Simulated validation of intron-less transgene detection using DELLY for gene-doping control in horse sports.

Gene doping is prohibited in horseracing. In a previous study, we developed a method for non-targeted transgene detection using DELLY, which is based on split-read (SR) and paired-end (PE) algorithms to detect structural variants, on WGS data. In this study, we validated the detection sensitivity of DELLY using artificially generated sequence data of 12 target genes. With DELLY, at least one intron was detected as a deletion in eight targeted genes using the 150 bp PE read WGS data, whereas all targeted genes were detected by DELLY using the 100 bp PE read data. The detection sensitivity was higher in 100 bp PE reads than in 150 bp PE reads, despite a lower total sequence coverage, probably because of mismatch tolerance between the mapped reads and reference genome. In addition, it was observed that the average intron size detected by SR alone was 293 bp and that that detected by both SR and PE was 8924 bp. Thus, we showed that transgenes with various intron-exon structures could be detected using DELLY, suggesting its application in gene-doping control in horses.

Age in months and birth order in infant nonfatal injuries: A retrospective cohort study.

Objective: To examine the age in months at which infants visited outpatient clinics or emergency rooms for the first time for nonfatal injuries and to identify risk factors for the occurrence of these injuries. Study design: Retrospective cohort study. Methods: We used a health insurance claims database in Japan. Infants born between April 2012 and December 2014 were identified and followed until 12 months of age. We identified their first visit to outpatient clinics or emergency rooms because of nonfatal injuries (wounds/fractures, foreign bodies, and burns). Cox regression analysis was used to examine the association of nonfatal injuries with infants' sex, birth order, and parental age. Results: We identified 46,431 eligible infants. Of these, 7606 (16.4%) were brought to an outpatient clinic or emergency room for nonfatal injuries within 12 months of birth. Of the 7,606, 21.7% were aged ≤4 months and 44.7% â€‹≤ â€‹7 months. First-born infants were more likely to have wounds/fractures and burns. Conclusion: One-fifth of first nonfatal infant injuries occurred within 4 months of age. Healthcare providers should provide early education about injury prevention, especially to caregivers of first-born infants.

A candidate-SNP retrospective cohort study for fracture risk in Japanese Thoroughbred racehorses.

Fractures are medical conditions that compromise the athletic potential of horses and/or the safety of jockeys. Therefore, the reduction of fracture risk is an important horse and human welfare issue. The present study used molecular genetic approaches to determine the effect of genetic risk for fracture at four candidate SNPs spanning the myostatin (MSTN) gene on horse chromosome 18. Among the 3706 Japanese Thoroughbred racehorses, 1089 (29.4%) had experienced fractures in their athletic life, indicating the common occurrence of this injury in Thoroughbreds. In the case/control association study, fractures of the carpus (carpal bones and distal radius) were statistically associated with g.65809482T/C (P = 1.17 x 10-8 ), g.65868604G/T (P = 2.66 x 10-9 ), and g.66493737C/T (P = 6.41 x 10-8 ). In the retrospective cohort study using 1710 racehorses born in 2000, the relative risk (RR) was highest for male horses at g.65868604G/T, based on the dominant allele risk model (RR = 2.251, 95% confidence interval 1.407-3.604, P = 0.00041), and for female horses at g.65868604G/T, based on the recessive allele risk model (RR = 2.313, 95% confidence interval 1.380-3.877, P = 0.00163). Considering the association of these SNPs with racing performance traits such as speed, these genotypes may affect the occurrence of carpus fractures in Japanese Thoroughbred racehorses as a consequence of the non-genetic influence of the genotype on the distance and/or intensity of racing and training. The genetic information presented here may contribute to the development of strategic training programs and racing plans for racehorses that improve their health and welfare.

Genetic diversity and relationships among native Japanese horse breeds, the Japanese Thoroughbred and horses outside of Japan using genome-wide SNP data.

