The people behind the papers – Masaki Kinoshita, Toshihiro Kobayashi, Hiroshi Nagashima, Ramiro Alberio and Austin Smith

The ability to derive and maintain pluripotent stem cells (PSCs) from livestock species in defined media conditions will contribute to many new research avenues, including comparative embryology and xenotransplantation. In a new paper in Development, Masaki Kinoshita, Toshihiro Kobayashi, Hiroshi Nagashima, Ramiro Alberio, Austin Smith and colleagues describe their three-component medium, which supports long-term propagation of PSCs in the absence of feeders or serum factors. We caught up with the authors to find out more about their research and their future plans.

Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species

ABSTRACT Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated ‘The people behind the papers’ interview.

A novel ex vivo model for critical illness neuromyopathy using freshly resected human colon smooth muscle

Patients suffering from critical illness are at risk to develop critical illness neuromyopathy (CINM). The underlying pathophysiology is complex and controversial. A central question is whether soluble serum factors are involved in the pathogenesis of CINM. In this study, smooth muscle preparations obtained from the colon of patients undergoing elective surgery were used to investigate the effects of serum from critically ill patients. At the time of blood draw, CINM was assessed by clinical rating and electrophysiology. Muscle strips were incubated with serum of healthy controls or patients in organ baths and isometric force was measured. Fifteen samples from healthy controls and 98 from patients were studied. Ratios of responses to electric field stimulation (EFS) before and after incubation were 118% for serum from controls and 51% and 62% with serum from critically ill patients obtained at day 3 and 10 of critical illness, respectively (p = 0.003, One-Way-ANOVA). Responses to carbachol and high-K+ were equal between these groups. Ratios of post/pre-EFS responses correlated with less severe CINM. These results support the existence of pathogenic, i.e. neurotoxic factors in the serum of critically ill patients. Using human colon smooth muscle as a bioassay may facilitate their future molecular identification.

Epicatechin ameliorative effects on methotrexate-induced hepatotoxicity in mice

Background Due to the fact that methotrexate is widely used both as an immunosuppressive drug and as a chemotherapy agent, many studies are needed to reduce the side effects of this drug on non-target organs. Purpose This study was designed to investigate the effects of epicatechin (Epi) on MTX (methotrexate)-induced hepatotoxicity in mice. Research Design After 1 week for adaptation, we randomly divided 42 male Naval Medical Research Institute mice into six groups: (I) control; (II) Epi (100 mg/kg, po); (III) MTX (20 mg/kg, i.p.) on the fifth day; and (IV, V, and VI) Epi (25, 50, and 100 mg/kg, po) + MTX (20 mg/kg, i.p.) on the fifth day. At day 10, the mice were sacrificed and serum factors, oxidative stress markers, and inflammatory cytokines were measured. Results MTX increased activity level of serum enzymes (alanine aminotransferase and aspartate aminotransferase), lipid peroxidation marker (malondialdehyde), and inflammatory factors including interleukin-1 beta, tumor necrosis factor-alpha, and nitric oxide. Furthermore, MTX decreased glutathione level and activity level of catalase, superoxide dismutase, and glutathione peroxidase. Epi was able to reduce the destructive effects of oxidative/antioxidant system imbalance and inflammatory reactions and also histopathological damage in MTX intoxicated mice. Epi pretreatment reduced liver dysfunction by improving the antioxidant defense system, anti-inflammatory effects, and alleviation of histopathological damage in MTX hepatotoxicity. Conclusions Accordingly, Epi can be used as a therapeutic agent in hepatotoxicity associated with MTX chemotherapy.

The Use of Human Serum Samples to Study Malignant Transformation: A Pilot Study

Obesity and excess adiposity account for approximately 20% of all cancer cases; however, biomarkers of risk remain to be elucidated. While fibroblast growth factor-2 (FGF2) is emerging as an attractive candidate biomarker for visceral adipose tissue mass, the role of circulating FGF2 in malignant transformation remains unknown. Moreover, functional assays for biomarker discovery are limited. We sought to determine if human serum could stimulate the 3D growth of a non-tumorigenic cell line. This type of anchorage-independent 3D growth in soft agar is a surrogate marker for acquired tumorigenicity of cell lines. We found that human serum from cancer-free men and women has the potential to stimulate growth in soft agar of non-tumorigenic epithelial JB6 P+ cells. We examined circulating levels of FGF2 in humans in malignant transformation in vitro in a pilot study of n = 33 men and women. Serum FGF2 levels were not associated with colony formation in epithelial cells (r = 0.05, p = 0.80); however, a fibroblast growth factor receptor-1 (FGFR1) selective inhibitor significantly blocked serum-stimulated transformation, suggesting that FGF2 activation of FGFR1 may be necessary, but not sufficient for the transforming effects of human serum. This pilot study indicates that the FGF2/FGFR1 axis plays a role in JB6 P+ malignant transformation and describes an assay to determine critical serum factors that have the potential to promote tumorigenesis.

