Two species of goby, Boleophthalmus pectinirostris and Scartelaos sp., as the new second intermediate hosts of heterophyid fluke in Korea

A survey was performed in order to determine the infection status of the metacercariae of heterophyid fluke in two goby species, Boleophthalmus pectinirostris and Scartelaos sp., collected from Gangjin-gun, and Shinangun, Sooncheon-shi, Jeollanam-do, Republic of Korea. A total of three metacercariae of Heterophyopsis continua was found in only one B. pectinirostris (10.0%) from Gangjin-gun. Heterophyes nocens metacercariae were detected in 24 B. pectinirostris (96.0%) and 14 Scartelaos sp. (63.6%) from Shinan-gun. Heterophyopsis continua metacercariae were found in 11 B. pectinirostris (44.0%) and 21 Scartelaos sp. (95.5%) from Shinan-gun. Stictodora fuscata metacercariae were detected in 18 B. pectinirostris (72.0%) from Shinan-gun. No metacercariae were detected in 20 B. pectinirostris from Sooncheon-shi. From the above results, this study is the first to prove that B. pectinirostris and Scartelaos sp. serve as the second intermediate hosts of some heterophyid flukes in Korea.     

Up-scaling single cell-inoculated suspension culture of human embryonic stem cells

We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only ～ 44% of the seeded cells were rescued, but an optimized heat shock treatment combined with Ri significantly increased cell survival to ～ 60%. Mechanistically, our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2, hES3) that were used at higher passages (> 86). In contrast, rapid down regulation of Oct4, Tra-1-60, and SSEA4 was observed for ESI049, a clinically compliant line, used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless, our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines.     

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi‐lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2‐DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin–avidin affinity column were separated by 2‐DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole‐time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin‐related proteins, F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF‐induced morphological change of MSCs.     

The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos

Preovulatory mouse oocytes and 2-cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33–34%) compared to that of 2-cell embryos (78–79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7–15% for oocytes and 79–80% for the embryos. Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2-cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze-thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.     

Characterization and in vitro selection for iron efficiency inPyrus andCydonia

Summary Responses to low Fe were characterized in tissue cultures ofPyrus amygdaliformis andCydonia oblonga (quince), two species used as rootstocks for pear. Cultured shoots and plantlets ofP. amygdaliformis had a higher chlorophyll concentration and Fe²⁺/total Fe ratio than those ofC. oblonga when grown under low Fe conditions. This tolerance to low Fe was correlated with high Fe³⁺-reducing ability and medium acidification. The adaptive responses were manifested in roots of plantlets, shoot bases, root cultures, and cell suspension cultures. Shoots were regenerated from leaves of quince and subjected to Fe-deficient conditions. Two somaclonal variants (IE-1 and IE-2) were recovered; each displayed higher ability to reduce Fe³⁺ and acidify the medium. These variants may be useful as rootstocks for regions with calcareous soils, which limit Fe availability.     

Evaluation of the Enhanced Antioxidant Activity of Curcumin within Exosomes by Fluorescence Monitoring

In this study, we compared the antioxidant activities of curcumin (Cur) and a Cur formulation using a fluorescence analysis assay. The Cur formulation was prepared by a simple incorporation of Cur into exosomes (EXO) to produce Cur/EXOs. Free Cur had a low fluorescence intensity in aqueous solution because of its poor stability as a result of its autoxidation, whereas a significantly higher fluorescence intensity was observed for Cur/EXOs. Compared to free Cur, the increased level of intact Cur in EXOs allowed for enhanced antioxidant activity in H₂O₂ scavenging activity and DPPH assays. Compared to Cur at high concentration (200 μM), Cur/ EXOs were significantly less cytotoxic. The antioxidant activity of Cur or Cur/EXOs in cells could be easily demonstrated by monitoring decreases in their fluorescence intensity. Following subcutaneous injection, the fluorescence intensities of Cur/EXOs were much higher than that of Cur, suggesting that Cur/EXOs improve Cur stability in vivo. Taken together, we have demonstrated the superiority of Cur/EXOs over free Cur in terms of aqueous stability and antioxidant activity using fluorescence monitoring both in vitro and in vivo.     

Development of Electrochemiluminescent Serology Assays to Measure the Humoral Response to Antigens of Respiratory Syncytial Virus

Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized “gold standard” panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the “gold standard” diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population.     

Examination of the potential role of the glycerophosphorylcholine (GPC) pathway in the biosynthesis of phosphatidylcholine by liver and lung

The potential involvement of the glycerophosphorylcholine (GPC) pathway for the synthesis of phosphatidylcholine (PC) has been examined in rat liver and lung and in a human line, the A549 cell which possesses characteristics representative of mature alveolar type II epithelial cells. Although mitochondrial and microsomal fractions from the above sources readily incorporated radioactive glycerophosphate into lipids, the only incorporation observed with radioactive GPC was a small variable labelling with the mitochondrial and microsomal fractions from rat lung. Even with these fractions, no radioactivity from GPC was incorporated into PC or lysoPC. Attempts to increase the incorporation of GPC into lipids by manipulating the incubation conditions were unsuccessful. It was concluded that the occurrence of the GPC pathway in liver and lung is unlikely.