Eight horse breeds-Hokkaido, Kiso, Misaki, Noma, Taishu, Tokara, Miyako and Yonaguni-are native to Japan. Although Japanese native breeds are believed to have originated from ancient Mongolian horses imported from the Korean Peninsula, the phylogenetic relationships among these breeds are not well elucidated. In the present study, we compared genetic diversity among 32 international horse breeds previously evaluated by the Equine Genetic Diversity Consortium, the eight Japanese native breeds and Japanese Thoroughbreds using genome-wide SNP genotype data. The proportion of polymorphic loci and expected heterozygosity showed that the native Japanese breeds, with the exception of the Hokkaido, have relatively low diversity compared to the other breeds sampled. Phylogenetic and cluster analyses demonstrated relationships among the breeds that largely reflect their geographic distribution in Japan. Based on these data, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The Japanese Thoroughbreds were distinct from the native breeds, and although they maintain similar overall diversity as Thoroughbreds from outside Japan, they also show evidence of uniqueness relative to the other Thoroughbred samples. This is the first study to place the eight native Japanese breeds and Japanese Thoroughbred in context with an international sample of diverse breeds.

Concentrations of indomethacin and its metabolite desmethylindomethacin in plasma and urine after repeated indomethacin topical application to Thoroughbreds.

BACKGROUND: Repeated topical application of indomethacin is common in Japanese racehorses, despite the lack of pharmacokinetic data. OBJECTIVES: To determine the concentrations of indomethacin and its metabolite, desmethylindomethacin, in plasma and urine of Thoroughbreds topically treated repeatedly with indomethacin. STUDY DESIGN: In vivo experimental. METHODS: Seven female Thoroughbreds were topically treated with 50 g of 1% indomethacin cream per horse to the back and hips (500 mg of indomethacin/head/2400 cm2 , 0.21 g/cm2 ) for 3 consecutive days. Samples were pretreated by protein precipitation for plasma and liquid-liquid extraction with ethyl acetate after hydrolysis with hydrochloric acid for urine. The concentrations of indomethacin and desmethylindomethacin in plasma and urine were measured by liquid chromatography-mass spectrometry. RESULTS: Indomethacin was quantifiable in plasma up to 48-72 h and in urine up to 96 h after the final application. Desmethylindomethacin was quantifiable in plasma up to 48 h and in urine up to 72-96 h after the final application. MAIN LIMITATIONS: The relationship between the local and systemic indomethacin concentrations after the topical application was not clarified. CONCLUSIONS: Pharmacokinetic data were acquired for repeated topical administration of 1% indomethacin cream to Thoroughbreds. Hydrolysing urine samples with hydrochloric acid was effective for the analysis of indomethacin and its metabolite, and indomethacin may be an excellent marker analyte for doping tests. The estimated withdrawal time based on the limit of detection was 342 h.

Right adrenal vein: comparison between adaptive statistical iterative reconstruction and model-based iterative reconstruction.

AIM: To compare right adrenal vein (RAV) visualisation and contrast enhancement degree on adrenal venous phase images reconstructed using adaptive statistical iterative reconstruction (ASiR) and model-based iterative reconstruction (MBIR) techniques. MATERIAL AND METHODS: This prospective study was approved by the institutional review board, and written informed consent was waived. Fifty-seven consecutive patients who underwent adrenal venous phase imaging were enrolled. The same raw data were reconstructed using ASiR 40% and MBIR. The expert and beginner independently reviewed computed tomography (CT) images. RAV visualisation rates, background noise, and CT attenuation of the RAV, right adrenal gland, inferior vena cava (IVC), hepatic vein, and bilateral renal veins were compared between the two reconstruction techniques. RESULTS: RAV visualisation rates were higher with MBIR than with ASiR (95% versus 88%, p=0.13 in expert and 93% versus 75%, p=0.002 in beginner, respectively). RAV visualisation confidence ratings with MBIR were significantly greater than with ASiR (p<0.0001, both in the beginner and the expert). The mean background noise was significantly lower with MBIR than with ASiR (p<0.0001). Mean CT attenuation values of the RAV, right adrenal gland, IVC, and hepatic vein were comparable between the two techniques (p=0.12-0.91). Mean CT attenuation values of the bilateral renal veins were significantly higher with MBIR than with ASiR (p=0.0013 and 0.02). CONCLUSION: Reconstruction of adrenal venous phase images using MBIR significantly reduces background noise, leading to an improvement in the RAV visualisation compared with ASiR.

