Genetic diversity among perennial wild rice Oryza rufipogon Griff., in the Mekong Delta

Oryza rufipogon Griff. is a perennial species of wild rice widely distributed along the channels and rivers of the Mekong Delta, Vietnam. This study attempted to find centers of diversity among wild rice populations in this area and their inter‐relationships. The highest genetic diversity was found in the Dong Thap population and the lowest in the Can Tho population. Maternal diversity evaluated using chloroplast INDELs detected ten plastid types, five of which were novel relative to other Asian countries. The mitochondrial genome suggested two unique deletions. One 699‐bp deletion via short tandem repeats was accompanied by another deletion including orf153. All accessions carrying the mitochondrial type were found in a particular plastid type. This unique maternal lineage was confined to specific channels where it showed vigorous vegetative growth in comparison to upstream areas where various maternal lineages and maximum genetic diversity occurred. This area along the Mekong Delta is a center of not only nuclear but also maternal diversity.     

Activation mechanism and steady state kinetics of Bruton's tyrosine kinase

Bruton's tyrosine kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr(551) within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr(551) causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH2) revealed that BTK employs a ternary complex mechanism with KmATP = 84 +/- 20 microM and KmS1 = 37 +/- 8 microM. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mm, suggesting that BTK may employ a "substrate clamping" type of kinetic mechanism whereby the substrate Kd is weaker than Km. This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.     

Contact analysis of a posterior cervical spine plate using a three-dimensional canine finite element model

The development of a three-dimensional finite element model of a posteriorly plated canine cervical spine (C3-C6) including contact nonlinearities is described. The model was created from axial CT scans and the material properties were derived from the literature. The model demonstrated sufficient accuracy from the results of a mesh convergence test. Significant steps were taken toward establishing model validation by comparison of plate surface strains with a posteriorly plated canine cervical spine under three-point bending. This model was developed to better characterize the contact pressures at the various interfaces under average physiologic canine loading. The analysis showed that the screw-plate interfaces had the highest values of all the mechanical parameters evaluated.     

Role of primary sensorimotor cortex and supplementary motor area in volitional swallowing: a movement-related cortical potential study

We investigated the role of the cerebral cortex, particularly the face/tongue area of the primary sensorimotor (SMI) cortex (face/tongue) and supplementary motor area (SMA), in volitional swallowing by recording movement-related cortical potentials (MRCPs). MRCPs with swallowing and tongue protrusion were recorded from scalp electrodes in eight normal right-handed subjects and from implanted subdural electrodes in six epilepsy patients. The experiment by scalp EEG in normal subjects revealed that premovement Bereitschaftspotentials (BP) activity for swallowing was largest at the vertex and lateralized to either hemisphere in the central area. The experiment by epicortical EEG in patients confirmed that face/tongue SMI and SMA were commonly involved in swallowing and tongue protrusion with overlapping distribution and interindividual variability. BP amplitude showed no difference between swallowing and tongue movements, either at face/tongue SMI or at SMA, whereas postmovement potential (PMP) was significantly larger in tongue protrusion than in swallowing only at face/tongue SMI. BP occurred earlier in swallowing than in tongue protrusion. Comparison between face/tongue SMI and SMA did not show any difference with regard to BP and PMP amplitude or BP onset time in either task. The preparatory role of the cerebral cortex in swallowing was similar to that in tongue movement, except for earlier activation in swallowing. Postmovement processing of swallowing was lesser than that of tongue movement in face/tongue SMI; probably suggesting that the cerebral cortex does not play a significant role in postmovement processing of swallowing. SMA plays a supplementary role to face/tongue SMI both in swallowing and tongue movements.     

17-epiestriol,an estrogen metabolite,is more potent than estradiol in inhibiting vascular cell adhesion molecule 1 (VCAM-1) mRNA expression

17-beta estradiol (17-beta E(2)) attenuates the expression of vascular cell adhesion molecule 1 (VCAM-1) in vivo at physiological levels (pg/ml), whereas supraphysiological concentrations of 17-beta E(2) (ng/ml) are required in vitro. We assessed whether a metabolite of estrogen, which could only be generated in vivo, might be a more potent inhibitor of VCAM-1 expression and thereby explain this discrepancy. We report here that 17-epiestriol, an estrogen metabolite and a selective estrogen receptor (ER) beta agonist, is approximately 400x more potent than 17-beta E(2) in suppressing tumor necrosis factor (TNF) alpha-induced VCAM-1 mRNA as well as protein expression in human umbilical vein endothelial cells. Genistein, an ERbeta agonist, at low concentrations (1 and 10 nm) also suppressed TNFalpha-induced VCAM-1 mRNA expression. These actions of 17-epiestriol and genistein were significantly attenuated in the presence of the estrogen receptor antagonist ICI-182780. Other estrogenic compounds such as ethinyl estradiol and estrone did not have any effect on TNFalpha-induced VCAM-1 expression at the concentrations tested. We further show that, 1) 17-epiestriol induces the expression of endothelial nitric-oxide synthase mRNA and protein, 2) 17-epiestriol prevents TNFalpha-induced migration of NFkappaB into the nucleus, 3) N(G)-nitro-l-arginine methyl ester, an inhibitor of NO synthesis, abolishes 17-epiestriol-mediated inhibition of TNFalpha-induced VCAM-1 expression and migration of NFkappaB from the cytoplasm to the nucleus. Our results indicate that 17-epiestriol is more potent than 17-beta E(2) in suppressing TNFalpha-induced VCAM-1 expression and that this action is modulated at least in part through NO.