Effects of sleeve gastrectomy on bone mass, microstructure of femurs and bone metabolism associated serum factors in obese rats

Background Sleeve gastrectomy (SG) is a profoundly effective operation for severe obese patients, but is closely associated with bone mass loss. Previous studies have reported changes of various serum factors which may be associated with bone mass loss after SG. However, those results are contradictory. In this study, we assessed the effects of SG on bone mass, microstructure of femurs, and changes in bone turnover markers (BTMs), serum adipokines, inflammatory factors and gastrointestinal hormones after SG in high-fat diet (HFD) induced obese rats. Methods Eight-week-old male Sprague–Dawley (SD) rats were fed with HFD to induce obesity. Then, SG and sham surgery were performed in anesthetized obese rats. SD rats in control group were fed with standard chow. Microstructure of femurs was scanned and analyzed by micro-computed tomography in control group, HFD sham group and HFD SG group. Serum inflammatory factors, adipokines markers, gastrointestinal hormones and BTMs were also measured. Results Bone mineral density (BMD) of trabecular bone in both HFD sham group and HFD SG group were remarkably decreased compared with control group. All serum BTMs were significantly higher in HFD SG group than HFD sham group. In the meantime, serum levels of several important inflammatory factors, gastrointestinal hormones and adipokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, monocyte chemoattractant protein-1(MCP-1), ghrelin, insulin and leptin in HFD SG group were remarkably reduced compared with HFD sham group, whereas glucagon-like peptide-1 (GLP-1), adiponectin, fibroblast growth factor (FGF)-19 and FGF-21 were dramatically increased after SG. Protein tyrosine phosphatase 1B (PTP1B) was significantly increased in the HFD sham group than control group. Spearman’s correlation analysis indicated that serum osteocalcin (OC) and 25-hydroxy vitamin D3 (25(OH)D3) were positively correlated with BMD of trabecular bone, whereas serum PTP1B and TNF-α were negatively related to BMD of trabecular bone. Conclusions SG aggravates bone mass loss and activates bone remodeling in obese rats. Levels of BTMs, adipokines, inflammatory factors, and gastrointestinal hormones could be affected by SG in obese rats. Serum PTP1B level might be associated with abnormal bone mass in obese rats.

Effects of Rapamycin on Insulin Brain Endothelial Cell Binding and Blood–Brain Barrier Transport

Rapamycin is an exogenous compound that has been shown to improve cognition in Alzheimer’s disease mouse models and can regulate pathways downstream of the insulin receptor signaling pathway. Insulin is also known to improve cognition in rodent models of Alzheimer’s disease. Central nervous system (CNS) insulin must first cross the blood–brain barrier (BBB), a specialized network of brain endothelial cells. This transport process is regulated by physiological factors, such as insulin itself, triglycerides, cytokines, and starvation. Since rapamycin treatment can alter the metabolic state of rodents, increase the circulating triglycerides, and acts as a starvation mimetic, we hypothesized rapamycin could alter the rate of insulin transport across the BBB, providing a potential mechanism for the beneficial effects of rapamycin on cognition. Using young male and female CD-1 mice, we measured the effects of rapamycin on the basal levels of serum factors, insulin receptor signaling, vascular binding, and BBB pharmacokinetics. We found chronic rapamycin treatment was able to affect basal levels of circulating serum factors and endothelial cell insulin receptor signaling. In addition, while acute rapamycin treatment did affect insulin binding at the BBB, overall transport was unaltered. Chronic rapamycin slowed insulin BBB transport non-significantly (p = 0.055). These results suggest that rapamycin may not directly impact the transport of insulin at the BBB but could be acting to alter insulin signaling within brain endothelial cells, which can affect downstream signaling.

