Mutation of nonessential cysteines shows that the NF-κB essential modulator forms a constitutive noncovalent dimer that binds IκB kinase-ß with high affinity.

NEMO (NF-κB essential modulator) associates with catalytic subunits IKKα and IKKß to form the IκB kinase (IKK) complex and is a key regulator of NF-κB pathway signaling. Biochemical and structural characterization of NEMO has been challenging, however, leading to conflicting data about basic biochemical properties such as the oligomeric state of active NEMO and its binding affinity for IKKß. We show that up to seven of NEMO's 11 cysteine residues can be mutated to generate recombinant full-length NEMO that is highly soluble and active. Using a fluorescence anisotropy binding assay, we show that full-length NEMO binds a 44-mer peptide encompassing residues 701-745 of IKKß with a K(D) of 2.2 ± 0.8 nM. The IKKß binding affinities of mutants with five and seven Cys-to-Ala substitutions are indistinguishable from that of wild-type NEMO. Moreover, when expressed in NEMO -/- fibroblasts, the five-Ala and seven-Ala NEMO mutants can interact with cellular IKKß and restore NF-κB signaling to provide protection against tumor necrosis factor α-induced cell death. Treatment of the NEMO-reconstituted cells with H2O2 led to the formation of covalent dimers for wild-type NEMO and the five-Ala mutant, but not for the seven-Ala mutant, confirming that Cys54 and/or Cys347 can mediate interchain disulfide bonding. However, the IKKß binding affinity of NEMO is unaffected by the presence or absence of interchain disulfide bonding at Cys54, which lies within the IKKß binding domain of NEMO, or at Cys347, indicating that NEMO exists as a noncovalent dimer independent of the redox state of its cysteines. This conclusion was corroborated by the observation that the secondary structure content of NEMO and its thermal stability were independent of the presence or absence of interchain disulfide bonds.

Comprehensive experimental and computational analysis of binding energy hot spots at the NF-κB essential modulator/IKKß protein-protein interface.

We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction (PPI) interface between nuclear factor kappa B (NF-κB) essential modulator (NEMO) and IκB kinase subunit ß (IKKß), an interaction that is critical for NF-κB pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NEMO binding domain (NBD) region of IKKß contains the highest concentration of hot-spot residues, the strongest of which are W739, W741, and L742 (ΔΔG = 4.3, 3.5, and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for ~16 Å to additionally include the regions that bind IKKß L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot-spot regions centered on IKKß residues L708/V709 and L719/I723. The computational approach successfully identified all three hot-spot regions on IKKß. Moreover, the method was able to accurately quantify the energetic importance of all hot-spot residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small-molecule inhibitors that target the NEMO/IKKß interaction. They additionally clarify the structural and energetic complementarity between "pocket-forming" and "pocket-occupying" hot-spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces.

Physician assistant students' perceptions of an entry-level doctorate degree.

PURPOSE: In recent years, a growing trend toward clinical doctorate degrees has emerged in several allied health professions. However, few studies have been conducted within the physician assistant (PA) profession related to changing the entry-level degree for PAs to a clinical doctorate. METHODS: A descriptive, quantitative study assessing PA students' perceptions of changing the entry-level credential for PAs to a clinical doctorate was conducted. Thirty randomly selected programs with 1,966 students were invited to participate in the survey. RESULTS: Of the programs invited, 25 (83%) participated, with 486 (25%) full-time students completing the survey. Of the respondents, 56.1% (272) were first-year students and 43.9% (213) were second-year students. Both groups had a negative perception of changing the entry-level degree for PAs to a clinical doctorate, indicating the doctorate will raise the cost of PA education and discourage some people from entering the profession (82.1%, n = 398); a doctorate for PAs will cause confusion for patients (71.3%, n = 346); and that there is no need for the profession-specific doctorate since the master's degree sufficiently prepares PAs to practice in today's health care setting (70.9%, n = 344). CONCLUSIONS: The present findings lend additional support to previous studies, endorsing the master's degree as the entry-level and terminal degrees.

Program evaluation: how faculty addressed concerns about the nursing program.

The goal of any nursing program is to graduate competent, compassionate future nurse leaders. Evaluating outcome data is also imperative. The authors realized that a more in-depth analysis of our program was needed to address low passing rates. As a result, we assessed and evaluated each course against the NCLEX-RN test plan to determine specific strengths as well as areas needing improvement. The process led to curriculum and teaching changes, resulting in a 42% increase in the NCLEX-RN pass rate in the first year.

Demography of gut symbiotic and aposymbiotic Nezara viridula L. (Hemiptera: Pentatomidae).

