Response of innate immune factors in abalone Haliotis diversicolor supertexta to pathogenic or nonpathogenic infection

Cell free hemolymph from Haliotis diversicolor supertexta was prepared from fluid collected at 1, 4, 8. 12. 24, 48 96 It after injection with either Escherichia coli, Vibrio parahaemolyticus or 0.9 NaCl solution (control group). The response of selected innate immune parameters (lysozyme, antibacterial activity, alkaline phosphatase, acid phosphatase, phenoloxidase, and superoxide dismutase) was investigated. Results showed that the activities of ACP (Acid Phosphatase) from abalone injected with V. parahaemolyticus were much higher than that of the control group at 24 h after injection. The ALP (Alkaline Phosphatase) activities of abalone challenged with V. parahaemolyticus were significantly higher than those of the control group at 8 h and increased further up to 48 h after the challenge. In contrast, the activities of ALP and ACP in the E. coli-challenged group showed no statistically significant differences at any of the sampling times. The activities of SOD (Superoxide Dismutase) in cell free hemolymph from the V. parahaemolyticus-exposed group were significantly lower than those of the control group at both I h and 24 h, whereas there was no difference in SOD activity observed in the group exposed to E. coli at any of the sampling times. The activities of lysozyme and phenoloxidase in Haliotis diversicolor were relatively low in both control and bacteria-ex posed groups when compared with reports for other invertebrates no significant difference was found between the infected groups and the control for these two parameters, due to the low activities and high individual variance

Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

Background: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffinembedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll TM Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and-RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA tha

Poplar GTL1 Is a Ca2+/Calmodulin-Binding Transcription Factor that Functions in Plant Water Use Efficiency and Drought Tolerance

Diminishing global fresh water availability has focused research to elucidate mechanisms of water use in poplar, an economically important species. A GT-2 family trihelix transcription factor that is a determinant of water use efficiency (WUE), PtaGTL1 (GT-2 like 1), was identified in Populus tremula × P. alba (clone 717-IB4). Like other GT-2 family members, PtaGTL1 contains both N- and C-terminal trihelix DNA binding domains. PtaGTL1 expression, driven by the Arabidopsis thaliana AtGTL1 promoter, suppressed the higher WUE and drought tolerance phenotypes of an Arabidopsis GTL1 loss-of-function mutation (gtl1-4). Genetic suppression of gtl1-4 was associated with increased stomatal density due to repression of Arabidopsis STOMATAL DENSITY AND DISTRIBUTION1 (AtSDD1), a negative regulator of stomatal development. Electrophoretic mobility shift assays (EMSA) indicated that a PtaGTL1 C-terminal DNA trihelix binding fragment (PtaGTL1-C) interacted with an AtSDD1 promoter fragment containing the GT3 box (GGTAAA), and this GT3 box was necessary for binding. PtaGTL1-C also interacted with a PtaSDD1 promoter fragment via the GT2 box (GGTAAT). PtaSDD1 encodes a protein with 60% primary sequence identity with AtSDD1. In vitro molecular interaction assays were used to determine that Ca2+-loaded calmodulin (CaM) binds to PtaGTL1-C, which was predicted to have a CaM-interaction domain in the first helix of the C-terminal trihelix DNA binding domain. These results indicate that, in Arabidopsis and poplar, GTL1 and SDD1 are fundamental components of stomatal lineage. In addition, PtaGTL1 is a Ca2+-CaM binding protein, which infers a mechanism by which environmental stimuli can induce Ca2+ signatures that would modulate stomatal development and regulate plant water use