach than with the PM approach, while maintaining the dose coverage to the CTV with organ deformation. The dose to the rectum and bladder were significantly reduced in the EE approach compared with the PM approach. We conclude that the EE approach is superior to the PM with regard to intrafraction organ deformation.

Background: The EGFR tyrosine kinase inhibitor gefitinib is used in therapy for non-small-cell lung cancer (NSCLC). However, its application is limited by resistance-accelerated disease progression, which is accompanied by the epithelial-to-mesenchymal transition (EMT). In the present study, we performed multiple expression analyses of microRNAs (miRNAs) and quantified the expression of several related EMT players in gefitinib-resistant NSCLC cells., Methods and Results: To establish gefitinib-resistant NSCLC cells, gefitinib-sensitive HCC827 cells, which exhibit an in-frame deletion [E746-A750] in EGFR exon 19, were exposed to gefitinib for at least 1.5 months. Next, to profile "gefitinib-resistant HCC827 (HCC827GR)" cells, which have a secondary T790M mutation in EGFR exon 20, a miRNA array analysis was performed in HCC827 and HCC827GR cells. The greatest differences were seen in the levels of miR-155 and miR-200c, which essentially disappeared in HCC827GR cells. In addition to these reductions, the levels of smad2 and zeb1, which are both key players in EMT and targets for miR-155 and miR-200c, respectively, were dramatically increased in HCC827GR cells. In HCC827GR cells, the expression of epithelial-cadherin (E-cadherin) was greatly reduced with repressive histone modifications, whereas vimentin, which is expressed in mesenchymal cells, was dramatically increased with active histone modifications. In another gefitinib-resistant NSCLC cell line (H1975 cells), similar to the findings in HCC827GR cells, both miR-155 and miR-200c were absent, and the EMT was induced along with epigenetic modifications. Interestingly, the inhibition of both miR-155 and miR-200c in HCC827 cells without gefitinib induced significant increases in smad2 and zeb1 along with a dramatic decrease in E-cadherin and a slight increase in vimentin. Furthermore, although the inhibition of these miRNAs in HCC827 cells decreased gefitinib sensitivity, this dual-inhibition in HCC827 cells without gefitinib did not produce a secondary T790M mutation in EGFR exon 20., Conclusion and Implications: These results suggest that chronic treatment of NSCLC cells with gefitinib changes the expression of miRNAs, including dramatic reductions in miR-155 and miR-200c along with an EGFR mutation. Furthermore, this depletion of miR-155 and miR-200c may be associated with the EMT along with histone modifications, and may contribute to the decrease in the sensitivity to gefitinib independent of a secondary EGFR mutation.

Background: Chest auscultation is commonly performed during respiratory physical therapy (RPT). However, the changes in breath sounds in children with atelectasis have not been previously reported. The aim of this study was to clarify the characteristics of breath sounds in children with atelectasis using acoustic measurements., Method: The subjects of this study were 13 children with right middle lobe atelectasis (3-7 years) and 14 healthy children (3-7 years). Lung sounds at the bilateral fifth intercostal spaces on the midclavicular line were recorded. The right-to-left ratio (R/L ratio) and the expiration to inspiration ratio (E/I ratio) of the breath sound sound pressure were calculated separately for three octave bands (100-200 Hz, 200-400 Hz, and 400-800 Hz). These data were then compared between the atelectasis and control groups. In addition, the same measurements were repeated after treatment, including RPT, in the atelectasis group., Result: Before treatment, the inspiratory R/L ratios for all the frequency bands were significantly lower in the atelectasis group than in the control group, and the E/I ratios for all the frequency bands were significantly higher in the atelectasis group than in the control group. After treatment, the inspiratory R/L ratios of the atelectasis group did not increase significantly, but the E/I ratios decreased for all the frequency bands and became similar to those of the control group., Conclusion: Breath sound attenuation in the atelectatic area remained unchanged even after radiographical resolution, suggesting a continued decrease in local ventilation. On the other hand, the elevated E/I ratio for the atelectatic area was normalized after treatment. Therefore, the differences between inspiratory and expiration sound intensities may be an important marker of atelectatic improvement in children., Competing Interests: The authors have declared that no competing interests exist.

