Ostrinia furnacalis PBP2 solution NMR structure: Insight into ligand binding and release mechanisms.

Ostrinia furnacalis is an invasive lepidopteran agricultural pest that relies on olfaction for mating and reproduction. Male moths have an extremely sensitive olfactory system that can detect the sex pheromones emitted by females over a great distance. Pheromone-binding proteins present in the male moth antenna play a key role in the pheromone uptake, transport, and release at the dendritic membrane of the olfactory neuron. Here, we report the first high-resolution NMR structure of a pheromone-binding protein from an Ostrinia species at pH 6.5. The core of the Ostrinia furnacalis PBP2 (OfurPBP2) consists of six helices, α1a (2-14), α1b (16-22), α2 (27-37), α3 (46-60), α4 (70-80), α5 (84-100), and α6 (107-124) surrounding a large hydrophobic pocket. The structure is stabilized by three disulfide bridges, 19-54, 50-108, and 97-117. In contrast to the unstructured C-terminus of other lepidopteran PBPs, the C-terminus of OfurPBP2 folds into an α-helix (α7) at pH 6.5. The protein has nanomolar affinity towards both pheromone isomers. Molecular docking of both pheromones, E-12 and Z-12-tetradecenyl acetate, to OfurPBP2 revealed that the residues Met5, Lys6, Met8, Thr9, Phe12, Phe36, Trp37, Phe76, Ser115, Phe118, Lys119, Ile122, His123, and Ala128 interact with both isomers, while Thr9 formed a hydrogen bond with the acetate head group. NMR structure and thermal unfolding studies with CD suggest that ligand release at pH 4.5 is likely due to the partial unfolding of the protein.

1H, 13C, and 15N resonance assignment and secondary structure of the pheromone-binding protein2 from the agricultural pest Ostrinia furnacalis (OfurPBP2).

Ostrinia furnacalis, a lepidopteran moth, is an invasive pest found in Asia, Australia, Africa, and parts of the United States. The O. furnacalis pheromone-binding protein2 (OfurPBP2), present in the male moth antenna, plays a role in the detection of female-secreted pheromone in a process that leads to mating. To understand the structural mechanism of pheromone binding and release in this pest, we have initiated characterization of OfurPBP2 by solution NMR. Here, we report the backbone resonance assignments and the secondary structural elements of OfurPBP2 at pH 6.5 using uniformly 13C, 15N-labeled protein with various triple resonance NMR experiments. The assignments are 97% completed for backbone and 88% completed for side-chain resonances. The secondary structure of OfurPBP2, based on backbone chemical shifts, consists of eight α-helices, including a well-structured C-terminal helix.

Pheromone Perception: Mechanism of the Reversible Coil-Helix Transition in Antheraea polyphemus Pheromone-Binding Protein 1.

Pheromone-binding protein (PBP) in male moth antennae transports pheromone to the olfactory receptor neuron by undergoing a pH-dependent conformational switch, from PBPB at higher pH to PBPA at lower pH, associated with ligand binding and release, respectively. The characteristic feature of the dramatic protein switch is the pH-dependent reversible coil-helix transition of the C-terminus. In the PBPB conformation at pH >6.0, the C-terminus is exposed to the solvent as a coil while the ligand occupies the hydrophobic pocket. However, in the PBPA conformation at acidic pH, the C-terminus switches to a helix and releases the ligand by outcompeting it for the hydrophobic pocket. In Antheraea polyphemus PBP1 (ApolPBP1), the C-terminus (P129-V142) is composed predominantly of hydrophobic residues except for three strategically located acidic residues: Asp132, Glu137, and Glu141. Here, we report for the first time on the consequences of the mutation of one or more acidic residues in the pH-driven reversible coil-helix transition of the ApolPBP1 C-terminus through biophysical characterization. Mutation of any single acidic residue in the C-terminus to its neutral counterpart destabilizes the helix formation at lower pH; these mutants exist as a mixture of both conformations. However, mutation of the two terminal acidic residues together knocks out the protein switch and adversely affects both ligand binding and release functions. Thus, these mutant proteins remain in the open (PBPB) conformation at all pH levels.

Structure and Function Studies of Asian Corn Borer Ostrinia furnacalis Pheromone Binding Protein2.

Lepidopteran male moths have an extraordinarily sensitive olfactory system that is capable of detecting and responding to minute amounts of female-secreted pheromones over great distances. Pheromone-binding proteins (PBPs) in male antennae ferry the hydrophobic ligand across the aqueous lymph to the olfactory receptor neuron triggering the response. PBPs bind ligands at physiological pH of the lymph and release them at acidic pH near the receptor while undergoing a conformational change. In Anthereae polyphemus PBP1, ligand binding to the hydrophobic pocket and its release is regulated by two biological gates: His70 and His95 at one end of the pocket and C-terminus tail at the other end. Interestingly, in Asian corn borer Ostrinia furnacalis PBP2 (OfurPBP2), critical residues for ligand binding and release are substituted in both biological gates. The impact of these substitutions on the ligand binding and release mechanism in OfurPBP2 is not known. We report here overexpression of soluble OfurPBP2 and structural characterization at high and low pH by circular dichroism (CD) and NMR. Ligand binding and ab initio model development were carried out with fluorescence and small-angle X-ray scattering (SAXS) respectively. OfurPBP2 in solution at pH 6.5 is homogeneous, well-folded and has a compact globular shape.

Tracking maternal mortality declines in Mongolia between 1992 and 2007: the importance of collaboration.