The effects of the Lactobacillus casei strain on obesity in children: a pilot study.

There are few data regarding the role of probiotics as a dietary intervention in the management of obesity in children. An open prospective examination was conducted to clarify the effects of Lactobacillus casei strain Shirota (LcS)-containing beverages in obese children. We compared the intestinal microbiota and organic acid levels between 12 obese (average age, 10.8 years; body mass index (BMI) Z score, 2.7±1.7) and 22 control children(average age, 8.5 years; BMI Z score, 0.1±0.7), and pre- and post-intervention in the obese children. The obese group underwent diet and exercise therapy for 6 months and then were given an LcS beverage daily for another 6 months and the body weight and serological markers were monitored. Significant reductions in the faecal concentrations of Bifidobacterium (obese group, 7.9±1.5 vs non-obese group, 9.8±0.5 Log10cells/g; P<0.01) along with a significant decline in the Bacteroides fragilis group, Atopobium cluster and Lactobacillus gasseri subgroup, and acetic acid (obese group, 45.1±16.9 vs non-obese group, 57.9±17.6 µmol/g; P<0.05) were observed in the obese group at baseline. A significant decline in body weight (-2.9±4.6%; P<0.05) and an elevation in the high density lipoprotein cholesterol level (+11.1±17.6%; P<0.05) were observed 6 months after ingestion of the LcS beverage compared to baseline. Furthermore, a significant increase in the faecal concentration of Bifidobacterium (7.0±1.2 before ingestion vs 9.1±1.2 Log10cells/g after ingestion; P<0.01) and an apparent increase in the acetic acid concentration (7.0±1.2 before ingestion vs 9.1±1.2 Log10cells/g after ingestion; P<0.01) were observed 6 months after ingestion. LcS contributed to weight loss while also improving the lipid metabolism in obese children via a significant increase in the faecal Bifidobacterium numbers and the acetic acid concentration.

Gut dysbiosis following C-section instigates higher colonisation of toxigenic Clostridium perfringens in infants.

Herein we investigated the intestinal carriage of α-toxigenic and enterotoxigenic Clostridium perfringens during infancy, focusing on its association with other gut microbes and mode of delivery and feeding. Faecal samples from 89 healthy term infants were collected at age 7 days, 1 month, 3 months, 6 months and 3 years. C. perfringens was quantified by qPCR; other gut bacteria were quantified by reverse-transcription-qPCR. Alpha-toxigenic C. perfringens was detected in 3.4% infants at day 7 but was present in 35-40% infants at subsequent time-points, with counts ranging from 103-107 cells/g faeces. Enterotoxigenic C. perfringens remained undetected at day 7 but was detected in 1.1, 4.5, 10.1 and 4.5% infants at 1 month, 3 months, 6 months and 3 years, respectively. Intriguingly, infants carrying α-toxigenic C. perfringens had lower levels of Bacteroides fragilis group, bifidobacteria, lactobacilli and organic acids as compared to non-carriers. Further analyses revealed that, compared to vaginally-born infants, caesarean-born infants had higher carriage of C. perfringens and lower levels of B. fragilis group, bifidobacteria, lactobacilli and faecal organic acids during first 6 months. Compared to formula-fed infants, breast-fed infants were slightly less often colonised with C. perfringens; and within caesarean-born infants, breast-fed infants had slightly lower levels of C. perfringens and higher levels of B. fragilis group, bifidobacteria, and lactobacilli than formula-fed infants. This study demonstrates the quantitative dynamics of toxigenic C. perfringens colonisation in infants during the early years of life. Caesarean-born infants acquire a somewhat perturbed microbiota, and breast-feeding might be helpful in ameliorating this dysbiosis. Higher carriage of toxigenic C. perfringens in healthy infants is intriguing and warrants further investigation of its sources and clinical significance in infants, particularly the caesarean-born who may represent a potential reservoir of this opportunistic pathogen and might be more prone to associated illnesses.

Nutritional Status and Habitual Dietary Intake Are Associated with Frail Skin Conditions in Community-Dwelling Older People.