Ephrin receptor A10 (EphA10), a transmembrane receptor that binds to ephrin, is a newly identified breast cancer marker protein that has also been detected in HER2-negative tissue. In this study, we report creation of a novel bispecific antibody (BsAb) binding both EphA10 and CD3, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells and redirection of cytotoxic T cells (CTL). This BsAb (EphA10/CD3) was expressed in supernatants of BsAb gene-transfected cells as monomeric and dimeric molecules. Redirected T-cell lysis was observed when monomeric and dimeric BsAb were added to EphA10-overexpressing tumor cells in vitro. Furthermore, dimeric BsAb (EphA10/CD3) was more cytotoxic than monomeric BsAb, with efficient tumor cell lysis elicited by lower concentrations (≤10(-1) μg/mL) and a lower effector to target (E/T) cell ratio (E/T = 2.5). Dimeric BsAb (EphA10/CD3) also showed significant anti-tumor effects in human xenograft mouse models. Together, these results revealed opportunities to redirect the activity of CTL towards tumor cells that express EphA10 using the BsAb (EphA10/CD3), which could be tested in future clinical trials as a novel and potent therapeutic for breast cancer tumors.

Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)-PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants.

We assessed brain abnormalities in rats exposed prenatally to radiation (X-rays) using magnetic resonance imaging (MRI) and histological experiments. Pregnant rats were divided into 4 groups: the control group (n = 3) and 3 groups that were exposed to different radiation doses (0.5, 1.0, or 1.5 Gy; n = 3 each). Brain abnormalities were assessed in 32 neonatal male rats (8 per group). Ex vivo T2-weighted imaging and diffusion tensor imaging (DTI) were performed using 11.7-T MRI. The expression of markers of myelin production (Kluver-Barrera staining, KB), nonpyramidal cells (calbindin-D28k staining, CaBP), and pyramidal cells (staining of the nonphosphorylated heavy-chain neurofilament SMI-32) were histologically evaluated. Decreased brain volume, increased ventricle volume, and thinner cortices were observed by MRI in irradiated rats. However, no abnormalities in the cortical 6-layered structure were observed via KB staining in radiation-exposed rats. The DTI color-coded map revealed a dose-dependent reduction in the anisotropic signal (vertical direction), which did not represent reduced numbers of pyramidal cells; rather, it indicated a signal reduction relative to the vertical direction because of low nerve cell density in the entire cortex. We conclude that DTI and histological experiments are useful tools for assessing cortical and hippocampal abnormalities after prenatal exposure to radiation in rats.

Tumor necrosis factor alpha (TNF-α), a central mediator of the inflammatory response, is released from basophilic cells and other cells in response to a variety of proinflammatory stimuli. Vialinin A is a potent inhibitor of TNF-α production and is released from RBL-2H3 cells. Ubiquitin-specific peptidase 5 (USP5), a deubiquitinating enzyme, was identified as a target molecule of vialinin A and its enzymatic activity was inhibited by vialinin A. Here we report production of TNF-α is decreased in USP5 siRNA-knockdown RBL-2H3 cells, compared with control cells. The finding of the present study strongly suggests that USP5 is one of the essential molecules for the production of TNF-α in RBL-2H3.

