Quantitative PCR Method for Enumeration of Cells of Cryptic Species of the Toxic Marine Dinoflagellate Ostreopsis spp. in Coastal Waters of Japan

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.     

Optimization of slow cooling cryopreservation for human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cell types of all three germ layers. Cryopreservation is a key process for successful application of hPSCs. However, the current conventional method leads to poor recovery of hPSCs after thawing. Here, we demonstrate a highly efficient recovery method for hPSC cryopreservation by slow freezing and single‐cell dissociation. After confirming hPSC survivability after freeze‐thawing, we found that hPSCs that were freeze‐thawed as colonies showed markedly decreased survival, whereas freeze‐thawed single hPSCs retained the majority of their viability. These observations indicated that hPSCs should be cryopreserved as single cells. Freeze‐thawed single hPSCs efficiently adhered and survived in the absence of a ROCK inhibitor by optimization of the seeding density. The high recovery rate enabled conventional colony passaging for subculture within 3 days post‐thawing. The improved method was also adapted to a xeno‐free culture system. Moreover, the cell recovery postcryopreservation was highly supported by coating culture surfaces with human laminin‐521 that promotes adhesion of dissociated single hPSCs. This simplified but highly efficient cryopreservation method allows easy handling of cells and bulk storage of high‐quality hPSCs. genesis 52:49–55, 2014. © 2013 Wiley Periodicals, Inc.     

Regulation of the ASC expression in response to LPS stimulation is related to IL-8 secretion in the human intestinal mucosa

Expression of the caspase-activating adaptor ASC was reported to be associated with the production of IL-8, a primary mediator of mucosal inflammation, in vitro. However, the significance of the ASC-mediated IL-8 production in primary tissues has remained poorly understood. Primary intestinal mucosa isolated from surgically resected ileum or colon was incubated with several concentrations of LPS or left untreated. ASC expression was up-regulated at 2 h after stimulation with low doses of LPS (1-10 ng/ml), and was associated with IL-8 secretion, and then was down-regulated later. In contrast, ASC expression remained at the basal level in mucosal tissue treated with a high dose of LPS (1000 ng/ml). Interestingly, in mucosa from several cases of Crohn's disease, ASC was highly expressed without stimulation, and IL-8 was stably secreted with no regulation by LPS. These findings revealed that ASC expression correlates with IL-8 secretion and may play an important role in maintaining mucosal homeostasis.     

In vivo diagnostic imaging using micro-CT: sequential and comparative evaluation of rodent models for hepatic/brain ischemia and stroke

Background

There is an increasing need for animal disease models for pathophysiological research and efficient drug screening. However, one of the technical barriers to the effective use of the models is the difficulty of non-invasive and sequential monitoring of the same animals. Micro-CT is a powerful tool for serial diagnostic imaging of animal models. However, soft tissue contrast resolution, particularly in the brain, is insufficient for detailed analysis, unlike the current applications of CT in the clinical arena. We address the soft tissue contrast resolution issue in this report.

Methodology

We performed contrast-enhanced CT (CECT) on mouse models of experimental cerebral infarction and hepatic ischemia. Pathological changes in each lesion were quantified for two weeks by measuring the lesion volume or the ratio of high attenuation area (%HAA), indicative of increased vascular permeability. We also compared brain images of stroke rats and ischemic mice acquired with micro-CT to those acquired with 11.7-T micro-MRI. Histopathological analysis was performed to confirm the diagnosis by CECT.

Principal Findings

In the models of cerebral infarction, vascular permeability was increased from three days through one week after surgical initiation, which was also confirmed by Evans blue dye leakage. Measurement of volume and %HAA of the liver lesions demonstrated differences in the recovery process between mice with distinct genetic backgrounds. Comparison of CT and MR images acquired from the same stroke rats or ischemic mice indicated that accuracy of volumetric measurement, as well as spatial and contrast resolutions of CT images, was comparable to that obtained with MRI. The imaging results were also consistent with the histological data.

Conclusions

This study demonstrates that the CECT scanning method is useful in rodents for both quantitative and qualitative evaluations of pathologic lesions in tissues/organs including the brain, and is also suitable for longitudinal observation of the same animals.     

