Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation

When buffalo embryonic stem (ES) cell–like cells that expressed surface markers SSEA‐4, TRA‐1‐60, TRA‐1‐81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX‐1 and NUCLEOSTEMIN as confirmed by RT‐PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three‐dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF‐68 and NESTIN (ectodermal lineage), BMP‐4 and α‐skeletal actin (mesodermal lineage), and α‐fetoprotein, GATA‐4 and HNF‐4 (endodermal lineage). When these EBs were cultured on gelatin‐coated dishes, they spontaneously differentiated to several cell types such as epithelial‐ and neuron‐like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10^?8 m or 10^?7 m retinoic acid for 25 days, ES cells could be directed to form muscle cell–like cells, the identity of which was confirmed by expression of α‐actinin by immunofluorescence and of MYF‐5, MYOD and MYOGENIN genes by RT‐PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell–like cells to undergo directed differentiation to cells of skeletal myogenic lineage.     

Quantitative risk assessment for the annual risk of exposure to Trichinella spiralis in imported chilled pork meat from New Zealand to Singapore

Abstract

AIMS: To determine the annual likelihood of exposure to an infectious dose of Trichinella spiralis from consuming imported pork meat from New Zealand to Singapore.

METHODS: Input values specific for chilled pork meat imported into Singapore from New Zealand were used in a quantitative risk-assessment model. The model, designed to allow any combination of importing and exporting countries, was divided into two components, viz the release assessment, and the exposure assessment that assessed the annual risk of exposure to the consumer (ARC). The former estimated the likelihood that a contaminated fresh meat product from New Zealand would arrive at Singapore's border, and took into consideration the prevalence of disease on different types of farms. The latter determined the likelihood over a year that a person in Singapore would consume one or more servings of imported fresh meat from New Zealand that contained a burden of greater than or equal to one larva(e) of T. spiralis per gram after preparation for consumption.

RESULTS: The ARC for offal was 2.41 × 10^?7, which was below the pre-selected safety threshold of 1.00 × 10^?6. The ARC for lean meat was 2.39 x 10^?5, which was above the acceptable safety threshold.

CONCLUSIONS: The study demonstrated that continued routine testing at slaughter is unnecessary for pig offal produced commercially, and provided a model with which to further assess management of the risk of exposure to T. spiralis in lean meat.

CLINICAL RELEVANCE: The potential of Trichinella species to cause disease in humans is a public health concern, and has created adverse effects on the international trade of fresh lean meat without regard to the surveillance measures employed by particular pork-producing countries.     

The behavior of clomazone in the soil environment

BACKGROUND: Clomazone is a herbicide used to control broadleaf weeds and grasses. Clomazone use in agriculturally important crops and forests for weed control has increased and is a potential water contaminant given its high water solubility (1100 µg mL^?1). Soil sorption is an environmental fate parameter that may limit its movement to water systems. The authors used model rice and forest soils of California to test clomazone sorption affinity, capacity, desorption, interaction with soil organic matter and behavior with black carbon. RESULTS: Sorption of clomazone to the major organic matter fraction of soil, humic acid (HA) (K_d = 29–87 L kg^?1), was greater than to whole soils (K_d = 2.3–11 L kg^?1). Increased isotherm non‐linearity was observed for the whole soils (N = 0.831–0.893) when compared with the humic acids (N = 0.954–0.999). Desorption isotherm results showed hysteresis, which was greatest at the lowest solution concentration of 0.067 µg mL^?1 for all whole soils and HA extracts. Aliphatic carbon content appeared to contribute to increased isotherm linearity. CONCLUSION: The results indicate that clomazone does not sorb appreciably to sandy or clay soils. Its sorption affinity and capacity is greater in humic acid, and consequently clomazone has difficulty desorbing from soil organic matter. Sorption appears to follow processes explained by the dual‐mode model, the presence of fire residues (black carbon) and a recently proposed sorption mechanism. Copyright © 2009 Society of Chemical Industry     

Short‐Term Antiandrogen Flutamide Treatment Causes Structural Alterations in Somatic Cells Associated with Premature Detachment of Spermatids in the Testis of Pubertal and Adult Guinea Pigs

In spite of widespread application of flutamide in the endocrine therapies of young and adult patients, the side effects of this antiandrogen on spermatogenesis and germ‐cell morphology remain unclear. This study evaluates the short‐term androgen blockage effect induced by the administration of flutamide to the testes of pubertal (30‐day old) and adult (65‐ and 135‐day old) guinea pigs, with an emphasis on ultrastructural alterations of main cell types. The testes removed after 10 days of treatment with either a non‐steroidal antiandrogen, flutamide (10 mg/kg of body weight) or a pharmacological vehicle alone were processed for histological, quantitative and ultrastructural analysis. In pubertal animals, flutamide androgenic blockage induces spermatogonial differentiation and accelerates testes maturation, causing degeneration and detachment of primary spermatocytes and round spermatids, which are subsequently found in great quantities in the epididymis caput. In post‐pubertal and adult guinea pigs, in addition to causing germ‐cell degeneration, especially in primary spermatocytes, and leading to the premature detachment of spherical spermatids, the antiandrogen treatment increased the relative volume of Leydig cells. In addition, ultrastructural evaluation indicated that irrespective of age antiandrogen treatment causes an increase in frequency of organelles involved with steroid hormone synthesis in the Leydig cells and a dramatic accumulation of myelin figures in their cytoplasm and, to a larger degree, in Sertoli cells. In conclusion, the transient exposition of the guinea pigs to flutamide, at all postnatal ages causes some degenerative lesions including severe premature detachment of spermatids and accumulation of myelin bodies in Leydig and Sertoli cells, compromising, at least temporarily, the spermatogenesis.     

Assessment of DNA Damage during In Vitro Development of Buffalo (Bubalus bubalis) Embryos: Effect of Cysteamine

Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 μm , respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8‐ to 16‐cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8‐ to 16‐ (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2‐cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8‐ to 16‐cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2‐cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H₂O₂ caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.     

Off-axis crustal thickness across and along the east pacific rise within the MELT area

Wide-angle seismic data along the Mantle Electromagnetic and Tomography (MELT) arrays show that the thickness of 0.5- to 1. 5-million-year-old crust of the Nazca Plate is not resolvably different from that of the Pacific Plate, despite an asymmetry in depth and gravity across this portion of the East Pacific Rise. Crustal thickness on similarly aged crust on the Nazca plate near a magmatically robust part of the East Pacific Rise at 17 degrees15'S is slightly thinner (5.1 to 5.7 kilometers) than at the 15 degrees55'S overlapping spreading center (5.8 to 6.3 kilometers). This small north-south off-axis crustal thickness difference may reflect along-axis temporal variations in magma supply, whereas the across-axis asymmetry in depth and gravity must be caused by density variations in the underlying mantle.