Efficient Imputation of Missing Markers in Low-Coverage Genotyping-by-Sequencing Data from Multiparental Crosses

We consider genomic imputation for low-coverage genotyping-by-sequencing data with high levels of missing data. We compensate for this loss of information by utilizing family relationships in multiparental experimental crosses. This nearly quadruples the number of usable markers when applied to a large rice Multiparent Advanced Generation InterCross (MAGIC) study.     

Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter

Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis might be reducing liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4⁺ T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.     

Direct Measurements of Drag Forces in C. elegans Crawling Locomotion

With a simple and versatile microcantilever-based force measurement technique, we have probed the drag forces involved in Caenorhabditis elegans locomotion. As a worm crawls on an agar surface, we found that substrate viscoelasticity introduces nonlinearities in the force-velocity relationships, yielding nonconstant drag coefficients that are not captured by original resistive force theory. A major contributing factor to these nonlinearities is the formation of a shallow groove on the agar surface. We measured both the adhesion forces that cause the worm’s body to settle into the agar and the resulting dynamics of groove formation. Furthermore, we quantified the locomotive forces produced by C. elegans undulatory motions on a wet viscoelastic agar surface. We show that an extension of resistive force theory is able to use the dynamics of a nematode’s body shape along with the measured drag coefficients to predict the forces generated by a crawling nematode.     

Genetic variation of three autosomal STR Loci D21S1435, D21S1411, and D21S1412 in Korean population

The autosomal tetranucleotide short tandem repeat loci D21S1435, D21S1411 and D21S1412 were analyzed in samples of unrelated 200 Korean individuals. The loci showed no significant deviations from Hardy–Weinberg equilibrium. Alleles were assigned according to the International Society for Forensic Haemogenetics (ISFH) recommendations. The power of discrimination of the analyzed markers was found to be high for the populations, thereby facilitating the validation and efficiency of these STR markers in forensic human identification and paternity testing. To our knowledge, this is the first report of the nomenclature and the allele frequency data for these three STR loci in Korean population.     

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.     

Matrix metalloproteinase sensitive gold nanorod for simultaneous bioimaging and photothermal therapy of cancer

Herein, we developed matrix metalloprotease (MMP) sensitive gold nanorods (MMP-AuNR) for cancer imaging and therapy. It was feasible to absorb NIR laser and convert into heat as well as visualize MMP activity. We showed the possibility of gold nanorods as a hyperthermal therapeutic agent and MMP sensitive imaging agent both in vitro and in vivo condition. The results suggested potential application of MMP-AuNR for simultaneous cancer diagnosis and therapy.     

Effects of cell-seeding methods of human osteoblast culture on biomechanical properties of porous bioceramic scaffold

Many studies have been performed to accelerate osteoinduction and osteoconduction into porous ceramic scaffolds by seeding them with cells. In this study, we compared available cell-seeding methods on a porous β-tricalcium phosphate (β-TCP) scaffold and evaluated the effects of cell-seeding on the mechanical properties of the porous β-TCP scaffold. Three types of porous bioceramic scaffolds were used: dry scaffold, scaffold wetted with media, and scaffold cultivated with normal human osteoblasts (NHOs). Cell-seeding into the porous β-TCP scaffolds was performed by conventional, centrifuge, high-density, and vacuum methods. After confirming cell proliferation with MTT assay and cell staining, a compressive test was performed after 2 and 4 weeks of cell culture. The vacuum method based on the high-density cell culture inserted effectively NHOs into the β-TCP scaffolds. The compressive elastic modulus of wetted β-TCP scaffolds decreased significantly (p < 0.05) about 20∼30% after 2 and 4 weeks of incubation in comparison with that of the dry scaffold. However, the compressive strength of the scaffolds cultivated with NHOs for 3 weeks was significantly (p < 0.05) higher than that of scaffolds without NHOs. The vacuum with the high-density of cell-seeding seems to be a suitable method for seeding cells into complex porous ceramic scaffolds. Cell proliferation and uniform distribution in the scaffolds can change the initial mechanical properties of porous ceramic scaffolds.     

A new thermolabile alkaline phospholipase D from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS628

A phospholipase D (PLD₆₂₈), constitutively secreted by Streptomyces sp. CS628, was purified by ion exchange with CM Trisacryl and gel filtration with Sepharose CL-6B. The enzyme production was highest with peptone and starch as nitrogen and carbon sources, and at 30°C with an initial medium pH of 7.5. Molecular weight, optimum pH, optimum temperature, pH stability, and thermostability of the enzyme were 50 kDa, pH 9.6, 30°C, pH 5.7 ∼ 10.6 and ≤30°C, respectively. Detergents and metal ions had varied effects on the enzyme activity. Importantly, PLD₆₂₈ could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol or ethanolamine, which are extensively used to assess the activity, suggesting that PLD₆₂₈ lacks the transphosphatidylation activity. PLD₆₂₈ could be a novel PLD based on its biochemical characteristics, which are significantly different from previously reported PLDs, such as thermolability, highest activity at alkaline pH, and lack of transphosphatidylation activity.     

Diversity of cold-active protease-producing bacteria from arctic terrestrial and marine environments revealed by enrichment culture

A new approach for enrichment culture was applied to obtain cold-active protease-producing bacteria for marine and terrestrial samples from Svalbard, Norway. The method was developed for the enrichment of bacteria by long-term incubation at low temperatures in semi-solid agar medium containing meat pieces as the main source of carbon and energy. ZoBell and 0.1× nutrient broth were added for marine and terrestrial microorganisms, respectively, to supply basal elements for growth. One to three types of colonies were observed from each enrichment culture, indicating that specific bacterial species were enriched during the experimental conditions. Among 89 bacterial isolates, protease activity was observed from 48 isolates in the screening media containing skim milk. Good growth was observed at 4°C and 10°C while none of the isolates could grow at 37°C. At low temperatures, enzyme activity was equal to or higher than activity at higher temperatures. Bacterial isolates were included in the genera Pseudoalteromonas (33 isolates), Arthrobacter (24 isolates), Pseudomonas (16 isolates), Psychrobacter (6 isolates), Sphingobacterium (6 isolates), Flavobacterium (2 isolates), Sporosarcina (1 isolate), and Stenotrophomonas (1 isolate). Protease activity was observed from Pseudoalteromonas (33 isolates), Pseudomonas (10 isolates), Arthrobacter (4 isolates), and Flavobacterium (1 isolate).