IL-33 mediates inflammatory responses in human lung tissue cells

IL-33 is a member of the IL-1 family and mediates its biological effects via the ST2 receptor, which is selectively expressed on Th2 cells and mast cells. Although polymorphic variation in ST2 is strongly associated with asthma, it is currently unclear whether IL-33 acts directly on lung tissue cells at sites of airway remodeling. Therefore, we aimed to identify the IL-33-responsive cells among primary human lung tissue cells. ST2 mRNA was expressed in both endothelial and epithelial cells but not in fibroblasts or smooth muscle cells. Correspondingly, IL-33 promoted IL-8 production by both endothelial and epithelial cells but not by fibroblasts or smooth muscle cells. Transfection of ST2 small interference RNA into both endothelial and epithelial cells significantly reduced the IL-33-dependent upregulation of IL-8, suggesting that IL-33-mediated responses in these cells occur via the ST2 receptor. Importantly, Th2 cytokines, such as IL-4, further enhanced ST2 expression and function in both endothelial and epithelial cells. The IL-33-mediated production of IL-8 by epithelial cells was almost completely suppressed by corticosteroid treatment. In contrast, the effect of corticosteroid treatment on the IL-33-mediated responses of endothelial cells was only partial. IL-33 induced activation of both ERK and p38 MAPK in endothelial cells but only ERK in epithelial cells. p38 MAPK was required for the IL-33-mediated responses of endothelial cells, whereas ERK was required for IL-33-mediated IL-8 production by epithelial cells. Taken together, these findings suggest that IL-33-mediated inflammatory responses of lung tissue cells may be involved in the chronic allergic inflammation of the asthmatic airway.     

Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells

Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human alpha-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of growth factors including hepatocyte growth factor (HGF), bone morphogenetic protein 4, fibroblast growth factor 4, all-trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and 20 ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.     

Characterization of mutations affecting embryonic hematopoiesis in the medaka, Oryzias latipes

In a genetic screen for mutations affecting organogenesis in the medaka, Oryzias latipes, we identified eight mutants with defects in embryonic hematopoiesis. These mutations were classified into seven complementation groups. In this paper, we characterize the five mutants that were confirmed in the next generation. The beni fuji mutant was defective in the generation of blood cells, exhibiting reduced blood cells at the initiation of circulation. Mutations in two genes, lady finger and ryogyoku, caused abnormal morphology of blood cells, i.e., deformation, along with a progressive decrease in the number of blood cells. The sekirei mutant exhibited photosensitivity with autofluorescent blood cells. Mutations in kyoho resulted in huge blood cells that were approximately three times longer than the wild-type blood cells. The spectrum of phenotypes identified in this study is similar to that of the zebrafish hematopoietic mutants except for the huge blood cells in kyoho. Our results demonstrate that medaka, as well as zebrafish, is a useful model to study hematopoiesis.     

Nitrogen dioxide air pollution near ambient levels is an atherogenic risk primarily in obese subjects: a brief communication

Ambient exposure to nitrogen dioxide, a critical air pollutant in developed countries, is positively associated with cardiovascular mortality and morbidity. Although its cardiovascular effects are predominantly shown in patients with high risk of atherogenesis, no studies have elucidated whether daily exposure to nitrogen dioxide air pollution enhances atherogenic metabolisms, primarily in obese subjects who are susceptible to atherogenesis and subsequent cardiovascular diseases. We used male Otsuka Long-Evans Tokushima Fatty (OLETF) rats as obese subjects and Long-Evans Tokushima (LETO) rats as nonobese controls. The animals were continuously exposed to nitrogen dioxide at a concentration of 0, 0.16, 0.8, or 4.0 ppm from 8 weeks of age through 32 weeks. At 40 weeks of age, levels of body weight, triglyceride, and total cholesterol were significantly greater in the OLETF rats than in the LETO rats. A ratio of high-density lipoprotein (HDL) to total cholesterol was significantly smaller in the former than in the latter. In the LETO rats, nitrogen dioxide exposure significantly decreased only the levels of HDL as compared with clean air exposure. In the OLETF rats, however, nitrogen dioxide exposure at a concentration of 0.16 ppm significantly elevated triglyceride concentration and decreased the ratio of HDL to total cholesterol as well as the levels of HDL. Nitrogen dioxide air pollution near ambient levels is an atherogenic risk primarily in obese subjects.     