OBJECTIVE: To describe the declining trend in maternal mortality observed in Mongolia from 1992 to 2007 and its acceleration after 2001 following implementation of the Maternal Mortality Reduction Strategy by the Ministry of Health and other partners. METHODS: We performed a descriptive analysis of maternal mortality data collected through Mongolia's vital registration system and provided by the Mongolian Ministry of Health. The observed declining mortality trend was analysed for statistical significance using simple linear regression. We present the maternal mortality ratios from 1992 to 2007 by year and review the basic components of Mongolia's Maternal Mortality Reduction Strategy for 2001-2004 and 2005-2010. FINDINGS: Mongolia achieved a statistically significant annual decrease in its maternal mortality ratio of almost 10 deaths per 100 000 live births over the period 1992-2007. From 2001 to 2007, the maternal mortality ratio in Mongolia decreased approximately 47%, from 169 to 89.6 deaths per 100 000 live births. CONCLUSION: Disparities in maternal mortality represent one of the major persisting health inequities between low- and high-resource countries. Nonetheless, important reductions in low-resource settings are possible through collaborative strategies based on a horizontal approach and the coordinated involvement of key partners, including health ministries, national and international agencies and donors, health-care professionals, the media, nongovernmental organizations and the general public.

Emergency medical services assessment and treatment of children with special health care needs before and after specialized paramedic training.

INTRODUCTION: This study evaluates whether a continuing education program for paramedics, focusing on Children with Special Health Care Needs, improved paramedics' assessment and management. METHODS: Emergency Medical Services responses for children, 21 years of age or younger, with a congenital or acquired condition or a chronic physical or mental illness, were identified. The responses before and after the specialized education program were reviewed by a multidisciplinary team to evaluate assessment and management of the children. Interreviewer agreement between the nurses on the team and between the physicians on the team was assessed. We also evaluated whether there was an improvement in assessment and care by paramedics completing our education program. RESULTS: Significant improvement was seen in appropriate assessment and overall care by paramedics who completed our specialized education program. Reviewers also noted an appropriate rating for the initial assessment category more often for responses involving paramedics who had the training. Agreement on whether assessment and treatment was appropriate for all five reviewers varied considerably, ranging from 32% to 93%. Overall there was a high percentage of agreement (>70%) between the nurses and between the physicians on most items. However, kappa statistics did not generally reflect good agreement except for most of the focused assessment items and some treatment and procedure items. CONCLUSION: Most of the documentation on the EMS records indicated appropriate assessment and treatment during all responses for Children with Special Health Care Needs. Nevertheless, the results indicate that paramedics may improve their assessment and management of these children after specialized continuing education.

Increasing paramedics' comfort and knowledge about children with special health care needs.

This study evaluated a continuing education program for paramedics about children with special health care needs (CSHCN). Pretraining, posttraining, and follow-up surveys containing two scales (comfort with CSHCN management skills and comfort with Pediatric Advanced Life Support [PALS] skills) were administered. Objective measures of knowledge were obtained from pre- and posttraining tests. Differences in average scores were assessed using t-tests. Response rates for paramedics completing the program ranged from 94% for the posttraining survey, 81% for the initial comfort survey, 56% for the knowledge pretest, and 56% for the follow-up survey. PALS comfort scores were significantly higher than CSHCN comfort scores both before and after training, both P < .01. Posttraining surveys showed an increase in CSHCN comfort, P < .01. The follow-up surveys showed a significant decline in CSHCN comfort, P = .05. Scores on the tests showed a similar pattern, with a significant increase in knowledge from pre- to posttraining (P = .02) and a significant decrease in knowledge from posttraining to follow-up (P < .01). Comfort was significantly higher for standard pediatric skills than for specialized management skills. Completion of the self-study program was associated with an increase in comfort and knowledge, but there was some decay over time.

Training paramedics: emergency care for children with special health care needs.

OBJECTIVE: To enhance knowledge and comfort related to the emergency care of children with special health care needs (CSHCN) through an innovative continuing education program for paramedics. METHODS: A self-study program presenting in-depth information about common problems that affect the assessment and management of a child's airway, breathing, circulation, disability, and environment (ABCDEs), regardless of the child's diagnosis, was developed. This program used a manual, a video, practice mannequins, and skills evaluations to teach skills to paramedics employed at a municipal fire department. RESULTS: Pre- and posttraining surveys found that the paramedics were significantly more comfortable with the assessment and management of CSHCN after the completion of the self-study program, with a pretraining average of 2.83 and posttraining average of 4.20 on a five-point Likert-type scale, t(37) = 12.87, p < 0.001. A skills evaluation showed that skills performance varied widely across 21 skills, ranging from skills mastery to low skills knowledge. On the posttraining survey, between 74% and 94% of the paramedics rated each topic (tracheostomies, indwelling central venous catheters, cerebrospinal fluid shunts, gastrostomies, child abuse, and latex allergy) as applicable to their practices as paramedics. CONCLUSION: Given the growing population of CSHCN, it is important to provide specialized education to increase an EMS provider's preparedness to respond to emergency situations involving children with special health care needs.

Unsuspected heterotopic pregnancy in a woman without risk factors.

A 27-year-old woman presented to the emergency department with abdominal pain. One week prior to this she had had an ultrasound which revealed an intrauterine pregnancy (IUP). A second ultrasound performed at our institution revealed an intrauterine pregnancy along with a concomitant ectopic pregnancy which had ruptured. This case emphasizes that coincident pregnancies may occur in women who are without risk of ectopic pregnancy or multiple gestations. In addition, ultrasound must be evaluated fully for heterotopic pregnancy in all women.