Background: miRNAs circulating in the blood in a cell-free form have been acknowledged for their potential as readily accessible disease markers. Presently, histological examination is the golden standard for diagnosing and grading liver disease, therefore non-invasive options are desirable. Here, we investigated if miRNA expression profile in exosome rich fractionated serum could be useful for determining the disease parameters in patients with chronic hepatitis C (CHC)., Methodology: Exosome rich fractionated RNA was extracted from the serum of 64 CHC and 24 controls with normal liver (NL). Extracted RNA was subjected to miRNA profiling by microarray and real-time qPCR analysis. The miRNA expression profiles from 4 chronic hepatitis B (CHB) and 12 non alcoholic steatohepatitis (NASH) patients were also established. The resulting miRNA expression was compared to the stage or grade of CHC determined by blood examination and histological inspection., Principal Findings: miRNAs implicated in chronic liver disease and inflammation showed expression profiles that differed from those in NL and varied among the types and grades of liver diseases. Using the expression patterns of nine miRNAs, we classified CHC and NL with 96.59% accuracy. Additionally, we could link miRNA expression pattern with liver fibrosis stage and grade of liver inflammation in CHC. In particular, the miRNA expression pattern for early fibrotic stage differed greatly from that observed in high inflammation grades., Conclusions: We demonstrated that miRNA expression pattern in exosome rich fractionated serum shows a high potential as a biomarker for diagnosing the grade and stage of liver diseases.

Background: There is an increasing need for animal disease models for pathophysiological research and efficient drug screening. However, one of the technical barriers to the effective use of the models is the difficulty of non-invasive and sequential monitoring of the same animals. Micro-CT is a powerful tool for serial diagnostic imaging of animal models. However, soft tissue contrast resolution, particularly in the brain, is insufficient for detailed analysis, unlike the current applications of CT in the clinical arena. We address the soft tissue contrast resolution issue in this report., Methodology: We performed contrast-enhanced CT (CECT) on mouse models of experimental cerebral infarction and hepatic ischemia. Pathological changes in each lesion were quantified for two weeks by measuring the lesion volume or the ratio of high attenuation area (%HAA), indicative of increased vascular permeability. We also compared brain images of stroke rats and ischemic mice acquired with micro-CT to those acquired with 11.7-T micro-MRI. Histopathological analysis was performed to confirm the diagnosis by CECT., Principal Findings: In the models of cerebral infarction, vascular permeability was increased from three days through one week after surgical initiation, which was also confirmed by Evans blue dye leakage. Measurement of volume and %HAA of the liver lesions demonstrated differences in the recovery process between mice with distinct genetic backgrounds. Comparison of CT and MR images acquired from the same stroke rats or ischemic mice indicated that accuracy of volumetric measurement, as well as spatial and contrast resolutions of CT images, was comparable to that obtained with MRI. The imaging results were also consistent with the histological data., Conclusions: This study demonstrates that the CECT scanning method is useful in rodents for both quantitative and qualitative evaluations of pathologic lesions in tissues/organs including the brain, and is also suitable for longitudinal observation of the same animals.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons. Recent studies have implicated that chronic hypoxia and insufficient vascular endothelial growth factor (VEGF)-dependent neuroprotection may lead to the degeneration of motor neurons in ALS. Expression of apelin, an endogenous ligand for the G protein-coupled receptor APJ, is regulated by hypoxia. In addition, recent reports suggest that apelin protects neurons against glutamate-induced excitotoxicity. Here, we examined whether apelin is an endogenous neuroprotective factor using SOD1(G93A) mouse model of ALS. In mouse CNS tissues, the highest expressions of both apelin and APJ mRNAs were detected in spinal cord. APJ immunoreactivity was observed in neuronal cell bodies located in gray matter of spinal cord. Although apelin mRNA expression in the spinal cord of wild-type mice was not changed from 4 to 18 weeks age, that of SOD1(G93A) mice was reduced along with the paralytic phenotype. In addition, double mutant apelin-deficient and SOD1(G93A) displayed the disease phenotypes earlier than SOD1(G93A) littermates. Immunohistochemical observation revealed that the number of motor neurons was decreased and microglia were activated in the spinal cord of the double mutant mice, indicating that apelin deficiency pathologically accelerated the progression of ALS. Furthermore, we showed that apelin enhanced the protective effect of VEGF on H(2)O(2)-induced neuronal death in primary neurons. These results suggest that apelin/APJ system in the spinal cord has a neuroprotective effect against the pathogenesis of ALS.

Normal cells, both in vivo and in vitro, become quiescent after serial cell proliferation. During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition.

During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.