OBJECTIVE: Prevention of frail skin is important in older people because frail skin is associated with a risk of injury in this population. In this study, we investigated the association of nutritional status and habitual dietary intake with skin conditions in community-dwelling older people. DESIGN: Cross-sectional study. SETTING: Three community settings in Japan from autumn to winter. PARTICIPANTS: Older people aged ≥65 years without care-need certification (n=118). MEASUREMENTS: Malnutrition and obesity were evaluated to assess the nutritional status. Nutrient and food group intakes per 1000 kcal were evaluated using a brief self-administered diet history questionnaire. Dietary patterns based on food groups were evaluated by principal component analysis. Skin condition parameters, including stratum corneum hydration, appearance of xerosis (specific symptom sum score [SRRC score]), and dermal intensity by high-frequency ultrasonography, were measured on a lower leg. Multiple linear regression analysis was performed with adjustment for confounders. RESULTS: The mean (standard deviation) age was 74.1 (4.8) years, and 83.1% of participants were female. A higher intake of plant fat (p=0.018) was associated with a lower SRRC score. Higher intakes of α-tocopherol (p=0.050) and vitamin C (p=0.017) were associated with increased dermal intensity. A body mass index ≥25 (p=0.016) was associated with decreased dermal intensity. A dietary pattern characterized by higher vegetable and fruit intake was associated with a better skin condition. CONCLUSION: Plant fat, antioxidant vitamins, and a dietary pattern characterized by vegetables and fruits showed positive and obesity showed negative associations for frail skin in community-dwelling older people.

MUC13 interaction with receptor tyrosine kinase HER2 drives pancreatic ductal adenocarcinoma progression.

Although MUC13, a transmembrane mucin, is aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC) and generally correlates with increased expression of HER2, the underlying mechanism remains poorly understood. Herein, we found that MUC13 co-localizes and interacts with HER2 in PDAC cells (reciprocal co-immunoprecipitation, immunofluorescence, proximity ligation, co-capping assays) and tissues (immunohistofluorescence). The results from this study demonstrate that MUC13 functionally interacts and activates HER2 at p1248 in PDAC cells, leading to stimulation of HER2 signaling cascade, including ERK1/2, FAK, AKT and PAK1 as well as regulation of the growth, cytoskeleton remodeling and motility, invasion of PDAC cells-all collectively contributing to PDAC progression. Interestingly, all of these phenotypic effects of MUC13-HER2 co-localization could be effectively compromised by depleting MUC13 and mediated by the first and second EGF-like domains of MUC13. Further, MUC13-HER2 co-localization also holds true in PDAC tissues with a strong functional correlation with events contributing to increased degree of disorder and cancer aggressiveness. In brief, findings presented here provide compelling evidence of a functional ramification of MUC13-HER2: this interaction could be potentially exploited for targeted therapeutics in a subset of patients harboring an aggressive form of PDAC.

Exposure of phosphatidylserine on the cell surface.

Phosphatidylserine (PtdSer) is a phospholipid that is abundant in eukaryotic plasma membranes. An ATP-dependent enzyme called flippase normally keeps PtdSer inside the cell, but PtdSer is exposed by the action of scramblase on the cell's surface in biological processes such as apoptosis and platelet activation. Once exposed to the cell surface, PtdSer acts as an 'eat me' signal on dead cells, and creates a scaffold for blood-clotting factors on activated platelets. The molecular identities of the flippase and scramblase that work at plasma membranes have long eluded researchers. Indeed, their identity as well as the mechanism of the PtdSer exposure to the cell surface has only recently been revealed. Here, we describe how PtdSer is exposed in apoptotic cells and in activated platelets, and discuss PtdSer exposure in other biological processes.

Pharmacokinetics of procaterol in thoroughbred horses.