Nezara viridula L. is a highly polyphagous and cosmopolitan pentatomid stink bug. Despite its economic importance, aspects of its biology are poorly understood. N. viridula has one primary bacterium associated with its gastric caeca, which females provide to offspring by smearing it on the surface of eggs during oviposition. We studied the impact of three temperatures and egg mass surface sterilization on N. viridula's nymphal development rate and reproductive performance. Our results show that maintenance of the symbiont is affected both by temperature and egg mass surface sterilization. We detected the symbiont in 100, 84, and 8.3% of the untreated control insects at 20, 25, and 30 degrees C, respectively, by using polymerase chain reaction. In insects originated from surface sterilized egg masses, the symbiont was never detected at 20 or 30 degrees C and was detected in only 1 of 21 insects at 25 degrees C. Nymphal mean development time decreased with increasing temperature, but there were no differences between the sterilized and control treatments. Sterilized insects at 20 degrees C lived longer than insects in any other treatment but never laid eggs. Life table analysis of N. viridula adults showed that net reproductive rate, intrinsic rate of increase, finite rate of increase, and gross reproductive rate were not significantly different among treatments except at 20 degrees C for the surface sterilized treatment. Mean generation time, however, was significantly longer at 20 degrees C (70.96 +/- 4.43 d), regardless of the surface sterilization treatment. Our results highlight the effect that temperature has on the maintenance of this symbiosis and its relationship with N. viridula host's development and reproduction.

Nezara viridula (Hemiptera: Pentatomidae) feeding patterns in macadamia nut in Hawaii: nut maturity and cultivar effects.

Nezara viridula L. (Hemiptera: Pentatomidae) is a serious pest of macadamia nuts, Macadamia integrifolia, in Hawaii. Using ruthenium red dye to stain stink bug feeding probes, feeding activity was determined for nuts of various maturity levels harvested from the tree and off the ground throughout the growing season in five commercial cultivars. Damage occurred in the tree and on the ground during all nut growth stages. Damage on the ground was often higher than in the tree. Cultivar 246 was more susceptible to attack than cultivars 333 and 800. It was previously thought that cultivar susceptibility was related to husk and shell thickness, but cultivar 246 showed higher damage than other cultivars even during early nut development when the nuts are small and before the shell has formed. This suggests that shell and husk thickness may play a secondary role in susceptibility to feeding by N. viridula. Monitoring N. viridula feeding activity during early nut development may help alert growers to potential problems later in the season, but early-season probing activity in immature nuts was not a good predictor of damage levels in mature nuts later in the season in our study.

Quantitative surface plasmon resonance imaging: a simple approach to automated angle scanning.

Here we present an automated angle-scanning surface plasmon resonance imaging (SPRi) instrument which provides multiplexed, quantitative reflectance data over a wide angular range. Angle-dependent artifacts, which arise from the simple optical setup, are corrected using software. This enables monitoring of significantly different surface coatings in many solvents, which would be outside the dynamic range of typical fixed-angle instruments. Operation in the visible to near-infrared range without the need for reconfiguration extends the instrument capabilities to increase sensitivity or to investigate the optical properties of surface films. This instrument provides maximum flexibility to study a wide range of systems with full exploitation of the quantitative capabilities of SPRi achieved by fitting data to the Fresnel model.

Regression of left ventricular hypertrophy in children with antihypertensive therapy.

Left ventricular hypertrophy (LVH) is a complication of hypertension that has received great attention in the adult population. Large-scale randomized control studies in adults have demonstrated that LVH regresses in response to pharmacologic intervention. While it is known that LVH occurs as a complication of hypertension in the pediatric population, few studies have focused on its regression with pharmacological intervention. We report on three cases of hypertension-induced LVH in the pediatric population and its regression after treatment with antihypertensive medications. This report brings to light the need for larger, prospective studies on the incidence, natural history, and treatment of LVH associated with hypertension in the pediatric population.

Assessing Nezara viridula (Hemiptera: Pentatomidae) feeding damage in macadamia nuts by using a biological stain.

Damage caused by southern green stink bug, Nezara viridula (L.), to macadamia nuts, Macadamia integrifolia Maiden & Betche, is normally determined after nuts are harvested and processed, which may be many months after damage occurred in the field. We developed a method using ruthenium red dye to stain stink bug feeding probes and indirectly assess feeding activity in macadamia nuts. By using the staining method, feeding probes were easily detected on the husk, shell, and kernel. Husk probing was highly correlated (0.80-0.90) with feeding and damage to the kernel. Failure rate to detect kernel damage from stained husk probes was generally <6%. The staining method was equally effective for immature and mature nuts; therefore, N. viridula feeding activity can be monitored throughout the season to evaluate pest management tactics and forecast outbreak populations.

Detection and quantification of low-molecular-weight aldehydes in pharmaceutical excipients by headspace gas chromatography.

The adverse effect of reactive chemical residues on the quality of drug products has necessitated the determination of low-molecular-weight aldehydes in pharmaceutical excipients. An analytical methodology for the detection of trace amounts of C1-C8 aliphatic aldehydes and benzaldehyde in excipients is described. The proposed procedure is based on the derivatization of aldehydes in aqueous solution with O-2,3,4,5,6-(pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA), followed by static headspace gas chromatographic (SHS-GC) analysis of PFBHA aldehyde oximes with negative chemical ionization (NCI) MS detection. The method developed was demonstrated to be simple, selective, sensitive, and was successfully applied to the screening of aldehydes at sub-microg/g levels in over 30 typical excipients. The most abundant aldehydes found in the samples were formaldehyde and acetaldehyde, for which a rapid and reliable routine quantification method by readily available SHS-GC instrumentation coupled with flame-ionization detection was also developed and validated.