Interaction Between Acute Exercise and Carnitine Supplementation on the Expression of Genes Involved in Liver Metabolism in Male Rats

Acute exercise induces rapid and dramatic induction of transcription in the liver. The beneficial effects of carnitine on serum factors and gene expression have been proven. This study examined the interaction between acute exercise and carnitine supplementation on the expression of genes involved in liver metabolism. Thirty-two male Wistar rats were randomly assigned into 4 groups (n = 8): Group 1 control, Group 2 received 200 mg/kg/day LCAR, Group 3 performed acute exercise, and Group 4 received LCAR and performed acute exercise. Gene expression in the liver was evaluated by Real-time PCR. Acute exercise significantly increased PDK4 expression compared to other groups. Also, carnitine administration, performing an acute exercise, and combination of LCAR-Acute significantly increased AMPK and PGC-1a expression compared with the control group. The expression of SREBP-1c and SCD1 was not significantly changed between studies. The combination of acute exercise and carnitine administration increased PGC-1a expression, indicating the importance of carnitine with exercise as a beneficial supplement.

Effects of a moderate intensity aerobic exercise on serum levels of BDNF, DCX, and DBHB in triathlon male adolescents

Background: Various serum factors in response to aerobic exercise improve cognitive function in healthy individuals; nonetheless, the effect of moderate intensity aerobic exercise on their levels has not been studied. Therefore, the present study aimed to assess the effect of a moderate intensity aerobic exercise on serum levels of Brain-derived neurotrophic factor (BDNF), Doublecortin (DCX), and D-β-hydroxybutyrate (DBHB) in triathlon male adolescents in Tuyserkan. Materials and Methods: A total of 22 triathlon male adolescents (age: 17.60±0.52 years, weight: 60.74±4.96 kg, height: 175.60±5.52 cm) voluntarily participated in this quasi-experimental study. Subjects in one session performed 60 minutes of running with an intensity of 12-13 on the Borg scale. Blood samples were taken from the subjects 5 min before and after training. Serum levels of BDNF, DCX and DBHB were measured by ELISA. To analyze the data, paired samples t-test was used, and a p-value less than 0.05 was considered statistically significant. In addition, the percentage of changes was compared to pre-test. Results: Based on the results, the serum levels of DCX (P=0.0005), BDNF (P=0.0005), and DBHB (P=0.001) increased significantly, compared to those in the pretest. Furthermore, serum levels of BDNF (21.30%), DBHB (12.95%) and DCX (12.01%) had the highest increase, as compared to the pretest, respectively. Conclusion: All three serum factors BDNF, DCX, and DBHB have a significant positive response to moderate-intensity aerobic exercise in triathlon male adolescents. Nevertheless, neurotrophin/growth factor BDNF demonstrates more changes and appears to play a greater role in neuroprotection, improving memory, learning, and cognitive function.

289 Serum Trace Minerals in Late Gestation Sows at Variable Risk for Pelvic Organ Prolapse

Within the last decade, pelvic organ prolapse (POP) resulting in sow mortality has become an increasing concern for the U.S. swine industry, contributing approximately 21% of all sow deaths. While little is known regarding the etiology preceding POP in sows, many have proposed an association with vitamin and trace minerals abundance to POP incidence. We tested the hypothesis that sows differing in POP risk would have differences in serum trace minerals and vitamins. A perineal scoring (PS) system (PS1 - presumed low risk; PS2 - presumed moderate risk; and PS3 - presumed high risk) to assess risk for POP during late gestation was used to score 213 individual sows. Blood was collected from sows of two different farms during late gestation (days 105–115) that scored a PS3 (n = 20) and a parity matched sow scored as PS 1 (n = 16). Subsequently, 1.5, 0.8, and 23.1% of sows scored as PS1, PS2, or PS3, respectively, experienced POP. Serum was analyzed at the Iowa State University Veterinary Diagnostic Lab for trace mineral content (Calcium, Copper, Iron, Potassium, Magnesium, Manganese, Molybdenum, Phosphorus, Selenium, and Zinc). Additionally, vitamin E was evaluated in serum via GC-MS. Differences (P < 0.05) in copper, potassium, molybdenum, phosphorus, and selenium was observed between farms. Further a PS x Farm interaction (P = 0.06) was observed for serum copper abundance which across all farms was 12% less (P < 0.04) in PS3 compared to PS1 sows. No impact on serum vitamin E was observed between PS sows. These data demonstrate sows with greater POP risk may have potential differences in serum factors although these data also underscore the importance of measuring vitamin and mineral quantities in a tissue specific manner. This project was supported by the National Pork Board and the Foundation for Food and Agriculture Research.