Prokaryotic homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase in the 2-nitrobenzoate degradation pathway of Pseudomonas fluorescens strain KU-7

The 2-nitrobenzoic acid degradation pathway of Pseudomonas fluorescens strain KU-7 proceeds via a novel 3-hydroxyanthranilate intermediate. In this study, we cloned and sequenced a 19-kb DNA locus of strain KU-7 that encompasses the 3-hydroxyanthranilate meta-cleavage pathway genes. The gene cluster, designated nbaEXHJIGFCDR, is organized tightly and in the same direction. The nbaC and nbaD gene products were found to be novel homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, respectively. The NbaC enzyme carries out the oxidation of 3-hydroxyanthranilate to 2-amino-3-carboxymuconate-6-semialdehyde, while the NbaD enzyme catalyzes the decarboxylation of the latter compound to 2-aminomuconate-6-semialdehyde. The NbaC and NbaD proteins were overexpressed in Escherichia coli and characterized. The substrate specificity of the 23.8-kDa NbaC protein was found to be restricted to 3-hydroxyanthranilate. In E. coli, this enzyme oxidizes 3-hydroxyanthranilate with a specific activity of 8 U/mg of protein. Site-directed mutagenesis experiments revealed the essential role of two conserved histidine residues (His52 and His96) in the NbaC sequence. The NbaC activity is also dependent on the presence of Fe(2+) but is inhibited by other metal ions, such as Zn(2+), Cu(2+), and Cd(2+). The NbaD protein was overproduced as a 38.7-kDa protein, and its specific activity towards 2-amino-3-carboxymuconate-6-semialdehyde was 195 U/mg of protein. Further processing of 2-aminomuconate-6-semialdehyde to pyruvic acid and acetyl coenzyme A was predicted to proceed via the activities of NbaE, NbaF, NbaG, NbaH, NbaI, and NbaJ. The predicted amino acid sequences of these proteins are highly homologous to those of the corresponding proteins involved in the metabolism of 2-aminophenol (e.g., AmnCDEFGH in Pseudomonas sp. strain AP-3). The NbaR-encoding gene is predicted to have a regulatory function of the LysR family type. The function of the product of the small open reading frame, NbaX, like the homologous sequences in the nitrobenzene or 2-aminophenol metabolic pathway, remains elusive.     

Co-operative effect of the isoforms of type III antifreeze protein expressed in Notched-fin eelpout, Zoarces elongatus Kner

We found that Notched-fin eelpout, which lives off the north east coast of Japan, expresses an antifreeze protein (AFP). The liver of this fish contains DNAs that encode at least 13 type III AFP isoforms (denoted nfeAFPs). The primary sequences of the nfeAFP isoforms were categorized into SP- and QAE-sephadex binding groups, and the latter were further divided into two subgroups, QAE1 and QAE2 groups. Ice crystals observed in HPLC-pure nfeAFP fractions are bipyramidal in shape with different ratios of c and a axes, suggesting that all the isoforms are able to bind ice. We expressed five recombinant isoforms of nfeAFP and analyzed the thermal hysteresis (TH) activity of each as a function of protein concentration. We also examined the change in activity on mixing the isoforms. TH was estimated to be 0.60 degrees C for the QAE1 isoform, 0.11 degrees C for QAE2, and almost zero for the SP isoforms when the concentrations of these isoforms was standardized to 1.0 mm. Significantly, the TH activity of the SP isoforms showed concentration dependence in the presence of 0.2 mm QAE1, indicating that the less active SP isoform becomes 'active' when a small amount of QAE1 is added. In contrast, it does not become active on the addition of another SP isoform. These results suggest that the SP and QAE isoforms of type III AFP have different levels of TH activity, and they accomplish the antifreeze function in a co-operative manner.