The Development and Characterization of Near-Isogenic and Pyramided Lines Carrying Resistance Genes to Brown Planthopper with the Genetic Background of Japonica Rice (Oryza sativa L.).

The brown planthopper (BPH: Nilaparvata lugens Stål.) is a major pest of rice, Oryza sativa, in Asia. Host plant resistance has tremendous potential to reduce the damage caused to rice by the planthopper. However, the effectiveness of resistance genes varies spatially and temporally according to BPH virulence. Understanding patterns in BPH virulence against resistance genes is necessary to efficiently and sustainably deploy resistant rice varieties. To survey BPH virulence patterns, seven near-isogenic lines (NILs), each with a single BPH resistance gene (BPH2-NIL, BPH3-NIL, BPH17-NIL, BPH20-NIL, BPH21-NIL, BPH32-NIL and BPH17-ptb-NIL) and fifteen pyramided lines (PYLs) carrying multiple resistance genes were developed with the genetic background of the japonica rice variety, Taichung 65 (T65), and assessed for resistance levels against two BPH populations (Hadano-66 and Koshi-2013 collected in Japan in 1966 and 2013, respectively). Many of the NILs and PYLs were resistant against the Hadano-66 population but were less effective against the Koshi-2013 population. Among PYLs, BPH20+BPH32-PYL and BPH2+BPH3+BPH17-PYL granted relatively high BPH resistance against Koshi-2013. The NILs and PYLs developed in this research will be useful to monitor BPH virulence prior to deploying resistant rice varieties and improve rice's resistance to BPH in the context of regionally increasing levels of virulence.

Structural characterization suggests models for monomeric and dimeric forms of full-length ezrin.

Ezrin is a member of the ERM (ezrin-radixin-moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ezrin. We observe a bistable array of phenylalanine residues in the core of subdomain F3 that is mobile in the active form and locked in the dormant form. As subdomain F3 is pivotal in binding membrane proteins and phospholipids, these transitions may facilitate activation and signaling. Full-length ezrin forms stable monomers and dimers. We used small-angle X-ray scattering to determine the solution structures of these species. As expected, the monomer shows a globular domain with a protruding helical coiled coil. The dimer shows an elongated dumbbell structure that is twice as long as the monomer. By aligning ERM sequences spanning metazoan evolution, we show that the central helical region is conserved, preserving the heptad repeat. Using this, we have built a dimer model where each monomer forms half of an elongated antiparallel coiled coil with domain-swapped FERM:C-terminal domain complexes at each end. The model suggests that ERM dimers may bind to actin in a parallel fashion.

Semiquantitative and quantitative analysis of protein-DNA interactions using steady-state measurements in surface plasmon resonance competition experiments.

One method commonly used to characterize protein-DNA interactions is surface plasmon resonance (SPR). In a typical SPR experiment, chip-bound DNA is exposed to increasing concentrations of protein; the resulting binding data are used to calculate a dissociation constant for the interaction. However, in cases in which knowledge of the specificity of the interaction is required, a large set of DNA variants has to be tested; this is time consuming and costly, in part because of the requirement for multiple SPR chips. We have developed a new protocol that uses steady-state binding levels in SPR competition experiments to determine protein-binding dissociation constants for a set of DNA variants. This approach is rapid and straightforward and requires the use of only a single SPR chip. Additionally, in contrast to other methods, our approach does not require prior knowledge of parameters such as on or off rates, using an estimate of the wild-type interaction as the sole input. Utilizing relative steady-state responses, our protocol also allows for the rapid, reliable, and simultaneous determination of protein-binding dissociation constants of a large series of DNA mutants in a single experiment in a semiquantitative fashion. We compare our approach to existing methods, highlighting specific advantages as well as limitations.

Characterization of a family of RanBP2-type zinc fingers that can recognize single-stranded RNA.

The recognition of single-stranded RNA (ssRNA) is an important aspect of gene regulation, and a number of different classes of protein domains that recognize ssRNA in a sequence-specific manner have been identified. Recently, we demonstrated that the RanBP2-type zinc finger (ZnF) domains from the human splicing factor ZnF Ran binding domain-containing protein 2 (ZRANB2) can bind to a sequence containing the consensus AGGUAA. Six other human proteins, namely, Ewing's sarcoma (EWS), translocated in liposarcoma (TLS)/FUS, RNA-binding protein 56 (RBP56), RNA-binding motif 5 (RBM5), RNA-binding motif 10 (RBM10) and testis-expressed sequence 13A (TEX13A), each contains a single ZnF with homology to the ZRANB2 ZnFs, and several of these proteins have been implicated in the regulation of mRNA processing. Here, we show that all of these ZnFs are able to bind with micromolar affinities to ssRNA containing a GGU motif. NMR titration data reveal that binding is mediated by the corresponding surfaces on each ZnF, and we also show that sequence selectivity is largely limited to the GGU core motif and that substitution of the three flanking adenines that were selected in our original selection experiment has a minimal effect on binding affinity. These data establish a subset of RanBP2-type ZnFs as a new family of ssRNA-binding motifs.