Procaterol (PCR) is a beta-2-adrenergic bronchodilator widely used in Japanese racehorses for treating lower respiratory disease. The pharmacokinetics of PCR following single intravenous (0.5 µg/kg) and oral (2.0 µg/kg) administrations were investigated in six thoroughbred horses. Plasma and urine concentrations of PCR were measured using liquid chromatography-mass spectrometry. Plasma PCR concentration following intravenous administration showed a biphasic elimination pattern. The systemic clearance was 0.47 ± 0.16 L/h/kg, the steady-state volume of the distribution was 1.21 ± 0.23 L/kg, and the elimination half-life was 2.85 ± 1.35 h. Heart rate rapidly increased after intravenous administration and gradually decreased thereafter. A strong correlation between heart rate and plasma concentration of PCR was observed. Plasma concentrations of PCR after oral administration were not quantifiable in all horses. Urine concentrations of PCR following intravenous and oral administrations were quantified in all horses until 32 h after administration. Urine PCR concentrations were not significantly different on and after 24 h between intravenous and oral administrations. These results suggest that the bioavailability of orally administrated PCR in horses is very poor, and the drug was eliminated from the body slowly based on urinary concentrations. This report is the first study to demonstrate the pharmacokinetic character of PCR in thoroughbred horses.

Evaluation of nanodispersion of iron oxides using various polymers.

In order to create Fe2O3 and Fe2O3·H2O nanoparticles, various polymers were used as dispersing agents, and the resulting effects on the dispersibility and nanoparticulation of the iron oxides were evaluated. It was revealed that not only the solution viscosity but also the molecular length of the polymers and the surface tension of the particles affected the dispersibility of Fe2O3 and Fe2O3·H2O particles. Using the dispersing agents 7.5% hydroxypropylcellulose-SSL, 6.0% Pharmacoat 603, 5.0% and 6.5% Pharmacoat 904 and 7.0% Metolose SM-4, Fe2O3 nanoparticles were successfully fabricated by wet milling using Ultra Apex Mill. Fe2O3·H2O nanoparticles could also be produced using 5.0% hydroxypropylcellulose-SSL and 4.0 and 7.0% Pharmacoat 904. The index for dispersibility developed in this study appears to be an effective indicator of success in fabricating nanoparticles of iron oxides by wet milling using Ultra Apex Mill.

Serum milk fat globule epidermal growth factor 8 elevation may subdivide systemic lupus erythematosus into two pathophysiologically distinct subsets.

OBJECTIVE: Impaired clearance of apoptotic cells is a potential trigger of systemic lupus erythematosus (SLE). Milk fat globule epidermal growth factor 8 (MFG-E8) plays an important role in the clearance of dying cells. Previously, we reported serum MFG-E8 was elevated in some SLE patients. Here we further investigated the prevalence of MFG-E8 in active SLE and other autoimmune diseases and also tried to clarify the characteristics of MFG-E8-positive and -negative SLE. METHODS: Serum MFG-E8 was measured in 40 active non-treated SLE patients, 104 disease controls and 104 healthy controls by ELISA. Clinical characteristics and serum cytokine profiles were compared between MFG-E8-positive and MFG-E8-negative SLE patients. RESULTS: Prevalence of MFG-E8 was significantly higher in SLE patients (40%) than in various controls (p < 0.05). MFG-E8 level became negative after treatment, and increased again upon relapse. When compared, MFG-E8-positive SLE patients showed higher immune complex (p = 0.021) and lower complement (p = 0.004 for CH50). In contrast, MFG-E8-negative SLE patients tended to show higher CRP (p = 0.094). There was a positive correlation between MFG-E8 level and immune complex level (r s = 0.49, p = 0.049). TNF-α (p = 0.019), IFN-γ (p = 0.031) and IL-10 (p = 0.013) were significantly higher in MFG-E8-positive SLE. CONCLUSION: MFG-E8-positive SLE and -negative SLE may have different clinical features, the one with stronger immunological response and the other with stronger inflammatory response, and those two groups may be two distinct subtypes of SLE driven by different mechanisms. Further, MFG-E8 could be used as a biomarker for diagnosis and monitoring of disease activity in certain SLE patients.

Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids.

