Sequence analysis of genomic DNA (680 Mb) by GS-FLX-Titanium sequencer in the monogonont rotifer, Brachionus ibericus

The monogonont rotifer, Brachionus ibericus (S type), is considered to be a promising model species for developmental biology, evolution, and environmental genomics. In an attempt to accelerate the molecular understanding of B. ibericus, we sequenced 680.5 Mb of genomic DNA using the genome sequencer GS-FLX-Titanium. We obtained 2,062,621 reads (average read length 329.9 bp) and 145,418 contigs (total contigs length 125.7 Mb) after excluding small reads (less than 200 bp) from the assembly, and finally obtained 10,133 unigenes (E value ?? 9.00E?04) after non-redundant (NR) BLAST search. In this article, we summarize the genomic DNA sequences of B. ibericus and discuss their potential use in the study of reproductive biology, endocrinology, environmental genomics, and ecotoxicological studies, and for providing insight into the genetic basis of mechanisms such as egg formation, antioxidant stress defense, and xenobiotic metabolism.     

Community-based colorectal cancer intervention in underserved Korean Americans

Background: Despite evidence of a decline in both incidence and prevalence of colorectal cancer nationwide, it remains the second most commonly diagnosed cancer and the third highest cause of mortality among Asian Americans, including Korean Americans. This community-based and theoretically guided study evaluated a culturally appropriate intervention program that included a bilingual cancer educational program among Korean Americans including information on CRC risks, counseling to address psychosocial and access barriers, and patient navigation assistance. Methods: A two-group quasi-experimental design with baseline and post-intervention assessment and a 12-month follow-up on screening was used in the study. Korean Americans (N = 167) were enrolled from six Korean churches. The intervention group received culturally appropriate intervention program addressing accessibility and psychosocial barriers, and navigation assistance for screening. The control group received general health education that included cancer-related health issues and screening. Results: There was a significant difference (p < 0.05) between the post-intervention and control groups in awareness of CRC risk factors. There was also a significant improvement in the pre–post across HBM measures in the intervention group for perceived susceptibility (p < 0.05) and benefits and barriers to screening (p < 0.001). At baseline, 13% of participants in the intervention group and 10% in control group reported having had a CRC cancer screening test in the previous year. At the 12-month post-intervention follow-up, 77.4% of participants in the intervention group had obtained screening compared to 10.8% in the control group. Conclusion: While health disparities result from numerous factors, a culturally appropriate and church-based intervention can be highly effective in increasing knowledge of and access to, and in reducing barriers to CRC screening among underserved Koreans.     

Evaluation of a fosmid-clone-based microarray for comparative analysis of swine fecal metagenomes

Glass slide arrayed with fosmid clone DNAs generated from swine feces as probes were fabricated and used as a metagenome microarray (MGA). MGA appeared to be specific to their corresponding target genomic fragments. The detection limit was 10 ng of genomic DNA (ca. 10(6) bacterial cells) in the presence of 1000 ng of background DNA. Linear relationships between the signal intensity and the target DNA (20-100 ng) were observed (r ( 2 )=0.98). Application of MGA to the comparison of swine fecal metagenomes suggested that the microbial community composition of swine intestine could be dependent on the health state of swine.     

Production of recombinant hirudin in galactokinase-deficientSaccharomyces cerevisiae by fed-batch fermentation with continuous glucose feeding

The artificial gene coding for anticoagulant hirudin was placed under the control of theGAL10 promoter and expressed in the galactokinase-deficient strain (Δgal1) ofSaccharomyces cereivisiae, which uses galactose only as a gratuitous inducer in order to avoid its consumption. For efficient production of recombinant hirudin, a carbon source other than galactose should be provided in the medium to support growth of the Δgal1 strain. Here we demonstrate the successful use of glucose in the fed-batch fermentation of the Δgal1 strain to achieve efficient production of recombinant hirudin, with a yield of up to 400 mg hirudin/L.     

Internalized group V secretory phospholipase A2 acts on the perinuclear membranes.

Mammalian secretory phospholipases A(2) (sPLA(2)) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA(2) mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA(2) acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA(2)s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA(2) hydrolysis products or cytokines, are necessary for the internalization of sPLA(2) into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA(2) internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA(2) induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA(2) acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis.     

Comprehensive Analysis to Improve the Validation Rate for Single Nucleotide Variants Detected by Next-Generation Sequencing

Next-generation sequencing (NGS) has enabled the high-throughput discovery of germline and somatic mutations. However, NGS-based variant detection is still prone to errors, resulting in inaccurate variant calls. Here, we categorized the variants detected by NGS according to total read depth (TD) and SNP quality (SNPQ), and performed Sanger sequencing with 348 selected non-synonymous single nucleotide variants (SNVs) for validation. Using the SAMtools and GATK algorithms, the validation rate was positively correlated with SNPQ but showed no correlation with TD. In addition, common variants called by both programs had a higher validation rate than caller-specific variants. We further examined several parameters to improve the validation rate, and found that strand bias (SB) was a key parameter. SB in NGS data showed a strong difference between the variants passing validation and those that failed validation, showing a validation rate of more than 92% (filtering cutoff value: alternate allele forward [AF]≥20 and AF<80 in SAMtools, SB<–10 in GATK). Moreover, the validation rate increased significantly (up to 97–99%) when the variant was filtered together with the suggested values of mapping quality (MQ), SNPQ and SB. This detailed and systematic study provides comprehensive recommendations for improving validation rates, saving time and lowering cost in NGS analyses.     

The in vitro anti-platelet,antioxidant and cellular immunity activity of <Emphasis Type="Italic">Phellinus gilvus</Emphasis> fractional extracts

The in vitro anti-platelet and antioxidant activities of various solvent extracts from Phellinus gilvus (PG), and the effects of hot water extract from PG (PGW) on murine cellular immunity were investigated. Chloroform extract (CE), methanol extract (ME) and butanol extract (BE) from PG could significantly inhibit platelet aggregation induced by thrombin. Ethyl acetate extract (EAE), BE, ME from PG had significant 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity compared with the control, and the EAE showed the highest effect with IC₅₀ values of 13.34 μg/ml, which is higher than that of ascorbic acid (40 μg/ml). In addition, EAE displayed the inhibition of xanthine oxidase (XO) activity with IC₅₀ value of 2.45 μg/ml. As to the cellular immunity activity, PGW could enhance both the lipopolysaccharide (LPS)-induced B lymphocyte proliferation and concanavalin A (Con A)-induced T lymphocyte proliferation in vitro. The phagocytosis of both peritoneal macrophages and RAW264.7 macrophage cells were also increased by the addition of PGW. Moreover, PGW was found to inhibit the nitric oxide (NO) production of RAW264.7 macrophages induced by LPS in a concentration-dependant manner.