A mass spectrometric investigation of the ability of metal complexes to modulate transcription factor activity.

ESI mass spectrometry was used to assess the ability of metal complexes to inhibit binding of a transcription factor to a DNA molecule containing its recognition sequence.

Effects of ventilation with 100% oxygen during early hyperdynamic porcine fecal peritonitis.

OBJECTIVE: Early goal-directed therapy aims at balancing tissue oxygen delivery and demand. Hyperoxia (i.e., pure oxygen breathing) has not been studied in this context, since sepsis increases oxygen radical production, which is believed to be directly related to the oxygen tension. On the other hand, oxygen breathing improved survival in various shock models. Therefore, we hypothesized that hyperoxia may be beneficial during early septic shock. DESIGN: Laboratory animal experiments. SETTING: Animal research laboratory at university medical school. SUBJECTS: Twenty domestic pigs of either gender. INTERVENTIONS: After induction of fecal peritonitis, anesthetized and instrumented pigs were ventilated with either 100% oxygen or supplemental oxygen as needed to maintain arterial hemoglobin oxygen saturation > or = 90%. Normotensive and hyperdynamic hemodynamics were achieved using hydroxyethyl starch and norepinephrine infusion. MEASUREMENTS AND MAIN RESULTS: Before and at 12, 18, and 24 hrs of peritonitis, we measured lung compliance; systemic, pulmonary, and hepatosplanchnic hemodynamics; gas exchange; acid-base status; blood isoprostanes; nitrates; DNA strand breaks; and organ function. Gluconeogenesis and glucose oxidation were calculated from blood isotope and expiratory 13CO2 enrichments during continuous intravenous 1,2,3,4,5,6-(13)C6-glucose. Apoptosis in lung and liver was assessed postmortem (TUNEL staining). Hyperoxia did not affect lung mechanics or gas exchange but redistributed cardiac output to the hepatosplanchnic region, attenuated regional venous metabolic acidosis, increased glucose oxidation, improved renal function, and markedly reduced the apoptotic death rate in liver and lung. CONCLUSIONS: During early hyperdynamic porcine septic shock, 100% oxygen improved organ function and attenuated tissue apoptosis without affecting lung function and oxidative or nitrosative stress. Therefore, it might be considered as an additional measure in the first phase of early goal-directed therapy.

Child behavior and quality of life in pediatric obstructive sleep apnea.

OBJECTIVE: To assess behavior and quality of life in children with obstructive sleep apnea (OSA) undergoing tonsillectomy and adenoidectomy compared with control children. DESIGN: Prospective controlled study. SETTING: Hospital-based pediatric otolaryngology practice. PARTICIPANTS: Forty-two children (25 boys and 17 girls; mean [SD] age, 5.8 [2.5] years) with OSA confirmed by positive findings on polysomnography undergoing tonsillectomy and adenoidectomy and 41 control children (29 boys and 12 girls; mean [SD] age, 7.3 [3.8] years) with no history of snoring undergoing unrelated elective surgery. INTERVENTIONS: Parents completed the standardized Child Behavior Checklist and a validated pediatric OSA quality-of-life survey before and 3 months after surgery. MAIN OUTCOME MEASURES: Child Behavior Checklist T scores and score classifications and quality-of-life survey mean scores. RESULTS: Change in mean total problem T score was significantly greater in the OSA group (from 51.6 at baseline to 48.3 at follow-up) than in controls (from 45.5 at baseline to 46.7 at follow-up) (P = .03). The improvement in total T score classification (normal vs borderline or abnormal) was significant for children with OSA compared with control children (P = .009). Children with OSA had significant improvements in the quality-of-life survey mean total score and all individual domain scores compared with controls (P<.001). CONCLUSIONS: Behavioral and emotional difficulties are found in children with documented OSA compared with control children, and they improve after treatment. Large improvements in disease-specific quality of life are also found. Scores on a standardized measure of behavior assessment demonstrated significant correlation with scores on a validated quality-of-life instrument.