The phagocytic clearance of apoptotic cells is essential to prevent chronic inflammation and autoimmunity. The phosphatidylserine-binding protein milk fat globule-EGF factor 8 (MFG-E8) is a major opsonin for apoptotic cells, and MFG-E8(-/-) mice spontaneously develop a lupus-like disease. Similar to human systemic lupus erythematosus (SLE), the murine disease is associated with an impaired clearance of apoptotic cells. SLE is routinely treated with glucocorticoids (GCs), whose anti-inflammatory effects are consentaneously attributed to the transrepression of pro-inflammatory cytokines. Here, we show that the GC-mediated transactivation of MFG-E8 expression and the concomitantly enhanced elimination of apoptotic cells constitute a novel aspect in this context. Patients with chronic inflammation receiving high-dose prednisone therapy displayed substantially increased MFG-E8 mRNA levels in circulating monocytes. MFG-E8 induction was dependent on the GC receptor and several GC response elements within the MFG-E8 promoter. Most intriguingly, the inhibition of MFG-E8 induction by RNA interference or genetic knockout strongly reduced or completely abolished the phagocytosis-enhancing effect of GCs in vitro and in vivo. Thus, MFG-E8-dependent promotion of apoptotic cell clearance is a novel anti-inflammatory facet of GC treatment and renders MFG-E8 a prospective target for future therapeutic interventions in SLE.

Growth of Japanese breastfed infants compared to national references and World Health Organization growth standards.

AIM: The aim of this study was to compare the growth of Japanese infants that were exclusively breastfed to those of national references and World Health Organization (WHO) standards. METHODS: Mothers, who delivered a normal term baby and had been exclusively breastfeeding for at least 4 months, were enrolled. The lengths, body weights and head circumferences of 647 children, aged 0-24 months, were obtained and compared to national references and WHO standards. RESULTS: Comparisons of the national references for both length and body weight indicated that breastfed infants were significantly shorter and lighter almost throughout the first 24 months. Conversely, head circumferences of breastfed infants were significantly larger at 1 and 6 months of age in boys and 6 months in girls. Compared to WHO standards, similar trends to the comparisons with national references were found. CONCLUSION: There were significant differences identified between the growth of breastfed infants and existing national references and WHO standards.

Apaf-1- and Caspase-8-independent apoptosis.

Two major apoptosis pathways, the mitochondrial and death receptor pathways, are well recognized. Here we established cell lines from the fetal thymus of Apaf-1-, Caspase-9-, or Bax/Bak-deficient mice. These cell lines were resistant to apoptosis induced by DNA-damaging agents, RNA or protein synthesis inhibitors, or stress in the endoplasmic reticulum. However, they underwent efficient apoptosis when treated with kinase inhibitors such as staurosporine and H-89, indicating that these inhibitors induce a caspase-dependent apoptosis that is different from the mitochondrial pathway. CrmA, a Caspase-8 inhibitor, did not prevent staurosporine-induced apoptosis of fetal thymic cell lines, suggesting that the death receptor pathway was also not involved in this process. The staurosporine-induced cell death was inhibited by okadaic acid, a serine/threonine phosphatase inhibitor, suggesting that dephosphorylation of a proapoptotic molecule triggered the death process, or that phosphorylation of an antiapoptotic molecule could block the process. Cells of various types (fetal thymocytes, bone marrows, thymocytes, and splenocytes), but not embryonic fibroblasts, were sensitive to the noncanonical staurosporine-induced apoptosis, suggesting that the noncanonical apoptosis pathway is tissue specific.

Cardiac metastasis of head and neck squamous cell carcinoma.

Two patients with cardiac metastasis from head and neck cancer are reported. Cardiac metastasis located in the left atrium was detected on a follow-up computed tomography (CT) scan 15 months after partial glossectomy for a tongue carcinoma in a 60-year-old man. The diagnosis was confirmed as cardiac metastasis of squamous cell carcinoma (SCC) by surgical excision of the cardiac lesion. The patient died 3 weeks after surgery. In a 69-year-old man with a partial maxillectomy for primary soft palate cancer, a follow-up CT scan 5 months after surgery revealed a mass in the right atrium and ventricle, and multiple lung metastases. He died of heart failure 3 weeks after the diagnosis of cardiac metastasis. Information on these cases should add to knowledge about rarely encountered cardiac metastasis.