Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.     

Very strong UV-A light temporally separates the photoinhibition of photosystem II into light-induced inactivation and repair

When organisms that perform oxygenic photosynthesis are exposed to strong visible or UV light, inactivation of photosystem II (PSII) occurs. However, such organisms are able rapidly to repair the photoinactivated PSII. The phenomenon of photoinactivation and repair is known as photoinhibition. Under normal laboratory conditions, the rate of repair is similar to or faster than the rate of photoinactivation, preventing the detailed analysis of photoinactivation and repair as separate processes. We report here that, using strong UV-A light from a laser, we were able to analyze separately the photoinactivation and repair of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803. Very strong UV-A light at 364 nm and a photon flux density of 2600 micromol photons m(-2) s(-1) inactivated the oxygen-evolving machinery and the photochemical reaction center of PSII within 1 or 2 min before the first step in the repair process, namely, the degradation of the D1 protein, occurred. During subsequent incubation of cells in weak visible light, the activity of PSII recovered fully within 30 min and this process depended on protein synthesis. During subsequent incubation of cells in darkness for 60 min, the D1 protein of the photoinactivated PSII was degraded. Further incubation in weak visible light resulted in the rapid restoration of the activity of PSII. These observations suggest that very strong UV-A light is a useful tool for the analysis of the repair of PSII after photoinactivation.     

Changes of cerebral blood oxygenation and optical pathlength during activation and deactivation in the prefrontal cortex measured by time-resolved near infrared spectroscopy

To determine the alterations in optical characteristics and cerebral blood oxygenation (CBO) during activation and deactivation, we evaluated the changes in mean optical pathlength (MOP) and CBO induced by a verbal fluency task (VFT) and driving simulation in the right and left prefrontal cortex (PFC), employing a newly developed time-resolved near infrared spectroscopy, which allows quantitative measurements of the evoked-CBO changes by determining the MOP with a sampling time of 1 s. The results demonstrated differences in MOP in the foreheads with the subjects and wavelength; however, there was no significant difference between the right and left foreheads (p > 0.05). Also, both the VFT and driving simulation task did not affect the MOP significantly as compared to that before the tasks (p > 0.05). In the bilateral PFCs, the VFT caused increases of oxyhemoglobin and total hemoglobin associated with a decrease of deoxyhemoglobin, while the driving simulation task caused decreases of oxyhemoglobin and total hemoglobin associated with an increase of deoxyhemoglobin; there were no significant differences in evoked-CBO changes between the right and left PFC. The present results will be useful for quantitative measurement of hemodynamic changes during activation and deactivation in the adults by near infrared spectroscopy.     

Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress

Vitamin C (l-ascorbate) is important for antioxidative and metabolic functions in both plants and humans. Ascorbate itself is oxidized to dehydroascorbate during the process of antioxidation, and dehydroascorbate reductase (DHAR, EC 1.8.5.1) re-reduces the oxidized ascorbate. Therefore, this enzyme is assumed to be critical for ascorbate recycling. Here we show that the expression of rice DHAR in transgenic Arabidopsis thaliana enhanced resistance to salt stress. Salt tolerance was remarkably improved despite slight increases in DHAR activity and total ascorbate. This study provides direct evidence for the importance of DHAR in salt tolerance.     

Modified substrate specificity of pyrroloquinoline quinone glucose dehydrogenase by biased mutation assembling with optimized amino acid substitution

A biased mutation-assembling method—that is, a directed evolution strategy to facilitate an optimal accumulation of multiple mutations on the basis of additivity principles, was applied to the directed evolution of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to reduce its maltose oxidation activity, which can lead to errors in blood glucose determination. Mutations appropriate for the reduction without fatal deterioration of its glucose oxidation activity were developed by an error-prone PCR method coupled with a saturation mutagenesis method. Moreover, two types of incorporation frequency based on their contribution were assigned to the mutations: high (80%) and evens (50%), in constructing a multiple mutant library. The best mutant created showed a marked reduction in maltose oxidation activity, corresponding to 4% of that of the wild-type enzyme, with 35% retention of glucose oxidation activity. In addition, this mutant showed a reduction in galactose oxidation activity corresponding to 5% of that of the wild-type enzyme. In conclusion, we succeeded in developing the PQQGDH-B mutants with improved substrate specificity and validated our method coupled with optimized mutations and their contribution-based incorporation frequencies by applying it to the development.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Periostin is an extracellular matrix protein required for eruption of incisors in mice

A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin-/- mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin-/- mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone.     

The cytochrome P450 gene family CYP157 does not contain EXXR in the K-helix reducing the absolute conserved P450 residues to a single cysteine

In this work, we have spectroscopically characterised CYP157C1 from Streptomyces coelicolor A3(2) which has the motif E(297)QSLW(301) rather than the invariant EXXR motif in the P450 K-helix. Site-directed mutagenesis of native E(297)QSLW(301) in CYP157C1 to E(297)ESLR(301) or E(297)QSRW(301) both containing standard EXXR motifs produced cytochrome P420 proteins thought to be inactive forms of P450 even though wild type CYP157C1 has the spectral properties of a normal P450. These results indicate that the EXXR motif is not required in all CYP tertiary architectures and only a single cysteine residue, which coordinates as the fifth thiolate ligand to the P450 haem iron, is invariant in all CYPs structures.     

Validation of an HPLC Analytical Method Coupled to a Multifunctional Clean-up Column for the Determination of Deoxynivalenol

To evaluate a method using a multifunctional clean-up column coupled with high performance liquid chromatography as an official analytical method for the determination of deoxynivalenol in wheat used as food or feed, an inter-laboratory study was performed in 12 laboratories using four naturally contaminated wheat samples and one spiked sample. The relative standard deviations for repeatability (RSD_r) and reproducibility (RSD_R) of naturally contaminated wheat were in the range 5.8–11.3% and 12.0–20.7%, respectively. The HORRAT was less than 1.0 in each sample. From the spiking test, the recovery rate, RSD_r, RSD_R and HORRAT value were 100.0%, 11.2%, 10.3% and 0.5, respectively. The limit of quantification is 0.10 mg/kg from the range obtained in a linear calibration. Thus, it should be useful as a sensitive and validated analytical method for the determination of deoxynivalenol in wheat intended for use in food and feed.     

Establishment of the primary imprint of the HYMAI/PLAGL1 imprint control region during oogenesis

Imprinting within domains occurs through epigenetic alterations to imprinting centers (ICs) that result in the establishment of parental-specific differences in gene expression. One candidate IC lies within the imprinted domain on human chromosome region 6q24. This domain contains two paternally expressed genes, the zinc finger protein gene PLAGL1 (ZAC/LOT1) and an untranslated mRNAcalled HYMAI. The putative IC overlaps exon 1 of HYMAI and is differentially methylated in somatic tissues. In humans, loss of methylation within this region is seen in some patients with transient neonatal diabetes mellitus, and hypermethylation of this region is found in ovarian cancer and is associated with changes in expression of PLAGL1, suggesting that it plays a key role in regulating gene expression. Differential methylation within this region is conserved in the homologous region on mouse chromosome 10A and is present on the maternal allele. In this paper, we report that DNA methylation is established during the growth phase of oogenesis and that this coincides with the establishment of monoallelic expression from this region lending further support to the hypothesis that this region functions as an IC.